R

R10 (Quail)

▶TPD52 (Tumor Protein D52)

R7 Binding Protein (R7BP)

▶R7BP/R9AP

R7BP/R9AP

Kirill A. Martemyanov1 and Pooja Parameswaran2

1Department of Neuroscience, The Scripps Research

Institute, Jupiter, Florida, USA2Department of Pharmacology, University of

Minnesota, Minneapolis, Minneapolis, MN, USA

Synonyms

R7 Binding Protein (R7BP); RGS9 Anchor Protein

(R9AP)

Introduction and Historical Background

Regulator of G protein signaling (RGS) proteins con-

stitute a diverse family with more than 30 members

that contain the hallmark RGS domain. Most members

serve as negative regulators of G protein signaling by

catalyzing the GTP hydrolysis on Ga subunits leading

to their inactivation (Ross and Wilkie 2000; Hollinger

S. Choi (ed.), Encyclopedia of Signalling Molecules, DOI 10.1007# Springer Science+Business Media, LLC 2012

and Hepler 2002). Based on their structural organiza-

tion and sequence homology RGS proteins are divided

into 5–6 families (Ross and Wilkie 2000; Hollinger

and Hepler 2002). The R7 RGS family (R7 RGS)

contains multidomain proteins conserved from

C. elegans to humans that, in mammals are represented

by four members: RGS6, RGS7, RGS9, and RGS11.

R7 RGS proteins play important roles in the nervous

system by controlling neurotransmitter action at rho-

dopsin, m-opioid, D2 dopamine, and GABA(B) recep-

tors (Anderson et al. 2009a).

The unique feature of this group is that they form

obligatory complexes with Gb5, an atypical member

of the G protein beta subunit family (Sondek and

Siderovski 2001; Slepak 2009). The stability of all

R7 RGS proteins crucially depends on this interaction

and knockout of Gb5 in mice leads to severe

downregulation in the levels of all four R7 RGS pro-

teins (Chen et al. 2003). Localization of R7 RGS-Gb5complexes in discrete membrane compartments in

native cells in parallel with cytoplasmic distribution

in heterologous expression systems have prompted

speculations that their membrane anchoring is medi-

ated by unidentified proteins (Hu and Wensel 2002;

Lishko et al. 2002). This led to searches for additional

binding partners.

For R7 RGS proteins, these studies were very pro-

ductive and resulted in the identification of two homol-

ogous binding partners: RGS9 Anchor Protein (R9AP)

and R7 Binding Protein (R7BP), novel proteins that

now constitute a two-member family. First, proteomics

search for RGS9 binding partners in the retina identi-

fied a transmembrane protein R9AP (Hu and Wensel

2002). Three years later, a homologous R7BP protein

was found as a binding partner of RGS9 in the brain

/978-1-4419-0461-4,

N R7BP R9AP

N

R 1524 R7BP/R9AP

using similar approach (Martemyanov et al. 2005).

R7BP was also independently discovered as a universal

anchor for all R7 RGS proteins by Ken Blumer’s group

by in silico BLAST searches (Drenan et al. 2005).

PBR

C

TM

C

CCCC

α α αα α α

R7BP/R9AP, Fig. 1 Schematic structure of R7BP and R9APproteins. Membrane attachment of R7BP is mediated by

palmitoylated cysteines (wavy lines) acting in conjunction with

polybasic region (PBR). R9AP is anchored to the membrane by

transmembrane segment (TM). Blue cylinders designate

predicted alpha helical regions. CC label designates heptad

repeats-containing region that is predicted to engage in coiled-

coil interactions

Structural Organization

Both R7BP and R9AP are small membrane proteins

that show distant homology to SNARE proteins

involved in the regulation of exocytosis (Keresztes

et al. 2003; Martemyanov et al. 2003). N-termini of

R9AP and R7BP are predicted to contain 4 alpha

helices organized in the bundle with the fourth helix

containing prominent heptad repeats, a feature under-

lying coiled coil interactions (Fig. 1). This helical

bundle region mediates binding to the R7 RGS proteins

(Anderson et al. 2009a; Jayaraman et al. 2009). From

the side of the RGS proteins, N-terminal DEP (Dishev-

eled, Egl-10, Pleckstrin) and DHEX (DEP Helical

Extension) domains were found to be essential for the

association (Anderson et al. 2009a; Jayaraman et al.

2009) although no high-resolution information of the

determinants involved in binding have been reported.

The C-termini of R7BP and R9AP contain membrane

anchoring elements. In R9AP this is presented by

transmembrane region, while R7BP is anchored

through palmitoylation of two conserved cysteine res-

idues which are additionally aided by a polybasic

stretch with a sequence resembling membrane attach-

ment motif of Ras proteins (Drenan et al. 2005; Song

et al. 2006). In summary, while differing in amino

acid composition, both R9AP and R7BP contain

two domains: the N-terminal RGS binding region and

C-terminal membrane localization domain.

Distribution, Subcellular Localization, andInteractions with R7 RGS Proteins

In mammals, expression of both R9AP and R7BP pro-

teins appears to be confined to the neuronal tissues

(Martemyanov et al. 2005; Grabowska et al. 2008).

However, only limited set of non-neuronal tissues

have been investigated and it remains possible that

the proteins could be expressed more broadly, as for

example, ample amounts of R9AP mRNA are found

across various tissues in birds (Keresztes et al. 2003).

While R7BP is expressed broadly in all regions of

central and peripheral nervous system, R9AP is more

restricted and appears to be reliably found only in the

retina, where it is found in three cell types: rod and

cone photoreceptors (Hu and Wensel 2002) and ON-

bipolar cells (Cao et al. 2009; Jeffrey et al. 2010).

At the subcellular level, R9AP is targeted to the disc

membranes of the outer segments, a ciliated compart-

ment of the photoreceptors and dendritic tips of the

ON- bipolar neurons (Hu and Wensel 2002; Cao et al.

2009; Jeffrey et al. 2010). Likewise, R7BP was also

found to be localized predominantly in the membrane

compartments. Its significant fraction is found in the

postsynaptic density and extrasynaptically at the

plasma membrane of the spines and dendrites (Ander-

son et al. 2007b; Grabowska et al. 2008). Although to

a lesser extent, some R7BP immunoreactivity is also

present pre-synaptically in axons (Grabowska et al.

2008). Subcellular tartgeting of R7BP and R9AP to

their membrane compartments requires the membrane

attachment sequence at the C-terminus (Drenan et al.

2006; Song et al. 2006).

Anchor R7 RGS Location

Broad range of neuronsMartemyanov et al. (2005)

Broad range of neuronsMartemyanov et al. (2005)Grabowska et al. (2008)

Striatal neuronsMartemyanov et al. (2005)Anderson et al. (2007b)

Rod and cone photoreceptorsHU et at. (2002)

Retina ON-bipolar cellsCao et al. (2009)Jeffrey et al. (2010)

R7BP

R9AP

RGS6

RGS7

RGS9-2

RGS9-1

RGS11

R7BP/R9AP,Fig. 2 Physiologically

relevant complexes of R7BP

and R9AP in vivo

R7BP/R9AP 1525 R

R

While plasma membrane compartment is the sole

localization site of R9AP, R7BP has been reported to

have an alternative destination – nucleus. It possesses

two active nuclear localization sequences that are

masked by palmitoylation (Drenan et al. 2005; Song

et al. 2006). When palmitoylation is abolished, R7BP

undergoes translocation to the nucleus, a phenomenon

most readily demonstrated in cultured cells (Drenan

et al. 2005; Song et al. 2006). However, the fraction of

R7BP in the nucleus is very small and no studies have

yet reported translocation in vivo under physiological

conditions.

R7BP and R9AP anchors show differential selec-

tivity in their association with individual R7 RGS

members. This is determined by both their interaction

specificity and co-expression patterns. At the biochem-

ical level, R9AP can bind only to RGS9 and homolo-

gous RGS11, but not to RGS6 or RGS7 (Martemyanov

et al. 2005). In contrast, R7BP associates with all R7

RGS proteins (Drenan et al. 2005; Martemyanov et al.

2005). However, the distribution of R7 RGS protein

subtypes does not always overlap with that of R7BP

and R9AP making some combinations physiologically

irrelevant. For example, although R9AP can bind to

RGS9-2, it is not expressed in the striatum, where

RGS9-2 is instead found in complex with R7BP. Sim-

ilarly, RGS11 can bind to R7BP, but because it is

present exclusively in bipolar cells where R9AP is

more abundant, most of it is found in complex with

R9AP (Cao et al. 2008). See Fig. 2 for details on

physiologically relevant configurations of R7BP and

R9AP complexes with R7 RGS proteins.

Regulation of the RGS Protein Localizationand Activity

Studies with transfected cells indicate that R7 RGS-

Gb5 complexes are predominantly cytoplasmic

(Drenan et al. 2005; Song et al. 2006). In contrast,

co-transfection with R7BP (or R9AP for RGS9-1)

targets R7 RGS proteins to the plasma membrane

(Hu and Wensel 2002; Drenan et al. 2005; Song et al.

2006). A similar situation is observed in striatal neu-

rons in vivo for RGS9-2 that becomes mis-localized

from post-synaptic densities and plasma membrane

compartments upon elimination of R7BP (Anderson

et al. 2007b). In striatal neurons, R7BP is also involved

GPCR

GTP

Response

GαGDP

Gα R7BPR9AP

R7RGS

R7RGS Gβ5

Gβ5γ

γ

ββ

R7BP/R9AP, Fig. 3 Role of

membrane anchors R7BP and

R9AP in G protein signaling

regulation

R 1526 R7BP/R9AP

in targetingRGS7 to the post-synaptic density (Anderson

et al. 2009b). Likewise, the role of R9AP in localiza-

tion of RGS9-1 to the disc membranes of the photore-

ceptor outer segments is also well established (Hu and

Wensel 2002). Nevertheless, mechanisms governing

localization of R7 RGS proteins appear to be complex

and anchor-independent targeting has been observed

for both RGS7 and RGS11 in the bipolar cells of the

retina (Cao et al. 2008; Cao et al. 2009)

The nucleus has been repeatedly reported to be an

alternative destination for relatively minor fraction of

several R7 RGS proteins (Burchett 2003). Consistent

with its nuclear shuttling, R7BP is capable of targeting

R7 RGS to the nucleus of the transfected cells upon de-

palmitoylation (Drenan et al. 2005). Furthermore,

knockout of R7BP abolishes nuclear localization of

a significant fraction of RGS7 in the central nervous

system neurons (Panicker et al. 2010). However, the

functional significance of plasma membrane – nuclear

shuttling of R7 RGS proteins or their functional role at

this location is currently unknown.

In addition to localization, association with R9AP

and R7BP influences the efficiency of R7 RGS to

catalyze G protein GTPase (GAP) activity. For

instance, R9AP has been shown to potentiate the abil-

ity of RGS9-1 and RGS11 to stimulate GTPase of Gatand Gao, respectively (Hu et al. 2003; Masuho et al.

2010). The most straightforward explanation for the

stimulatory effects is facilitation of the R7 RGS com-

plex compartmentalization with membrane bound

G proteins and receptors. The restriction of the diffu-

sion of the complex from the three-dimensional cyto-

plasm to the two-dimensional plane of the plasma

membrane is expected to speed up Ga-GTP encounter.

However, the mechanism is likely to be more complex,

as at least R9AP action was shown to provide an

allosteric modulation of the RGS9 and RGS11 com-

plexes (Baker et al. 2006; Masuho et al. 2010).

Effects on the Proteolytic Stability of theR7 RGS Complexes

Perhaps the most pronounced effects of membrane

anchors are on regulation of post-translational stability

of R7 RGS proteins. These effects are observed only

with two RGS proteins: RGS9 and RGS11. Studies

with genetic knockouts indicate that elimination of

R9AP severely compromises proteolytic stability of

RGS9 (Keresztes et al. 2004) and RGS11 (Cao et al.

2008) in the retina. Likewise, knockout of R7BP leads

to destabilization of RGS9-2 in the brain (Anderson

et al. 2007a). This explains why loss-of-function muta-

tions in R9AP produce the same phenotype as RGS9-1

R7BP/R9AP 1527 R

mutations – slow adaptation to both light and dark

conditions and difficulty in tracking moving objects

(Nishiguchi et al. 2004). Similarly, knockout of R7BP

causes motor co-ordination deficits characteristic of

severe reduction in levels of RGS9-2 (Anderson et al.

2010). Loss of R7BP has been shown to facilitate

recruitment of the destabilizing chaperone Hsc70

(Posokhova et al. 2010) to RGS9-2 and trigger its

proteolysis by cellular cysteine proteases (Anderson

et al. 2007b). Association of RGS9-Gb5 with R7BP

is controlled dynamically and is sensitive to changes in

oxygenation and neuronal excitability (Anderson et al.

2009b). Because the abundance of R7 RGS proteins

controls the extent of the G protein signaling and has

direct behavioral implications, regulation of R7 RGS

degradation and coupling to R7BP can be viewed as

a plasticity mechanism.

Summary and Conclusions

In summary, R7BP and R9AP proteins play very

important roles in controlling stability, localization

and functional activity of R7 RGS proteins and as

a result critically contribute to the regulation of

G protein signaling (Fig. 3). There are currently a num-

ber of outstanding questions pertaining to the role of

R7BP and R9AP in regulation of (1) G protein signal-

ing selectivity, (2) RGS catalytic activity and (3) phys-

iological processes in the nucleus. Finding answers to

these questions would help gain a better understanding

of G protein signaling.

R

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RA70 (Retinoic Acid-Induced Protein 70)

▶ SKAP-HOM

RAB18

Irene Aligianis and Mark Handley

MRC Human Genetics Unit, Western General

Hospital, Edinburgh, Scotland, UK

Synonyms

AtRabC1-C2b (Arabidopsis thaliana); Rab18b (Danio

rerio); Rab-RP4 (Drosophila melanogaster)

Historical Background

Interest in the RAB proteins stems from early

work on yeast that identified essential roles for the

RABs Ypt1p (Rab1) and Sec4p in pre- and post-

Golgi membrane trafficking (Salminen and Novick

1987; Segev et al. 1988). These findings prompted

efforts to clone other members of the RAB gene

family and it was quickly established that this family

had undergone significant expansion in mammals.

As each new RAB was discovered, it also became

clear that different RAB proteins could adopt spe-

cific subcellular localizations, associating with par-

ticular membrane compartments and regulating the

functions of these organelles. RAB18 was partially

cloned in 1992, and then fully cloned in 1993

(Chavrier et al. 1992; Yu et al. 1993). Initial char-

acterization showed that it was widely expressed in

different tissues and localized to endosomes in

polarized epithelia (Lutcke et al. 1994). Subsequent

research has suggested, however, that it adopts var-

ious different subcellular localizations and can func-

tion in a cell-type-specific manner (Dejgaard et al.

2008; Hashim et al. 2000; Hashimoto et al. 2008;

Martin et al. 2005; Ozeki et al. 2005; Vazquez-

Martinez et al. 2007). Currently, little is known

about the protein-protein interactions that regulate

and mediate RAB18 activity, but genetic evidence

has linked it to the regulation of RAB3 isoforms. In

a recent study, loss-of-function mutations in RAB18

were found to cause Micro syndrome, a disorder

previously associated with mutations in RAB3GAP1

or RAB3GAP2 (Aligianis et al. 2005, 2006; Bem

et al. 2011).

RAB18 1529 R

R

RAB18 in Disease

Micro syndrome is a rare autosomal recessive disor-

der with both developmental and degenerative fea-

tures. Patients present with micropthalmia, atonic

pinpoint pupils, and congenital cataracts. Even with

early cataract surgery, most have only light percep-

tion due to severe cortical visual impairment charac-

terized by normal electroretinogram (ERG), but

virtually absent visually evoked potentials (VEPs).

Affected individuals have severe developmental

delay and do not learn to sit independently, walk,

or talk. They suffer from postnatal growth retardation,

microcephaly, and hypothalamic hypogenitalism

and can have cerebral anomalies including

polymicrogyria and hypoplasia or agenesis of the

corpus callosum. In addition to initial muscular hypo-

tonia, they go on to develop ascending spastic para-

plegia and contractures.

The pathogenesis ofMicro syndrome indicates that

RAB18 function is important in eye and lens devel-

opment, neurotransmission and neuronal migration

and homeostasis. Knockdown of rab18b in zebrafish

produces a phenotype reminiscent of Micro syn-

drome, suggesting that RAB18 has conserved roles

in these processes and that animal models might offer

a valuable resource in future studies (Bem et al. 2011).

Further, as it is now clear that specific physiological

systems are particularly susceptible to loss of func-

tional RAB18, characterization of this deficit at

a cellular and molecular level can be better targeted.

In turn, this work may inform the development of

therapeutics for Micro syndrome and related disor-

ders. However, it is important to note that the physi-

ological roles of RAB18 may in fact be much broader

than can be inferred from the effects of its absence.

RAB proteins can show a varying degree of functional

redundancy in different tissues, either as a result of the

different expression patterns of RABs with

overlapping cellular roles, or because a given RAB

may mediate responses through tissue-specific effec-

tor proteins. Furthermore, RAB18 expression may be

regulated in concert with other factors. Dysregulation

of RAB18 expression has been observed in a number

of different cancers and has been extensively

studied in the context of pituitary tumors (Vazquez-

Martınez and Malagon 2011). In these tumors, its

downregulation has been linked to the hypersecretion

of human growth hormone (hGH) that contributes to

acromegaly in sufferers (Vazquez-Martinez et al.

2008). However, Micro syndrome patients who do

not express any functional RAB18 do not show abnor-

mal hGH secretion.

Rab18 Expression and SubcellularLocalization

Several reports show that RAB18 mRNA is ubiqui-

tously expressed, implying that it has a general cellular

role (Lutcke et al. 1994; Schafer et al. 2000; Yu et al.

1993). However, other evidence suggests that its role is

more specialized. It appears to be expressed at differ-

ent levels in the different tissues examined, with it

being highly expressed in the brain and the heart.

Immunohistochemistry suggests that the protein is

enriched in polarized epithelia (Lutcke et al. 1994).

Furthermore, in a number of situations, its expression

is reported to be inducible. For example, it is induced

in endothelial cells stimulated with histamine (Schafer

et al. 2000), in differentiating adipocytes (Pulido et al.

2011), and in the brains of alloxan-treated rats (Karthik

and Ravikumar 2011). The most convincing evidence

that the protein serves discrete cellular roles, though,

comes from a series of divergent reports on its cellular

localization and function.

RAB18 has been reported to associate with

endosomes in polarized epithelia where it has been

suggested to function in endocytosis (Lutcke et al.

1994). In a macrophage cell line, it localized to a

specialized phagocytic compartment and was suggested

to function in immune evasion (Hashim et al. 2000).

Several reports have found that the protein can localize

to lipid droplets in adipocyte, fibroblast, and epithelial

cell lines (Martin et al. 2005; Ozeki et al. 2005; Pulido

et al. 2011) and this has been linked to roles in lipogen-

esis (Pulido et al. 2011) and lipolysis (Martin et al. 2005;

Pulido et al. 2011). However, in the same cells, it can

localize to endoplasmic reticulum (ER) and Golgi under

some circumstances (Dejgaard et al. 2008; Martin et al.

2005), and one report has suggested that it functions in

Golgi to ER trafficking (Dejgaard et al. 2008). In endo-

crine cell lines and in pituitary melanotropes, it has been

found associated with secretory granules, and suggested

to function in modulation of the secretory response

(Vazquez-Martinez et al. 2007). These reports are

R 1530 RAB18

difficult to reconcile. However, a common feature of

several of them is that the recruitment of RAB18 to

intracellular organelles can be enhanced by cellular

stimulation. In adipocytes, for example, recruitment of

RAB18 to lipid droplets was promoted by treatment

with insulin, which stimulates lipogenesis (Pulido

et al. 2011), or by the b-adrenoceptor agonist isoproter-enol, which stimulates lipolysis (Martin et al. 2005;

Pulido et al. 2011). Similarly, in PC12 and AtT20

cells, stimulation with KCl led to redistribution of

RAB18 from the cytosol to a subpopulation of secretory

granules (Vazquez-Martinez et al. 2007). It will be

important to establish the extent to which different

molecular interactions underlie the functions of

RAB18 in different cell types, and reciprocally, that

to which common molecular interactions underlie its

function at diverse intracellular compartments.

Regulator and Effector Proteins

Following synthesis and posttranslational prenylation,

RAB proteins are regulated by four classes of protein:

GDP-dissociation inhibitors (GDIs), GDI displace-

ment factors (GDFs), Guanine-nucleotide exchange

factors (GEFs), and GTPase-activating proteins

(GAPs). GDIs and GDFs are thought to be general

regulators of multiple RABs, while GEFs and GAPs

are thought to show more specificity for particular

RAB proteins. GDIs can sequester GDP-bound RABs

in the cytosol by binding to their hydrophobic prenyl

groups, but also coordinate with GDFs in the delivery

of RABs to membranes. GEFs catalyze the exchange

of bound GDP for GTP, and so can serve to concentrate

RABs on a particular cellular compartment because

RAB proteins are not susceptible to GDI-mediated

membrane extraction when GTP-bound. GAPs stimu-

late a RAB’s intrinsic GTP-hydrolysis activity, which

converts bound GTP to GDP, thus rendering them

susceptible to extraction once again.

The GEF(s) and GAP(s) that regulate RAB18 are

not known. However, the identification of these

proteins may be key to understanding how it can

be recruited to a wide range of organelles in a cell-

type-specific manner. It is possible that a single

RAB18GEF might be differentially localized in differ-

ent cells. However, in some cases, specific RABs are

the substrates for multiple GEFs, and so differential

expression of RAB18GEFs with different localizations

might offer another explanation for this phenomenon.

Alternatively, RAB18 localization might be regulated

by its GAP(s). The presence of a RABGAP on

a particular membrane compartment can effectively

exclude its target RAB from this compartment by

promoting GTP hydrolysis and GDI-mediated mem-

brane extraction at this location. Thus, the transloca-

tion of the RABGAP away from a given compartment

can indirectly promote RAB recruitment there.

The function of RAB18 is genetically linked to that

of RAB3GAP1 and RAB3GAP2, mutations in which

can also cause Micro syndrome (Aligianis et al. 2005,

2006; Bem et al. 2011). RAB3GAP1 and RAB3GAP2

form a heterodimeric RAB3GAP complex that regu-

lates the activity of RAB3 isoforms (Fukui et al. 1997;

Nagano et al. 1998). However, the activity of

Rab3GAP against many other RABs, including

RAB18, is unknown. Work is underway to determine

whether RAB3GAP is a regulator of RAB18 function.

However, it is also possible that it is a mediator of this

function. RAB proteins mediate cellular responses via

interacting partners generically called “effectors,” and

it has been proposed that RABGAPs acting on one

RAB protein are frequently effectors of another

(Kanno et al. 2010).

While it remains to be established whether

RAB3GAP is a regulator and/or an effector of RAB18

function, several candidate RAB18-interacting proteins

have previously been identified in the literature. A weak

interaction between RAB18 andmammalian suppressor

of Sec4 (Mss4) was found in a screen for RABs that

interact with this protein (Wixler et al. 2011). Investi-

gators used copurification of exogenously expressed

proteins from HEK293 cells to identify this interac-

tion. However, though Mss4 is proposed to be either

a GEF or a RAB chaperone, they found that the protein

showed no GEF activity toward RAB18 and that it

interacted much more strongly with other RAB pro-

teins. Therefore, the physiological relevance of this

finding is unclear. In another screen, this time for

novel RAB-binding proteins, a pull-down assay with

GST-Rab18 identified a potential interaction with

N-ethylmaleimide-sensitive factor (NSF), a protein

involved in the disassembly of cis-SNARE complexes

following membrane fusion (Kanno et al. 2010). This

finding may implicate RAB18 in the fusion process,

though it should be noted that NSF is thought to regu-

late fusion even in the absence of RAB proteins. In

a third study, the Arabidopsis RAB18 orthologue

RAB18 1531 R

AtRabC2a was shown by yeast 2-hybrid (Y2H) and by

in vitro binding assays to interact with a class

V myosin, Arabidopsis myosin XI (Hashimoto et al.

2008). Since myosins function as molecular motors,

transporting cargoes along actin filaments, this inter-

action may suggest a mechanism by which RAB18

could direct organelle mobility. However, because of

sequence divergence of both RABs and myosins

between plants and mammals and the presence of

multiple RAB18 orthologues in Arabidopsis, it will

be necessary to show that the interaction is conserved

in mammals before any such conclusions can be

drawn. Clearly, the validation of proposed effectors,

and the identification of novel effectors, will help to

better define the role of RAB18 in cells.

R

Structure

A RAB18 orthologue is not present in yeast, but its

orthologues are found in plants, nematodes, and flies

(Pereira-Leal and Seabra 2001). Despite this high

degree of conservation, however, attempts to classify

the protein on the basis of phylogeny and active con-

formation have not yielded any clues as to its function

(Pereira-Leal and Seabra 2001). It has been suggested

in the literature that RAB18 is one of a group of RAB

proteins with a putative exocytotic role (Vazquez-Mar-

tınez andMalagon 2011;Wixler et al. 2011). However,

the systematic application of phylogenetic algorithms

and principal components analysis to the RAB protein

family did not place the protein in this group (Collins

2005). Thus, it remains to be shown whether RAB18 is

part of some larger functional subgroup of RAB pro-

teins or possesses a distinct role.

One feature that sets RAB18 apart from most other

RAB proteins is its differential posttranslational mod-

ification. Most RAB proteins have a C-terminal di-

cysteine motif that is subject to modification by two

geranylgeranyl lipid groups. In contrast, RAB18, like

RAB8, RAB13, and ▶RAB23, has a C-terminal

CAAX motif like that of RAS and RHO proteins.

This motif is monoprenylated, and then sequentially

cleaved and carboxymethylated by ER-resident

enzymes (Leung et al. 2007). As methylation affects

the susceptibility of RAB proteins to GDI-mediated

membrane extraction, and is a potentially reversible

modification, this may represent an additional level of

RAB18 regulation.

Summary

To summarize, many questions remain to be answered

about RAB18. In the context of the other members of

the RAB gene family, it appears unusual in terms of the

sequence determinants that aid classification, and it is

subject to atypical posttranslational modification

(Leung et al. 2007; Pereira-Leal and Seabra 2001).

However, it is conserved in all but the simplest eukary-

otes, and so future work should determine whether it is

truly novel, or an unrecognized member of an

established subgroup.

The function of RAB18 has been strongly liked to

that of RAB3GAP1 and RAB3GAP2, as loss-of-

function mutations in each of these proteins causes

clinically indistinguishable Micro syndrome in humans

(Aligianis et al. 2005, 2006; Bem et al. 2011). Therefore,

a primary aim of future work will be to define the

relationship between these proteins. Further, since this

finding implicates RAB18 in a primarily neurological

pathology, work in animal models and neuronal cell

lines should seek to establish its role in these processes.

References

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Hoffmann K, et al. Mutations of the catalytic subunit of

RAB3GAP cause Warburg Micro syndrome. Nat Genet.

2005;37:221–3.

Aligianis IA, Morgan NV, Mione M, Johnson CA, Rosser E,

Hennekam RC, et al. Mutation in Rab3 GTPase-activating

protein (RAB3GAP) noncatalytic subunit in a kindred with

Martsolf syndrome. Am J Hum Genet. 2006;78:702–7.

Bem D, Yoshimura S, Nunes-Bastos R, Bond FC, Kurian MA,

Rahman F, et al. Loss-of-function mutations in RAB18

cause Warburg micro syndrome. Am J Hum Genet. 2011;

88:499–507.

Chavrier P, Simons K, Zerial M. The complexity of the Rab and

Rho GTP-binding protein subfamilies revealed by a PCR

cloning approach. Gene. 1992;112:261–4.

Collins RN. Application of phylogenetic algorithms to assess

Rab functional relationships. Methods Enzymol. 2005;

403:19–28.

Dejgaard SY, Murshid A, Erman A, Kizilay O, Verbich D,

Lodge R, et al. Rab18 and Rab43 have key roles in ER-Golgi

trafficking. J Cell Sci. 2008;121:2768–81.

Fukui K, Sasaki T, Imazumi K, Matsuura Y, Nakanishi H,

Takai Y. Isolation and characterization of a GTPase activat-

ing protein specific for the Rab3 subfamily of small

G proteins. J Biol Chem. 1997;272:4655–8.

Hashim S, Mukherjee K, Raje M, Basu SK, Mukhopadhyay A.

Live Salmonella modulate expression of Rab proteins to

persist in a specialized compartment and escape transport to

lysosomes. J Biol Chem. 2000;275:16281–8.

R 1532 Rab18b (Danio rerio)

Hashimoto K, Igarashi H, Mano S, Takenaka C, Shiina T,

Yamaguchi M, et al. An isoform of Arabidopsis myosin XI

interacts with small GTPases in its C-terminal tail region.

J Exp Bot. 2008;59:3523–31.

Kanno E, Ishibashi K, Kobayashi H, Matsui T, Ohbayashi N,

Fukuda M. Comprehensive screening for novel rab-binding

proteins by GST pull-down assay using 60 different mam-

malian Rabs. Traffic. 2010;11:491–507.

Karthik D, Ravikumar S. Characterization of the brain proteome

of rats with diabetes mellitus through two-dimensional

electrophoresis and mass spectrometry. Brain Res. 2011;

1371:171–9.

Leung KF, Baron R, Ali BR, Magee AI, Seabra MC. Rab

GTPases containing a CAAX motif are processed post-

geranylgeranylation by proteolysis and methylation. J Biol

Chem. 2007;282:1487–97.

Lutcke A, Parton RG, Murphy C, Olkkonen VM, Dupree P,

Valencia A, et al. Cloning and subcellular localization of

novel rab proteins reveals polarized and cell type-specific

expression. J Cell Sci. 1994;107(Pt 12):3437–48.

Martin S, Driessen K, Nixon SJ, Zerial M, Parton RG. Regulated

localization of Rab18 to lipid droplets: effects of lipolytic

stimulation and inhibition of lipid droplet catabolism. J Biol

Chem. 2005;280:42325–35.

Nagano F, Sasaki T, Fukui K, Asakura T, Imazumi K, Takai Y.

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protein. J Biol Chem. 1998;273:24781–5.

Ozeki S, Cheng J, Tauchi-Sato K, Hatano N, Taniguchi H,

Fujimoto T. Rab18 localizes to lipid droplets and induces

their close apposition to the endoplasmic reticulum-derived

membrane. J Cell Sci. 2005;118:2601–11.

Pereira-Leal JB, Seabra MC. Evolution of the Rab family

of small GTP-binding proteins. J Mol Biol. 2001;313:

889–901.

Pulido MR, Diaz-Ruiz A, Jimenez-Gomez Y, Garcia-Navarro S,

Gracia-Navarro F, Tinahones F, et al. Rab18 dynamics in

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Golgi event in yeast secretion. Cell. 1987;49:527–38.

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and characterisation of the human rab18 gene after stimula-

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Rab18b (Danio rerio)

▶RAB18

Rab23

Marga Gual-Soler, Tomohiko Taguchi, Jennifer L.

Stow and Carol Wicking

Institute for Molecular Bioscience, The University of

Queensland, Brisbane, QLD, Australia

Synonyms

Opb; Opb2; Open brain; RAB23, member RAS onco-

gene family

Historical Background

Rab23 (Ras-related protein Rab 23) belongs to the Rab

family of monomeric small guanosine triphosphatases

(GTPases) involved in the regulation of membrane

traffic. Rab GTPases are conserved from yeast to

humans and coordinate the delivery of cargo to its

correct destination within eukaryotic cells. Rab pro-

teins regulate many membrane trafficking steps,

including vesicle formation, budding, motility along

the cytoskeleton, docking, and membrane fusion

(Zerial and McBride 2001; Stenmark 2009). More

than 60 members of the Rab family have been identi-

fied in humans to date (Zerial and McBride 2001). Rab

proteins function as molecular switches cycling from

an active GTP (guanosine triphosphate)-bound form to

an inactive GDP (guanosine diphosphate)-bound form.

The GDP/GTP exchange factors (GEFs) catalyze the

conversion from GDP to GTP-bound forms, whereas

GTP hydrolysis to GDP is catalyzed by GTPase-

activating proteins (GAPs). Once in their active state,

GTP-bound Rabs can recruit specific effector

Rab23 1533 R

molecules to transduce signals in the transport path-

way. These effectors include sorting adaptors, tether-

ing factors, kinases, phosphatases, motor proteins,

GEFs, and GAPs. Crosstalk between Rab GTPases

through their effectors allows the spatiotemporal reg-

ulation of vesicle trafficking (Zerial and McBride

2001; Stenmark 2009). Post-translational modification

by protein prenylation of C-terminal cysteines is gen-

erally required for membrane association and biologi-

cal function of Rab proteins. (Zerial and McBride

2001; Stenmark 2009).

Rab23 was first identified in 1994 using a

PCR-based homology cloning approach, and although

it is expressed ubiquitously in many tissues, its

predominant site of expression is the brain (Olkkonen

et al. 1994). The human RAB23 gene localizes to

chromosome 6p11, is conserved in evolution back to

Drosophila, and encodes a 237 amino acid protein. The

Rab23 protein contains a CAAX-motif (“C” is Cyste-

ine, “A” is an aliphatic amino acid, and “X” is variable)

in its C-terminus, which acts as substrate for the post-

translational prenylation modifications required for

membrane anchoring (Fig. 1) (Olkkonen et al. 1994;

Leung et al. 2007). As described below, several

mutations in the Rab23 gene have been described in

both mouse and human (Fig. 1), providing insight into

the physiological function of Rab23 (Eggenschwiler

et al. 2001; Jenkins et al. 2007; Alessandri et al. 2010).

R

Rab23 and Hedgehog Signaling

Homozygous mutation of the Rab23 gene is responsi-

ble for the mouse open brain (opb) phenotype

(Eggenschwiler et al. 2001). There are two indepen-

dent opb alleles, both of which encode truncated

proteins (Eggenschwiler et al. 2001). The opb1 allele

is a natural mutation, while opb2 was experimentally

induced by N-ethyl-N-nitrosurea (ENU). Opbmice are

characterized by severe defects in neural tube closure

and patterning (Gunther et al. 1994), related to a failure

in the correct specification of neurons along the dorso-

ventral axis of the neural tube. This process is highly

dependent on the correct activity of▶Sonic hedgehog

(SHH), a morphogen secreted from the notochord and

floorplate (Ericson et al. 1997). The hedgehog (HH)

pathway is one of the most pivotal signaling pathways

directing embryonic development, and at the cellular

level is regulated by trafficking events at the primary

cilium (Huangfu et al. 2003). Genetic studies in mice

revealed that the opbmutation rescues the Shhmutant

phenotype, demonstrating that Rab23 acts as a cell

autonomous negative regulator of HH signaling

(Eggenschwiler et al. 2001). Since Rab proteins typ-

ically regulate vesicle trafficking, Rab23 was initially

presumed to have a role in the trafficking of HH

pathway components. Localization studies in a range

of mammalian cell types showed distribution of exog-

enously expressed GFP-tagged Rab23 at the plasma

membrane and on the endocytic pathway in transfer-

rin-positive endosomes, where it co-localized with

the HH receptor patched (PTCH1), but not with the

co-receptor smoothened (SMO) (Evans et al. 2003).

However, further genetic studies suggested a regula-

tory role for Rab23 downstream of both PTCH and

SMO and upstream of the transcriptional regulators of

the HH pathway, the Gli proteins (named after glio-

blastoma from where they were first isolated)

(Eggenschwiler et al. 2006; Yang et al. 2008). Of the

three vertebrate Gli proteins, Gli1 and Gli2 generally

act as transcriptional activators, while Gli3 is primar-

ily processed to a truncated transcriptional repressor.

Studies in the mouse neural tube led to the suggestion

that Rab23 negatively regulates HH signaling through

trafficking of a molecule that mediates the effects of

SMO on the formation of Gli2 activators and Gli3

repressors (Eggenschwiler et al. 2006). To date the

identity of such a factor remains elusive, but increas-

ing evidence suggests that the activation and

processing of Gli proteins, and hence possibly

Rab23 function, is intricately linked to the primary

cilium.

Rab23 and Primary Cilia

The discovery that the primary cilium is an essential

organelle for mammalian HH signaling (Corbit et al.

2005; Huangfu et al. 2003) is arguably one of the most

significant findings in the fields of cell and develop-

mental biology over the past decade. The primary

cilium is a microtubule-based organelle that projects

from the surface of virtually every vertebrate cell type.

The major components of the HH pathway, including

the Gli proteins, localize to the ciliary axoneme exten-

sion and shuttle in and out in a dynamic fashion

(Rohatgi et al. 2007; Kim et al. 2009). The

intraflagellar transport (IFT) system is primarily

Y29X

E48fsX7

Y78fsX30

C85R

E137X

L145X

K39X

R80X

Carpentersyndrome

Openbrain

GTP/Mg2+

Switch domain I

Switch domain II

Prenylation signal

Rab23 protein

1

10

20

30

40

50

60

70

80

90

100

110

120

130

140

150

160

170

180

190

200

210

220

230

237

Rab23, Fig. 1 Rab23domains and mutations.Functional domains of the

237 amino acid protein Rab23.

Human mutations responsible

for Carpenter syndrome are

located on the right column,

and those causing the mouse

open brain phenotype are

situated on the left. All

mutations produce truncated

proteins except C85R, which

causes a non-conservative

substitution from uncharged to

charged amino acid, possibly

impairing normal folding of

the protein. Rab23 structure

was analyzed with Cn3D 4.1

software from NCBI.

(http://www.ncbi.nlm.nih.

gov/Structure/cdd/cddsrv.cgi)

R 1534 Rab23

responsible for trafficking of cargo between the cell

body and the cilia tip, but a number of other accessory

proteins also mediate ciliary trafficking. A screen for

Rab GTPases involved in primary cilium formation

identified Rab8 as the sole Rab localized on primary

cilia. However, in the same study, biochemical

analysis of Rab GTPase-activating proteins (Rab

GAPs) and their attendant Rabs suggested a role

for Rab23 and Rab17 in primary cilia formation

(Yoshimura et al. 2007). More recently, exogenously

expressed wild-type Rab23 was shown to localize to

the primary cilium in Madin-Darby Canine Kidney

(MDCK) epithelial cells (Boehlke et al. 2010). In

these cells, shRNA-mediated depletion of Rab23 or

expression of a GDP-bound form of Rab23 decreased

the steady state level of SMO at the cilium, suggesting

a role for Rab23 in ciliary turnover. Thus, the precise

role of Rab23 at the primary cilium has yet to be

determined, but the findings to date hint at a potential

role in trafficking cargo to or from the cilium.

Rab23 1535 R

Rab23 and Planar Cell Polarity

The planar cell polarity (PCP) pathway coordinates

cell polarization in a given plane across a cell layer,

a process essential for the correct formation of certain

highly ordered differentiated tissues during develop-

ment (Fanto and McNeill 2004). A recent study in

Drosophila provides new evidence for Rab23 as

a PCP regulator (Pataki et al. 2010). Mutations in the

Drosophila Rab23 gene resulted in abnormal trichome

orientation and the formation of multiple hairs on the

wing, leg, and abdomen. This work also showed that

Rab23 associates with the PCP protein Prickle, likely

contributing to its asymmetric cellular accumulation

to regulate the hexagonal packing of Drosophila

wing cells and the orientation of cuticular hairs. Since

components of the PCP pathway are important for the

correct formation and positioning of cilia in vertebrate

cells (Park et al. 2006), these data potentially provide

a further link between Rab23 and cilia. However, cilia

do not appear to be important for PCP or HH signaling

in Drosophila, and a role for Rab23 in vertebrate PCP

signaling has not yet been elucidated.

R

Rab23 in Human Disease

The physiological relevance of Rab23 has been

highlighted by its involvement in a number of human

disorders. Homozygous loss-of-function mutations in

RAB23 are responsible for Carpenter syndrome,

a pleiotropic disorder with autosomal recessive inheri-

tance, characterized by premature closure of the cranial

sutures, polysyndactyly, obesity, and cardiac defects

(Jenkins et al. 2007). Six independent mutations in

RAB23 have been identified in Carpenter syndrome

patients (Alessandri et al. 2010; Jenkins et al. 2007).

Five of the six mutations are predicted to result in

premature protein truncation, and one is a missense

mutation thought to interfere with protein folding

(Fig. 1). They show no apparent clustering to specific

domains within the RAB23 protein and all are likely to

represent loss-of-function alleles. While craniosynosto-

sis and obesity are not classic features of perturbed HH

signaling, obesity in particular has been associated with

disrupted cilia in humans and mice (Sheffield 2010),

again reinforcing a role for Rab23 in ciliogenesis.

Rab23 appears to have functions beyond mamma-

lian embryonic development, as overexpression of

Rab23 has been associated with human cancers. In

the gastric cancer cell line Hs746T, siRNA-mediated

silencing of Rab23 significantly reduced cellular

migration and invasion, whereas overexpression

of Rab23 enhanced invasion in gastric epithelial

(AGS) cells (Hou et al. 2008). Rab23 expression is

also upregulated in hepatocellular carcinoma

(Liu et al. 2007). The finding that Rab23 is upregulated

in a number of human cancers seems contradictory

given that Rab23 antagonizes HH signaling, and

enhanced expression would be expected to result in

pathway inhibition. In a wide range of tumor types,

activation of HH signaling, rather than inhibition, is

generally associated with tumorigenesis. It is possible

that the role of Rab23 in cancer is unrelated to its

regulation of HH signaling, or alternatively that a fine

balance of Rab23 is required for correct functioning in

the tumor environment. Future elucidation of the pre-

cise role of Rab23 in regulating HH signaling,

ciliogenesis, and other cellular events will shed light

on its involvement in cancer and other disease states.

Summary and Perspectives

Genetic and biochemical studies have implicated

Rab23 in embryonic development, thus highlighting

the role of vesicular trafficking in regulating embryo-

genesis at the cellular level. However, no Rab23 effec-

tors or interacting partners have been identified to date

in vertebrates, and as a result no molecular mechanism

for Rab23 action has yet been elucidated. It may be that

Rab23 acts indirectly to inhibit HH signaling. The key

to Rab23 function might be found at the primary cil-

ium, as the two signaling pathways in which Rab23 is

involved (PCP and HH), converge at this organelle

(Veland et al. 2009). Rab23 is not essential for Dro-

sophila development (Pataki et al. 2010) but it is pos-

sible that, unlike Rab23 involvement in HH signaling,

the Rab23-PCP link is evolutionarily conserved. PCP

genes are required for neural tube closure,

a characteristic phenotype of Rab23 and ciliogenesis

mouse mutants (Doudney and Stanier 2005). Taken

together, these observations suggest a key role for

Rab23 at the intersection of the cilia-related HH and

PCP pathways. Elucidation of such a link, along with

the more precise definition of Rab23 function, is likely

to come from future detailed studies at both the whole

organism and cellular levels.

R 1536 RAB23, Member RAS Oncogene Family

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RAB23, Member RAS Oncogene Family

▶Rab23

RAB7a

▶Rab7a in Endocytosis and Signaling

Rab7a in Endocytosis and Signaling

Soumik BasuRay1, Jacob O. Agola1, Patricia A. Jim1,

Matthew N. Seaman2 and Angela Wandinger-Ness1

1Department of Pathology and Cancer Center,

MSC08-4640, University of New Mexico Health

Sciences Center, Albuquerque, NM, USA2Department of Clinical Biochemistry, Cambridge

Institute for Medical Research, Cambridge, UK

Synonyms

BRL-Ras; CMT2B; FLJ20819; PRO2706; RAB7a;

Ras-related protein Rab-7a; Ypt7p (yeast ortholog)

Rab7a in Endocytosis and Signaling 1537 R

Rab7 Historical Background and Function

Mammalian Rab7 was first identified in a rat liver cell

line as BRL-Ras [X12535; NM_023950] and subse-

quently named Rab7 when it was recognized to be

a member of an emerging, separate branch of Ras-

related GTPases now well known as the Rab family

of GTPases [NP_004628.4; P51149; P09527]. Rab7a is

the most widely studied form and encoded on human

chromosome 3q21.3 (mouse chromosome 6) as two

splice variants differing in the 30 untranslated region.

The most intensively studied mammalian forms of

Rab7a (mouse, canine, rat, and human) are 99.5% iden-

tical with only a single conservative change among the

207 amino acids (D/E 196). Amore recently discovered

homolog, Rab7b/Rab7L1, is encoded on human and

mouse chromosome 1q32 and functions in late endo-

some to Golgi trafficking [Q96AH8; Q8VEA8].

Human Rab7b is only 47% identical and 82% homol-

ogous to human Rab7a across its 199 aa length. Fol-

lowing the initial demonstration of Rab7a function in

regulating membrane transport from early to late

endosomes, Rab7a has been found to have critical

roles in autophagy, lipid metabolism, growth factor

signaling, bone resorption, and phagolysosome biogen-

esis (Fig. 1) (Agola et al. 2011).

R

Rab7a Activation and Localization

Typical of Ras-related GTPases, Rab7a undergoes

a cycle of membrane association and dissociation that

is closely linked to nucleotide binding and hydrolysis

(Fig. 2) (Agola et al. 2011). In the membrane-

associated state, Rab7a is GTP bound and active,

while upon hydrolysis the GDP-bound Rab7a is inac-

tive and recycles to the cytoplasm. The GDP-/GTP-

dependent activation cycle is regulated by two sets of

proteins, the guanine nucleotide exchange factors

(GEFs) that catalyze the GTP binding and conversion

to the active GTP-bound state and the GTPase-

activating proteins (GAPs) that stimulate the GTPase

activity of the Rab protein to convert it to the inactive

GDP-bound state. Early reports suggested Vps39 func-

tioned as a Rab7a GEF. More recently, studies in yeast

and C. elegans indicate that Ypt7p/Rab7 activation is

linked to endosome conversion involving coordinate

inactivation and loss of the early endosomal Rab5 and

acquisition and activation of the late endosomal Rab7a

through large multimeric complexes with overlapping

components (Fig. 3a) (Wang et al. 2011a). SAND-1

(Mon1a-Mon1b in vertebrates) binds to the CORVET

components (Vps11, VPS16A, Vps18, and Vps33) and

functions to displace the Rab5 GEF (RABX-5). Sub-

sequent recruitment of Ccz1 through Mon1 and coop-

eration with Vps39 is central to the endosomal

recruitment and activation of Ypt7p in yeast and

Rab7 C. elegans enabling binding to the HOPS com-

plex (Vps11, Vps16A, Vps18, Vps33, and Vps41)

(Fig. 2). Mon1 and Ccz1 are conserved in mammals

(Wang et al. 2011a), though mammalian Rab7a GEF

activity remains to be demonstrated.

The activation of Rab7a is dynamically regulated

through differential interactions of proteins first iden-

tified to be important in autophagy called Rubicon

(RUN domain and cysteine-rich domain containing

Beclin 1-interacting protein) and UVRAG (Liang

et al. 2008; Zhong et al. 2009) (Fig. 2). Rubicon,

a regulatory component of the ▶ phosphatidylinositol

3-kinase complex (PI3KC3; hVps34/hVps15), can

bind and sequester UVRAG and thereby block

Rab7a-mediated transport (Sun et al. 2010, 2011; Lin

and Zhong 2011). Conversely, membrane-bound,

active Rab7a can relieve the inhibition of its activation

by binding Rubicon (Sun et al. 2010).

Proteins of the Tre-Bub-CDC16 (TBC) family

function as GTPase-activating proteins that stimulate

nucleotide hydrolysis. Three family members

(TBC1D2/Armus, TBC1D5, and TBC1D15) have all

been shown to stimulate Rab7a nucleotide hydrolysis

and may regulate Rab7a involvement in discrete func-

tions in coordination with specific signaling (Seaman

et al. 2009; Frasa et al. 2010; Peralta et al. 2010). For

example, on endosomes the recycling of mannose

6-phosphate receptor to the Golgi via retromer is

thought to be regulated by Rab7a/TBC1D5, while the

disassembly of adherens junctions and degradation of

E-cadherin depends on signal integration of Arf6 and

a Rac1/TBC1D2/Rab7a complex. As illustrated by the

specific examples given, the facilitated nucleotide

binding and hydrolysis cycle brings about conforma-

tional changes in Rab7a that modulate its activity and

localization.

Membrane localization is dependent on posttrans-

lational modification with a lipid anchor (prenylation)

(Fig. 2). Nascent Rab7a synthesized on cytosolic ribo-

somes is inactive and GDP bound. Prenylation on two

C-terminal cysteine residues is mediated by the

Rab7a in Endocytosis and Signaling, Fig. 1 Rab7a-regulated pathways. Rab7a regulates endocytic transport from

early to late endosomes in a process requiring Rab5 to Rab7a

conversion. Rab7a also cooperates with other Rab GTPases to

facilitate late endosome-lysosome fusion and phagolysosome

formation (see Table 1). Key Rab7a effectors involved on indi-

vidual pathways are noted in parentheses. Rab7a cooperates with

Rac1 in epithelia to promote internalization of cell adhesion

molecules and in osteoclasts to promote localized hydrolase

secretion for bone resorption

R 1538 Rab7a in Endocytosis and Signaling

universal Rab geranylgeranyl transferase, through rec-

ognition of the last nine amino acid residues of Rab7a

(Wu et al. 2009). The Rab escort protein (REP) serves

as the intermediary for Rab7a presentation to the

prenylating enzyme and first-time membrane associa-

tion (Zhang et al. 2009; Agola et al. 2011). GDP

dissociation inhibitor (GDI) functions as a universal

Rab recycling factor, binding preferentially to doubly

prenylated, GDP-bound Rab7a (Wu et al. 2007). GDI

binding masks the isoprenyl anchor in the cytosol and

renders Rab7a membrane association a reversible pro-

cess that is closely linked to the nucleotide bound

status, based on the fact that GDI has a 3 order of

magnitude higher affinity for Rab7a-GDP than

Rab7a-GTP (Wu et al. 2010). A GDI-displacement

factor (GDF) has been implicated in GDI release dur-

ing endosomal Rab membrane association, though

GEF proteins may also perform this function in con-

junction with nucleotide exchange (Wu et al. 2010).

Once on the membrane and in the GTP-bound state,

Rab7a interacts with diverse effectors to carry out

specific functions.

Rab7a Effectors in the Control of EndocyticTrafficking

Over the years, many effector proteins have been iden-

tified to interact specifically with active GTP-bound

Rab7a (Table 1). Rab7a effectors orchestrate events

ranging from cargo selection to microtubule transloca-

tion to downstream membrane tethering and

endosomal membrane fusion (Fig. 3). Emerging con-

cepts are that Rab7a activation contributes to the

dynamic assembly of large protein complexes in

a spatially and temporally regulated manner (Wang

et al. 2011a). Specific protein complexes serve discrete

functions in the transport process, yet handoffs and

RE

P

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ab7

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ab7

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Rab7ain

EndocytosisandSignaling,Fig.2

Rab7

aactivation

cycle.New

lysynthesized

Rab7aisprenylatedbygeranylgeranyltransferase(G

GT)anddelivered

toendosomal

mem

branes

byrabescortprotein

(REP),thereafter

Rab7amem

branecyclingisfacilitatedbyGDPdissociationinhibitor(G

DI);pathwaysthat

arecommonto

allRab

GTPases.

AGDIdisplacementfactor(G

DF)has

beenim

plicatedin

mem

branetransfer

oflate

endocyticRab9andRab7a.

Rab7aactivationis

closely

linked

toRab5inactivationin

aconversionprocess

that

involves

Mon1.A

Vps39-M

on1-Ccz1complexlikelyacts

asaguaninenucleotideexchangefactor(G

EF)to

promote

activation,whileTBC1D5or

TBC1D15actas

GTPase-activatingproteins(G

APs)topromotehydrolysisandinactivation.A

ctive,GTP-boundRab7aactsas

ascaffoldforsequentiallybindingmultipleeffectors

phosphoinositide3-kinase(PI3K,V

ps34/Vps15),HOPS,R

ubicon,U

VRAG,amongothers(see

Table1)topromotecargoselection,cytoskeletaltranslocation,andmem

branefusion

Rab7a in Endocytosis and Signaling 1539 R

R

Rab7ain

EndocytosisandSignaling,Fig.3

(continued)

R 1540 Rab7a in Endocytosis and Signaling

Rab7a in Endocytosis and Signaling 1541 R

R

multiple layers of regulation are common. The impor-

tance of maintaining transport fidelity is evidenced by

the increasing numbers of human diseases attributable

to defects in endosomal trafficking and Rab7a specif-

ically (Charcot-Marie-Tooth disease type 2) (Cogli

et al. 2009; Zhang et al. 2009; Agola et al. 2011).

On early endosomes, Rab7a functions in cargo

sorting by recruiting the retromer complex (Vps26/

29/35), which enables retrieval of cation-independent

mannose 6-phosphate receptor, TGN38, Wntless

among other cargo from early endosomes to the

Golgi (Rojas et al. 2008; Seaman et al. 2009;McGough

and Cullen 2011) (Fig. 3a). Interaction of retromer with

Snx proteins and actin-binding proteins couples sorting

with membrane tubulation (Harbour et al. 2010;

McGough and Cullen 2011). Dysregulation of retromer

is associated with neurologic diseases, including

Alzheimer’s disease, underscoring the importance of

the Rab7a-retromer link.

On multivesicular bodies and late endosomes,

Rab7a facilitates coordinate cargo sorting and bidirec-

tional transport on microtubules through interactions

with effectors that differentially associate with dynein

or kinesin motors (Fig. 3b–c). Lysosomal sorting and

perinuclear transport are mediated by the Rab7a-

interacting lysosomal protein (RILP) effector (Zhang

et al. 2009; Wang et al. 2011b). RILP interacts with

components of the endosomal sorting complex

required for transport (ESCRT-II) (Vps22 and

Vps36) and based on depletion studies, RILP is

shown to participate in the sorting of ubiquitinated

receptors into intraluminal vesicles (Zhang et al.

2009; Wang et al. 2011b). In this manner, RILP sorts

and sequesters the receptors from the cytosolic

��

Rab7a in Endocytosis and Signaling, Fig. 3 Rab7a traffick-ing and signaling complexes. (a) Recycling from early

endosomes to the Golgi. Cargo recycling from early endosomes

to the Golgi entails sequential Rab5 (via CORVET) and Rab7a

activation and Rab7a-mediated recruitment of a complex of Vps

proteins (26/29/35) known as retromer. Rab5 activation can be

positively modulated by EGF receptor signaling. (b) Transportto lysosomes. Rab7a cooperates with the Dyn2-CIN85 complex

to regulate signaling and lysosomal degradation of the ligand-

receptor (EGF-EGF receptor) complex. Bidirectional transport

on microtubules depends on kinesin and dynein motors. (c)Transport from early to late endosomes. Growth factor receptor

signaling is intimately coupled to endocytic transport. As illus-

trated, nerve growth factor receptor TrkA and EGF receptor

associate with MAPK on endosomes are translocated bidirec-

tionally on microtubules. Transport toward perinuclear late

signaling machinery and targets them for lysosomal

degradation. RILP is also targeted by bacterial patho-

gens to create a specialized intracellular niche for

replication (Zhang et al. 2009). In a tripartite complex,

Rab7a, RILP, and a second effector known as

oxysterol-binding protein-related protein 1 L

(ORP1L) serve to recruit a dynein/dynactin motor

complex that in association with betaIII spectrin facil-

itates the perinuclear transport of endosomes on micro-

tubules (Wang et al. 2011b) (Fig. 3b).

Dynein-/dynactin-mediated perinuclear positioning

of late endosomes has also been shown to depend

on the membrane-associated scaffolding protein,

Huntingtin (Htt), which when mutant causes

Huntington’s disease, though the link between Htt

and Rab7a remains unclarifed (Agola et al. 2011;

Caviston et al. 2011). Htt and the Huntingtin-

associated protein of 40 kDa (HAP40) are known

effectors of Rab5 that facilitate transfer between

microtubule-and actin-based networks (Agola et al.

2011). Parallel in vitro studies testing Rab7a did not

provide evidence for a direct Rab7a-Htt or a Rab7a-

HAP40 interaction, although Huntingtin-associated

protein 1 (HAP1) binds dynactin p150Glued (Agola

et al. 2011). Therefore, it is speculated that an Htt

interaction with the Rab7a dynein/dynactin complex

may occur through HAP1 (Fig. 3c). In light of the

disease relevance, further study of the potential inter-

faces between Htt, HAP1, and Rab7a is warranted.

Anterograde movement of endosomes to the

cell periphery along the microtubular network is

incompletely characterized. Plus-end motility of

autophagosomes is dictated by the recently identified

Rab7a effector FYVE and coiled-coil domain protein 1

endosomes occurs through association with the Rab7a effector

RILP and the p150/dynein motor complex. Association with

HAP1/Htt contributes to perinuclear late endosome positioning.

Transport to the cell periphery is mediated in association with

kinesin motor complexes (Kif3a/Kinesin-2 via FYCO1 or

Kinesin-1 via SKIP). (d) Distinct multi-protein complexes reg-

ulate transport to and from late endosomes. Rab5-Rab7a conver-

sion involves coordinate inactivation of Rab5 and activation of

Rab7a, transition of CORVET complex to HOPS complex,

which ensures seamless cargo transport to late endosomes.

Handoff to ESCRT machinery enables membrane invagination

and sequestration of growth factor receptors on intraluminal

vesicles of multivesicular bodies. Rab7a-retromer complex

enables Golgi recycling. Rab7a HOPS complex enables late

endosome-lysosome fusion

R 1542 Rab7a in Endocytosis and Signaling

(FYCO1) and an unknown kinesin (Wang et al.

2011a). Late endosome movement is known to depend

on kinesin-2 KIF3A heavy chain, while the Rab7a link

and effector remain enigmatic (Loubery et al. 2008).

Evidence from studies on Salmonella suggest that

Rab9 and Rab7a associate with distinct domains on

SifA and kinesin-interacting protein (SKIP), implicat-

ing kinesin-1 in anterograde motility and late

endosomal sorting (Jackson et al. 2008). In sum, the

function of the Rab7a-RILP complex in sorting and

cytoskeletal transport is best characterized, while other

Rab7a effector interactions including those with

kinesins and disease relevant proteins (Htt and

HAP1) await further characterization. Bacterial pro-

teins from Salmonella with identified functions in

interfering with Rab7a motor proteins or linkers may

offer unique tools for further dissecting Rab7a motor

protein interactions.

Endosomal lipids such as cholesterol and phosphoi-

nositides are critical regulators of cargo sorting and

transport on the late endosomal pathway that are inte-

grated through Rab7a and associated motor proteins.

In particular, cholesterol sensing is integrated with

transport through the Rab7a effector ORP1L (Wang

et al. 2011a). When cholesterol levels are low, ORP1L

promotes the association of late endosomes with the

endoplasmic reticulum via the dissociation of minus-

end motor proteins. The ER protein VAPB contributes

to motor dissociation and the peripheral movement of

late endosomes. Being more peripherally localized,

late endosomes are poised to receive cholesterol and

other cargo internalized through early endosomes or

association with the endoplasmic reticulum (ER). Con-

versely, when cholesterol levels are high, the confor-

mation of ORP1L is altered and perinuclear transport is

favored. In Niemann-Pick type C disease, where

endosomal cholesterol levels are constitutively high,

the bidirectional motility of endosomes/phagosomes

and activation of Rab7a are perturbed (Chen et al.

2008; Zhang et al. 2009). The perturbations contribute

to disease pathology and can be reversed by

overexpression of Rab9 or Rab7a (Zhang et al. 2009).

Similar to Rab5 on early endosomes, GTP-bound

Rab7a is required for class-III ▶ phosphatidylinositol

3-kinase (consisting of the hVps34 catalytic, the

hVps15/p150 Rab7a-binding adaptor, and the Rubicon

regulatory subunits) activation on late endosomes

(Agola et al. 2011; Ho et al. 2012). The local synthesis

of PI(3)P on late endosomes enables the recruitment of

FYVE domain–containing proteins that promote mem-

brane remodeling (including intraluminal vesicle for-

mation) and eventually terminate the signal. FYVE

domain–containing factors include the PI(3,5)P(2)-

producing kinase PIKfyve, myotubularin lipid phos-

phatases, among others (Table 1). Together these

downstream effectors control endolysosome morphol-

ogy, membrane trafficking, acidification, among other

functions. Rab7a together with the early endosomal

myotubularin lipid phosphatases (MTM1) and late

endosomal myotubularin-related protein 2 (MTMR2)

acts as a molecular switch controlling the sequential

synthesis and degradation of endosomal PI(3)P (Cao

et al. 2008). Direct binding of the phosphatases to the

phosphatidylinositol 3-kinase complex leads to inacti-

vation of the myotubularins. The lipid kinase-

myotubularin interaction also precludes the interaction

of the activated Rab7a with the lipid kinase, illustrating

the importance of protein handoffs in phosphoinositide

3-phosphate homeostasis on late endosomes. Together,

the examples cited provide evidence for Rab7a func-

tion in endosomal lipid homeostasis in both metabo-

lism and signaling, the disruption of which leads to

human disease.

Two Rab7a effectors participate directly in the reg-

ulation of cargo degradation. Rabring7 (Rab7a-

interacting ring finger protein) functions as an E3

ligase in conjunction with the Ubc4 and Ubc5 as E2

proteins (Zhang et al. 2009; Wang et al. 2011a). Func-

tionally, overexpression of Rabring7 increases epider-

mal growth factor receptor degradation and lysosome

biogenesis. The proteasome alpha-subunit XAPC7 or

PSMA7 in mammals has been found to interact

specifically with Rab7a and is recruited to late

multivesicular endosomes (Zhang et al. 2009; Agola

et al. 2011). Overexpression of XAPC7 impairs late

endocytic transport of EGF receptor and hence is

a negative regulator of trafficking. Together, Rabring7

and XAPC7 may coordinate the degradation of

ubiquitinated growth factor receptors via a link to the

proteasomal degradation machinery though further

studies are required to elucidate mechanistic details.

In addition to the described Rab7a effectors whose

functional activities have been detailed, there are many

more putative effectors whose characterization

remains to be documented (Table 1). Therefore, further

complexity in Rab7a-mediated regulation of cargo

Rab7a in Endocytosis and Signaling, Table 1 Rab7 GTPase regulators and effectors, and their functions

Rab7 isoform and

nucleotide-bound state Rab7 effector/binding partner Regulator or effector functiona

Rab7a ANKFY1 (ankyrin repeat and FYVE

domain containing 1)/ANKHZN/

Rabankyrin-5

Possible role in vesicular trafficking. Novel interactor of

Rab7. Specific role yet to be established

Rab7a ATP6V0A1 Component of vacuolar ATPase that regulates organelle

acidification required for protein sorting, receptor-mediated

endocytosis, zymogen activation, and synaptic vesicle proton

gradient. Novel interactor of Rab7. Specific role yet to be

established

Rab7a-GDP Ccz1 (vacuolar protein trafficking and

biogenesis-associated homolog)

Recruited to endosomes by Mon1a/Mon1b and acts as Rab7

GEF in yeast. Possible human homolog C7orf28B also some

similarity to HPS4 involved in biogenesis of lysosome-

related organelles

Rab7a-GTP FYCO1 (FYVE and coiled-coil domain

containing 1)

Promotes microtubule plus end transport of autophagosomes

presumably by functioning as a kinesin adapter

Rab7a GNB2L1 (guanine nucleotide binding

protein (G protein), beta polypeptide)

Role in intracellular signaling and activation of protein

kinase C and possible interaction with Rab7 via WD40

domain. Novel interactor of Rab7. Specific role yet to be

established

Ypt7p/ Rab7a-GTP HOPS complex (Vps11,-16,-18,-33,-39,

and-41)

Involved in vacuolar tethering and fusion in yeast and

conserved mammalian homologs function in mammalian

endolysosomal fusion. Interfaces with CORVET complex to

promote rab5 to rab7 conversion in yeast. Vps39 subunit

binds Mon1-Ccz1 complex that serves as a Rab7 GEF in

yeast and C. elegans

Rab7a hVps39 In yeast Vps39p, cooperates with Mon1-Ccz1 complex to

promote Ypt7p nucleotide exchange, function of mammalian

protein remains to be determined

Rab7a IMMT (Mitofilin) Maintains mitochondrial morphology and suggested role in

protein import. Novel interactor of Rab7. Specific role yet to

be established

Rab7a KIF3A (kinesin + adapter?) Kinesin2 heavy chain associates with late endosomes along

with dynein, Rab7, and dynactin. Possible mediator of Rab7-

regulated anterograde transport coordinated by Rab7-

interacting adapter such as FYCO1 or other as-yet-

unidentified protein

Rab7a-GDP Mon1a-Mon1b Mammalian homologs ofC. elegans SAND1. Mon1a-Mon1b

causes Rab5 GEF displacement and Mon1b interacts with the

HOPS complex. Mon1 is an effector of Rab5, but only

interacts with Rab7 when complexed to Ccz1

Rab7a-GTP ORP1L ([oxysterol-binding protein,

OSBP]-related protein 1)

Required for cholesterol sensing and regulation of dynein/

dynactin motor with Rab7 and RILP, regulates late

endosome/lysosome morphogenesis and transport

Rab7a-GTP Phosphoinositide 3-kinase complex

(hVps34/hVps15)

Type III ▶ PI 3-kinase that generates phosphoinositide 3-

phosphate to control endosomal trafficking and signaling.

Forms complex with myotubularins for negative regulation

Rab7a-GTP Plekhm1 (pleckstrin homology domain

containing, family M [with RUN domain]

member)

Regulates lysosomal secretion in osteoclasts for bone

resorption by interacting with LIS1 to control microtubule

transport and Rab7 and ▶ PI 3-kinase to recruit effectors for

fusion

Rab7a Prohibitin Negative regulator of cell proliferation and a possible tumor

suppressor. Novel interactor of Rab7, specific role yet to be

established

(continued)

Rab7a in Endocytosis and Signaling 1543 R

R

Table 1 (continued)

Rab7 isoform and

nucleotide-bound state Rab7 effector/binding partner Regulator or effector functiona

Rab7a-GTP Rabring7 Rab7-interacting ring finger protein, functions as E3 ligase

that ubiquitinates itself and controls EGF receptor

degradation

Rab7a-GDP REP1 (Rab escort protein 1) Presents Rab7 to Rab geranylgeranyl transferase for addition

of prenyl group that acts as a membrane anchor

Rab7a-GTP Retromer (Vps26, Vps29, Vps35) Regulates retrograde transport from late endosome to trans-

Golgi network (TGN) through direct interaction with Vps26

Rab7a-GTP RILP (Rab7-interacting lysosomal

protein)

Involved in late endosomal/lysosomal maturation. Recruits

dynein-dynactin motor protein complex

Rab7a-GTP Rubicon Regulates endosome maturation through differential

interaction with UVRAG and Rab7. Rubicon binding inhibits

UVRAG. Rubicon binding to active Rab7 frees UVRAG to

activate the hVps34/hVps15 ▶ PI 3-kinase and HOPS,

thereby simultaneously increasing the active pool of Rab7

and PI3P signaling

Rab7a-GTP SKIP (SifA and kinesin-interacting

protein)

Homolog of PLEKHM1 that binds Rab7, Rab9 and kinesin-1,

and may regulate anterograde motility of late endosomes.

Target of Salmonella SifA protein

Rab7a Spg21 Loss of function causes autosomal recessive hereditary

spastic paraplegia. Involved in vesicular transport. Novel

interactor of Rab7. Specific role yet to be established

Rab7a STOML2 (Stomatin-like 2) Negatively modulates mitochondrial sodium calcium

exchange. Novel interactor of Rab7. Specific role yet to be

established

Rab7a-GTP TBC1D2 ([tre-2/USP6, BUB2, cdc16]

domain family, member 5)/Armus and

Rac1

Regulates cytoskeleton organization, ruffled border

formation in osteoclasts, and E-cadherin/adherens junction

degradation in conjunction with Rac1, inactivates Rab7

through C-terminal GAP activity

Rab7a-GTP TBC1D5 ([tre-2/USP6, BUB2, cdc16]

domain family, member 5)

Negatively regulates retromer recruitment and causes Rab7

to dissociate from membrane and may have Rab7 GAP

activity

Rab7a-GTP TBC1D15 ([tre-2/USP6, BUB2, cdc16]

domain family, member 15)

Functions as Rab7 GAP and reduces interaction with RILP,

fragments lysosomes, and confers resistance to growth factor

withdrawal-induced cell death

Rab7a-GTP TrkA (neurotrophic tyrosine kinase

receptor)

Interacts with Rab7 and regulates endocytic trafficking and

nerve growth factor signaling as well as influencing neurite

outgrowth

Rab7a-GTP UVRAG (UV radiation resistance-

associated gene)/Beclin1

UVRAG/C-Vps complex positively regulates Rab7 activity

via ▶ PI 3-kinase (PI3KC) during autophagic and endocytic

maturation

Rab7a-GTP VapB ([vesicle-associated membrane

protein]-associated protein B)

Involved in mediating endosome-ER interaction in response

to ORP1L conformation sensing low cholesterol levels

Rab7a Vps13c (vacuolar protein sorting 13c) Vacuolar protein sorting and novel interactor of Rab7.

Specific role yet to be established

Rab7a-GDP, GTP XAPC7/PSMA7 (proteasome subunit,

alpha type 7)

Negative regulator of late endocytic transport.

Overexpression inhibits EGF receptor degradation

Rab7b SP-A (Surfactant protein A) Transiently enhances the expression of Rab7 and Rab7b and

makes them functionally active to increase the

endolysosomal trafficking in alveolar macrophages

aAgola, JO thesis provides reference listing for effectors

R 1544 Rab7a in Endocytosis and Signaling

Rab7a in Endocytosis and Signaling 1545 R

sorting, cytoskeletal transport, and membrane fusion

will emerge through continued study. An important

area for investigation is how Rab7a interactions with

tethering factors and SNARE proteins control late

endosomal fusion events which have primarily been

characterized for the yeast homolog Ypt7p (Zhang

et al. 2009; Wang et al. 2011a).

R

Rab7a in Endosomal Signaling

At the late endosome, Rab7a coordinately regulates

intracellular signaling through special scaffolds, selec-

tive endosome positioning, and control of growth fac-

tor receptor trafficking. Epidermal growth factor

receptor (EGF receptor), vascular endothelial growth

factor receptor (VEGFR2), and nerve growth factor

receptor (TrkA) all depend on Rab7a for their signaling

and downregulation (Agola et al. 2011). For example,

upon EGF stimulation, K-Ras is endocytosed and

sorted to late endosomes where Rab7a and the p14-

MP1-p18 scaffolding proteins recruit and activate

▶MEK-Erk on late endosomes (Lu et al. 2009; Nada

et al. 2009). ▶MAP kinase signaling is further regu-

lated by Rab7a-dependent late endosome positioning

through dynactin such that peripheral mislocalization

results in prolonged EGF receptor activation and

downstream Erk and p38 signaling. The localization

of two signaling mediators of the TGF-b superfamily

to Rab7-positive late endosomes (p-Smad1 and p-

Smad2) is also suggested to be critical for regulation

of growth factor signaling (Rajagopal et al. 2007). At

the conclusion of signaling, Rab7a may act coopera-

tively with dynamin 2 and CIN85 (cbl-interacting pro-

tein of 85 kDa) to promote the transfer of signaling

receptors from late endosomes to lysosomes for deg-

radation (Fig. 3b) (Schroeder et al. 2010).

In neuronal cells, the receptor tyrosine kinase, TrkA

is activated by nerve growth factor (NGF). On NGF

stimulation, Rab7a interacts with TrkA as it transits

through early and late endosomes. Cells expressing

Rab7a T22N, which is predominantly GDP bound,

showed prolonged Erk1/2 signaling due to impaired

trafficking of activated TrkA (Agola et al. 2011). Dis-

ease-causing Rab7a mutants that are constitutively

activated have also been shown to exhibit enhanced

NGF-stimulated Erk1/2 signaling (BasuRay et al.

2010). This apparently contradictory result can be

explained by the duality of Rab7a in regulating transfer

of cargo to lysosomes and interacting with scaffold

proteins. Thus, Rab7a plays a significant role in growth

factor transport by controlling both signaling scaffolds

and trafficking to degradative compartments.

Phosphorylation of Rab7a in response to growth

factor suggests a further layer of regulation. Large-

scale proteomics analyses have identified Rab7a to be

both serine and tyrosine phosphorylated. In mouse

liver extracts, Rab7a was found phosphorylated on

serine 72 within a highly conserved sequence near

the GTP-binding pocket (Villen et al. 2007). Rab7a

was phosphorylated in response to EGF stimulation

on tyrosine 183 in the C-terminal region. Enhanced

tyrosine 183 phosphorylation of Rab7a was also

associated with mutant EGF receptor and HER2

overexpression in non–small cell lung carcinoma and

mammary epithelia, respectively (Guo et al. 2008).

The functional consequences of Rab7a serine and tyro-

sine phosphorylation with respect to membrane traf-

ficking, GTP binding, and hydrolysis remain to be

established.

Ubiquitination of activated growth factor receptors

plays a crucial role in the endosomal sorting and

lysosomal targeting to downregulate receptor levels.

Such ubiquitination may depend on interaction with

Rab7a and the Rabring7 E3 ubiquitin ligase. The

sorting of ubiquitinated receptors into luminal vesicles

of multivesicular bodies depends on the ESCRT0,

ESCRTI, ESCRTII, and ESCRTIII complexes

(Raiborg and Stenmark 2009). EGF receptor and

TrkA endolysosomal degradation are both ubiquitin

and proteasome dependent. The K63-linked

polyubiquitin chain on activated TrkA receptors gets

shuttled by the p62 scaffolding protein, possibly in

association with Rab7a/XAPC7, to the proteasome

for deubiquitination prior to degradation in lysosomes

(Geetha and Wooten 2008). TrkA deubiquitination

prior to lysosomal degradation may allow crucial

recycling of ubiquitin since the ubiquitin tagging is

essential for optimum interaction of activated TrkA

with the transport machinery and its delivery along

the long axonal route from the tip to the cell body.

Only after TrkA reaches the cell body, the termination

of signaling calls for deubiquitination of the cargo

prior to its lysosomal degradation. As illustrated, the

reversible ubiquitination is an important component of

growth factor receptor downregulation.

R 1546 Rab7a in Endocytosis and Signaling

The Rab7a-regulated, interdependent late endocytic

trafficking, and signaling pathways are indispensible

for translating growth factor signals into appropriate

cell responses. The development of suitable in vivo

models will therefore be crucial to elucidate how

impaired trafficking of growth factor receptors and

consequent alterations in signaling lead to neurodegen-

erative diseases and cancer.

Summary

Since its discovery over 20 years ago, Rab7a and its

functions in late endocytic trafficking and signaling

have remained under active investigation. Unflagging

interest is attributed to the diverse processes that are

regulated by Rab7a together with a demonstrated role

in human disease. The list of Rab7a effector proteins

continues to grow, though the exact functions of many

recent interacting partners remains to be elucidated.

Rab7a helps to coordinate signaling through the tempo-

ral and localized assembly of signaling scaffolds and

a close coupling to degradative pathways. Elucidating

how Rab7 nucleotide exchange and hydrolysis are reg-

ulated and how Rab7 is selectively recruited to specific

macromolecular complexes to regulate individual path-

ways remain important areas for further investigation.

Acknowledgments We acknowledge PREP fellowship from

NIGMS (R25GM075149) to PJ and research support from NSF

(MCB0956027) to AWN and MRC (G0701444) to MNM.

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Rab8

Heather H. Ward and Angela Wandinger-Ness

Department of Pathology and Cancer Center, MSC08-

4640, University of New Mexico Health Sciences

Center, Albuquerque, NM, USA

Synonyms

Dmel; Mel transforming oncogene (RAB8 homolog);

MEL; MGC124948; Oncogene c-mel; Rab8a;

Ras-related Rab8; Ras-associated protein RAB8;

Ras-related protein Rab-8A

Historical Background

Ras-like Rab GTPases (guanosine triphosphatases)

are regulators of membrane trafficking. Identification

of two Ras-like GTPases, Sec4p and Ypt1p, which are

involved in regulating secretion in yeast, rapidly led

to the discovery of additional small GTPases in mam-

malian cells. Rab8 was among the first group of mam-

malian Rab GTPases to be identified and is a close

functional and sequence homolog of the yeast pro-

teins Ypt2 and Sec4p. A second isoform, Rab8b (b for

basophil), was cloned from mast cells in 1996. Rab8b

shares 83% sequence identity with MEL/Rab8 (now

also termed Rab8a) primarily over amino acids 1–

152. The two isoforms display significant overlap in

tissue distribution (coexpressed in liver, skeletal mus-

cle, and testis), though Rab8 is much more abundant

in lung and kidney, while Rab8b is more prevalent

in heart, brain, and spleen. This review presents

a synopsis of recent developments on Rab8 function.

As detailed in the following sections, Rab8 regulates

transport from the trans-Golgi network to the

basolateral plasma membrane of epithelia, neuronal

dendrites, and the ciliary membrane. Rab8 is required

in cellular polarization, cellular signaling and devel-

opment. Consequently, alterations in Rab8 expression

and localization affect numerous cellular events and

organ systems.

Rab8 Function

Analyses of Rab8 function across diverse cell types

demonstrate that Rab8 plays a pivotal role in exocytic

and endocytic pathway interfaces and is particularly

important in exocytic events (Fig. 1). Rab8 predomi-

nately localizes to budding vesicles at the trans-Golgi

network, recycling endosomes, exocytic vesicles, and

ruffling plasma membrane domains. From the trans-

Golgi network, Rab8 regulates delivery of newly syn-

thesized lysosomal enzymes to endosomes (del Toro

et al. 2009), exocytosis of regulated secretory vesicles

and melanosomes (Faust et al. 2008; Wandinger-Ness

and Deretic 2008; Sun et al. 2010), and plasma mem-

brane export to neuronal dendrites, as well as to the

basolateral, apical and ciliary membrane domains of

epithelia (Bravo-Cordero et al. 2007; Wandinger-Ness

and Deretic 2008). An emerging theme is that Rab8-

mediated transport of newly synthesized ciliary

Rab8, Fig. 1 Rab8 trafficking routes. Rab8 plays an important

role in both exocytic and endocytic pathway interfaces and is

particularly important in exocytic events. Through interactions

with different protein complexes, Rab8 regulates delivery of

newly synthesized lysosomal enzymes to endosomes, exocytosis

of secretory vesicles, and cargo export basolateral, apical and

ciliary membrane domains of epithelial cells. RE recycling

endosome, EE/SE early endosome/sorting endosome, LE late

endosome

R 1548 Rab8

membrane proteins, basolateral and apical cargo fre-

quently occurs via recycling endosomes in coordina-

tion with Rab10 and Rab11a (Schuck et al. 2007;

Cramm-Behrens et al. 2008; Wandinger-Ness and

Deretic 2008; Knodler et al. 2010; Ward et al. 2011;

Westlake et al. 2011). On the endosomal circuit, Rab8

may cooperate with Arf6, Rab11a or Rab13 to regulate

externalization of recycling receptors (transferrin and

glutamate), cell adhesion molecules (integrins and E-

cadherin), and lipid regulators (ABCA1), often to spe-

cialized domains such as membrane protrusions, junc-

tions and postsynaptic membranes (Hattula et al. 2006;

Nagabhushana et al. 2010; Rahajeng et al. 2010;

Roland et al. 2011).

Rab8 function in membrane trafficking is intimately

connected to the cytoskeleton. Rab8 is associated with

microtubules, actin and intermediate filaments, and

expression of mutant Rab8 variants results in signifi-

cant perturbations of both microtubule and actin net-

works (Omori et al. 2008; Wandinger-Ness and

Deretic 2008). Rab8 has not been shown to bind micro-

tubule-based motors, and the perturbation of microtu-

bules induced by mutant Rab8 may be linked to

Rabin8-dependent activation of Rab8 at centrosomes

and the dependence of microtubule-based cilia on

Rab8 membrane transport (Wandinger-Ness and

Deretic 2008; Knodler et al. 2010; Westlake et al.

2011). On the other hand, Rab8-dependent actin

Rab8 1549 R

R

filament association depends on direct Rab8 interac-

tions with myosin V(a,b,c) or myosin VI motor pro-

teins, which in turn facilitate directed vesicle

trafficking (Wandinger-Ness and Deretic 2008; Roland

et al. 2009, 2011). Rab8-myosin VI interactions direct

basolateral cargo to the plasma membrane

(Wandinger-Ness and Deretic 2008). Rab8/Rab27/

myosin Va regulate the final stages of melanosome

docking to the plasma membrane via actin filaments

(Wandinger-Ness and Deretic 2008). Rab8-Rab11-

myosin Vb and Rab8-myosinVb interactions are

involved in apical lumen formation and insulin-

dependent GLUT4 exocytosis, respectively (Ishikura

and Klip 2008; Roland et al. 2011). Rab8 and myosin

Vc likely cooperate in the early stages of regulated

secretion involving tubule-dependent transport to the

cell periphery of exocrine epithelia followed by hand-

off in the cell periphery to Rab27a vesicles whose

cortical actin anchoring depends on myosin Vc (Jacobs

et al. 2009). The three isoforms of myosin V also act as

scaffolds for the binding of multiple Rab GTPases

cooperating on linked pathways, including Rab8,

Rab10, Rab11a, and Rab27 (Roland et al. 2009). For

example, Rab8 as well as Rab10 interact via an alter-

natively spliced exon D in myosin Va and Vb or an

exon D-like domain in myosin Vc, while Rab27a inter-

acts via exon F in myosin Va (Roland et al. 2009).

Thus, regulated interactions of distinct myosin

isoforms with distinct combinations of Rab GTPases

may partially explain cell-type-specific regulation of

Rab8-dependent pathways (Roland et al. 2009, 2011).

However, questions remain as to how Rab8 functions

cooperatively with other GTPases and how specific

functions are regulated, for example: Is Rab8 bound

simultaneously with multiple Rab GTPases to individ-

ual myosin motors? Are there hand-offs between Rab8

and other GTPases at discrete locations and how do

they occur? How are specific effectors involved in

discrete transport steps enriched at Rab8-positive

membranes?

As illustrated by the given examples, Rab8 serves as

a key regulator in communication between membrane

transport circuits, the cytoskeleton and cellular signal-

ing. Due to the critical role that Rab8 plays in multiple

cellular pathways, loss of Rab8-protein interactions

and regulation can result in numerous human diseases

including open angle glaucoma, retinitis pigmentosa,

microvillus inclusion disease, Huntington’s disease,

and cystic kidney disease.

Rab8 Regulation

As with other Rab family members, Rab8 cycles

through membrane-associated, GTP-bound “on” states

and cytoplasmic, GDP-bound “off” states. Membrane

association depends on isoprenylation by protein

geranylgeranyl transferases (primarily REP/GGTase

II or GGTase I, minor pathway) [reviewed in

(Wandinger-Ness and Deretic 2008)]. Activation of

membrane-bound Rab8 through GTP binding may be

facilitated by one of several guanine nucleotide

exchange factors (GEFs) such as MSS4, Rabin8/

Rabin3, Rabin 3-like GRAB, or retinitis pigmentosa

GTPase regulator (RPGR). In the activated state, Rab8

is a scaffold for numerous effectors and kinases that

cooperatively serve in the temporal and spatial regula-

tion of Rab8 transport. Upon completion of the trans-

port cycle, nucleotide hydrolysis and Rab8

inactivation can be mediated by two known GTPase

activating proteins (GAPs), AS160 (Akt substrate)/

Tbc1d4 (Tre-2/Bub2/Cdc16) domain containing pro-

tein or Tbc1d30/XM_037557, for which multiple

isoforms may exist (Wandinger-Ness and Deretic

2008). Interestingly, AS160 also serves as a GAP for

Rab10 and Rab13 suggesting that GTPases that func-

tion on interrelated pathways may also be coregulated

(Ishikura and Klip 2008; Sun et al. 2010). Inactive

Rab8-GDP is bound and recycled from membranes to

the cytosol by GDP dissociation inhibitor-2 (GDI-2)

(Shisheva et al. 1999). Given Rab8 involvement in

multiple transport pathways, some of which are highly

cell type specific, it remains to be clarified if and how

Rab8 function is regulated in a site-specific manner.

Rab8 Effectors, Signaling Integration, and Disease

Spatial and temporal regulation of Rab8 trafficking is

partially dependent on signaling receptors, as well as

effector and cargo protein interactions, thereby confer-

ring specificity to Rab8-mediated targeting in accor-

dance with cell need and cell type (Wandinger-Ness

and Deretic 2008). For example, Rab8 interactions

differentially regulate a2B- and b2-adrenergic receptortransport from the trans-Golgi network to the plasma

membrane (Dong et al. 2010). Triggered by integrin-

mediated adhesive events, Rab8 controls the polarized

and regulated exocytosis of matrix metalloprotease

MT1-MMP/MMP-14, which is critical for collagen

degradation and tumor cell invasion (Bravo-Cordero

et al. 2007). Specific cell types use conserved

Rab8, Fig. 2 Rab8 exocytic trafficking in ciliated and polariz-ing epithelial cells. (a) Ciliogenesis and cilial transport is regu-

lated in part by Rab8-mediated vesicle trafficking. Vesicular

transport of membranous protein cargo such as polycystin-1

begins with formation of a Golgi-exit complex consisting of

Arf4 and Rab11 and recruitment of the Arf4 GAP (ASAP1)

and Rab11 effector FIP3. TRAPPII components and Rab11 are

required for recruitment and vesicular trafficking of Rabin8.

Near the centrosome Rabin8 recruits and activates Rab8,

which facilitates transit and docking with BBSome. Vesicle

fusion is thought to require Rab8, Rab10, the exocyst complex

and Cdc42. Kinesin and dynein motor protein isoforms transport

proteins within the primary cilium, though the link between Rab

GTPases and motor proteins within the cilium has not been

defined. (b) Rab8, Rabin8, exocyst components and Cdc42

also participate in Rab11-mediated vesicle transport (recycling

endosome transition to exocytic vesicle) in polarizing epithelial

during the formation of tubule lumens. These vesicles interact

with myosin Vb to transport to the apical surface via the actin

cytoskeleton. Rab11 interacts with Rabin8, which in turn recruits

and activates Rab8. Rab8 recruits Tuba, the Cdc42 GEF, which

activates Cdc42. Cdc42 and Par6 recruit aPKC, which then

recruits Par3-Sec8 and Sec10. Interactions with the exocyst

and Rab GTPases promote association with the plasma

membrane

R 1550 Rab8

trafficking mechanisms during different stages of

polarization. For example, Rab8, Rab11, Rabin8, and

the exocyst complex are used with different effectors

to traffic apical protein cargo during lumen formation

and ciliary protein cargo during ciliogenesis upon

reaching a fully polarized, ciliated state (Fig. 2). Loss

of cargo, especially signaling proteins, to the proper

target membrane results in aberrant signaling and dis-

ease. Although an assortment of diverse Rab8a

effectors and cargo are known, further study is needed

to determine if Rab8a activity is regulated by distinct

sets of regulators within specific cellular

compartments.

Rab8-mediated trafficking is required to maintain

cellular polarity and homeostasis, which when altered

can lead to tumor formation and malignancy. Mem-

brane type 1-matrix metalloproteinase (MT1-MMP,

also known as MMP-14) plays a key role in tumor

Rab8 1551 R

R

invasiveness by participating in proteolytic degrada-

tion of surrounding tissues through activation of

MMP2. MT1-MMP is exocytosed via Rab8, and traf-

ficking to the plasma membrane is enhanced with

expression of constitutively active Rab8 mutants,

whereas RNAi knockdown of Rab8 prevents MT1-

MMP from reaching the plasma membrane and thus

prevents collagen degradation (Bravo-Cordero et al.

2007). Of note, MT1-MMP activation of MMP2 and

MMP9 from pro- to active form is dependent upon

interaction between MSS4 (a Rab8 GEF) and

a-integrin chains (Knoblauch et al. 2007). The MSS4

binding sites for a-integrin chains and Rab8 are likely

competitive. Decreased expression of MSS4 affects

assembly and remodeling of the extracellular matrix.

Rab8 expression can be up-regulated in breast cancer

malignancies and associated lymph node metastases,

and further studies on Rab8 mechanisms in tumorigen-

esis and malignancy may pave the way for the devel-

opment of small molecule treatments with Rab8 or

associated effectors as therapeutic targets.

Rab8 mediates basolateral trafficking and protein

transport to adherens and tight junctions. Rab8 and

Rab13 interact with effector MICAL-L2 (Molecule

Interacting with CasL-Like 2) via competitive binding

and formation of distinct independent Rab8a-MICAL-

L2 and Rab13-MICAL-L2 protein complexes. Rab-

MICAL-L2 protein interactions dictate trafficking

specificity by regulating E-cadherin recycling to

adherens junction (further evidenced by Rab8 knock-

down causing delay of E-cadherin delivery to adherens

junctions in calcium switch assays), and Rab13-

MICAL-L2 interactions mediate recycling of occludin

to tight junctions (Rahajeng et al. 2010). MICAL-1,

MICAL-2, MICAL-3, MICAL-L1, and MICAL-L2

make up a family of large proteins with two to three

common domains that mediate vesicular transport and

cytoskeleton organization (Rahajeng et al. 2010). Rab8

interacts with at least three MICAL family members.

Rab6-dependent recruitment of Rab8 to exocytic ves-

icles involves a MICAL3 intermediate in Rab8-ELKS

interactions (Grigoriev et al. 2011). A related MICAL-

like protein, MICAL-L1, regulates Rab8 in endocytic

recycling. MICAL-L1 is proposed to serve as a Rab8

effector that serves to stably link Rab8 and EHD1

(Eps15 homology domain 1) on endocytic recycling

vesicles (Rahajeng et al. 2010). EHDATPase scaffold-

ing proteins localize to tubular and vesicular mem-

branes, regulate endocytic trafficking, and coordinate

activity with Rab GTPases through interaction with

Rab effectors. EHD also mediates GLUT4 recycling.

Thus, Rab8 in conjunction with MICAL and EHD pro-

teins form scaffolds that are crucial for integrating

basolateral and endocytic pathways.

Over the last 3 years insulin-dependent signaling

has been linked with Rab-mediated vesicle trafficking.

Insulin-dependent glucose uptake is mediated by reg-

ulation of the surface recycling of GLUT4 trans-

porters. AS160/TBC1D4 serves as a Rab GAP and

AKT signaling target and is phosphorylated in

response to insulin. Upon phosphorylation AS160

GAP activity is likely inhibited to allow activated

Rab targets to function, thereby releasing the brakes

on vesicle docking and fusion and allowing GLUT4

insertion at the plasma membrane of myoblasts

(Randhawa et al. 2008). TBC1D1 is a second GAP

that coregulates Rab activation in response to insulin

stimulation. Rab8a and Rab14 are targets of the GAP

TBC1D1 in skeletal myotubes and AS160, along with

Rab13, in myoblasts (Ishikura and Klip 2008; Sun et al.

2010). The Rab8, Rab10, and Rab14 GTPases are

required for insulin-induced GLUT4 trafficking, with

GTP loading stimulated by insulin in a cell-type-

specific manner (Ishikura and Klip 2008; Sun et al.

2010). Rab8 and Rab13 act as a Rab regulatory cascade

that is activated sequentially to promote GLUT4 trans-

location in muscle cells, while Rab10 functions in

adipocytes. Downregulation of GLUT4 translocation

is thought to be achieved through the interaction of

active Rab8a with the myosin Vb motor protein,

thereby altering Rab8a localization and negatively

impacting GLUT4 translocation (Ishikura and Klip

2008; Sun et al. 2010). This example is illustrative of

how signaling and Rab8-regulated membrane traffick-

ing are closely intertwined and modulated through

cell-type-specific processes.

Noc2, rabphilin, Rim2 and Slp4/granuphilin are

members of the ▶ synaptotagmin-like family and all

bind to multiple Rab GTPases, including Rab3a,

Rab8a, and Rab27a. Noc2 and rabphilin bind to active,

GTP-bound Rab8a suggesting that both may serve as

Rab8a effector proteins. Rim2 has been predominately

studied in regulated secretion within presynaptic nerve

terminals and insulin-secreting cells, where it func-

tions in regulated exocytosis (Yasuda et al. 2010).

Rabphilin is expressed in neuronal, neuroendocrine,

intestinal goblet cells and kidney podocytes. Slp4 is

also expressed in neuroendocrine cell dense core

R 1552 Rab8

vesicles and additionally localizes to the insulin-

containing vesicles of pancreatic beta cells. All the

members of the synaptotagmin-like family are impli-

cated in regulated secretion and are of interest due to the

fact that synaptotagmin-like proteins also bind plus-end

directed myosin motors and thus, may bridge Rab8-

regulated vesicle docking and fusion to cytoskeletal

translocation.

Further evidence for a link between Rab8 and

myosin motors is provided by studies in enterocytes.

Rab8a is essential for localization of apical proteins

and maintenance of the small intestine (Fig. 1) (Sato

et al. 2007). Microvillus inclusion disease of the small

intestine is characterized by microvillar atrophy and

malabsorption. Rab8 conditional knockout in mice

causes microvillus inclusion disease and one case of

human disease has been linked to decreased Rab8

mRNA and protein expression in the enterocytes of

the small intestine. To date, no further work has been

reported on the role of Rab8 in microvillus inclusion

disease. However, several reports have linked myosin

Vb mutations to microvillus inclusion disease, and

myosin Vb mutations cause disruption of epithelial

cell polarity, as evidenced by loss of microvilli on the

surface of intestinal absorptive cells and microvilli

present within intracellular inclusion (Ruemmele

et al. 2010). Given that both Rab8a and myosin Vb

defects can result in microvillus inclusion disease, it is

interesting to speculate that, as is the case in insulin-

dependent signaling, the motor protein myosin Vb

may interact with Rab8a and direct trafficking to the

apical surface, which when perturbed, inhibits surface

expression of microvilli. Further roles for Rab8 and

apical protein targeting were brought to light through

the study of zymogen granules in the exocrine pan-

creas. Rab8 localizes to zymogen granules and facil-

itates delivery of digestive enzymes to the apical

surface, evidenced by the fact that Rab8 knockdown

decreases granule numbers and causes granule pro-

teins to accumulate in the Golgi (Wandinger-Ness and

Deretic 2008). The authors speculate that Rab8-

zymogen trafficking may depend on a clathrin/AP-1/

dynamin association at the Golgi.

At the Golgi, Rab8a interfaces with optineurin

(FIP-2) to promote cargo export, which is mediated

by clathrin adaptor complex AP-1 (del Toro et al.

2009). Optineurin localizes to the cytosol, Golgi, and

recycling endosomes and participates in vesicular traf-

ficking. Optineurin binds active, but not GDP-bound,

Rab8, thus suggesting that optineurin serves as

a downstream effector. Optineurin interacts with myo-

sin VI, a minus-end directed motor, to link Rab8 pos-

itive membranes to the actin cytoskeleton [reviewed in

(Wandinger-Ness and Deretic 2008)]. The optineurin

E50K mutation causes glaucoma and has been shown

to impair endocytic trafficking (as demonstrated by

impaired transferrin uptake) and slows the velocity of

Rab8-GFP positive vesicles (Nagabhushana et al.

2010). Interestingly, mutant E50K optineurin

completely abolishes optineurin-Rab8 interactions at

the Golgi (Chi et al. 2010). In mice, the E50Kmutation

causes massive apoptosis and degeneration of the ret-

ina, but not broader neuronal degeneration (Chi et al.

2010). The composite data suggest there may be

a conserved mechanism for zymogen and optineurin-

mediated trafficking in exocrine cells and neurons.

Optineurin-Rab8 interactions also come into play in

Huntington’s disease. Huntington’s disease results

from abnormal expansion of a polyglutamine tract in

the N-terminus of the huntingtin protein and has

defects in lysosome function. Huntingtin regulates

post-Golgi trafficking of secreted proteins and inter-

acts with the optineurin-Rab8 complex. Huntingtin

links Rab8/optineurin vesicles to microtubules via

interactions with via HAP1 (a Trio-like protein with

a Rac1 GEF domain), dynactin (p150glued) and

dynein [reviewed in (Wandinger-Ness and Deretic

2008)]. Expression of huntingtin mutants or decreased

huntingtin expression decreases Rab8 and optineurin

localization at the Golgi and inhibits clathrin and

optineurin/Rab8-dependent trafficking to lysosomes

(del Toro et al. 2009). Loss of huntingtin causes loss

of Rab11 in isolated membranes, and Rab11-GDP was

shown to interact with huntingtin �30-fold greater

than Rab11-GTP. Rab8 and Rab11 both play a role in

polarized outgrowth and are preferentially located in

the somatodendritic domain of neurons, and Rab8

functions in neuronal maturation and polarized trans-

port. For example, Rab8 is required for the transport

and insertion of (AMPA)-type glutamatergic recep-

tors, which mediate the fast synaptic transmission

throughout the nervous system, into the postsynaptic

compartment. AMPA receptors are recycled back to

the membrane via Rab11 recycling endosomes,

suggesting a potential Rab11/Rab8 trafficking path-

way similar to those found in neuronal photoreceptor

cells. However, the generalizability of the mechanism

remains to be studied further.

Rab8 1553 R

R

Rab8a in Cilial Transport

The role of Rab8 in cilial transport and cilial-mediated

signaling was suggested approximately 16 years ago

(Wandinger-Ness and Deretic 2008), and the body of

literature supporting this hypothesis has grown signif-

icantly over the last 5 years. The primary cilium is

a specialized organelle that sits atop most cell types

in the body and serves as an antenna to sample the

extracellular space and transmit signals to the cell

body. Accordingly, components of several signaling

pathways such as hedgehog, WNT and JAK-STAT,

which regulate cellular growth, proliferation, differen-

tiation, and polarization, are found within the primary

cilium. Cilial structures are evolutionarily conserved

but can have cell-type-specific modifications to detect

and respond to various stimuli such as mechanical,

physical, chemical, or temperature sensation. For

example, the modified cilium in photoreceptor cells

responds to light, whereas the primary cilia in kidney

epithelia are thought to be chemo-mechano sensors.

Loss or aberrant localization of membrane-bound and

cytoplasmic ciliary proteins at the site of the primary

cilia is associated with a group of inherited diseases

known as the ciliopathies. Ciliopathies encompass

a broad range of genetic mutations and phenotypes.

Some genetic mutations cause single organ, adult onset

and progressive disease such as autosomal dominant

polycystic kidney disease (ADPKD) and retinitis

pigmentosa, whereas other genetic mutations affect

multiple organs. An example of a multiorgan disorder

is Bardet-Biedl Syndrome (BBS), which exhibits phe-

notypes that include (but are not limited to) obesity,

polydactyly, retinopathy, mental impairment, and kid-

ney abnormalities.

Rab8 function aligns strongly with the defined

responsibilities of primary cilia, namely, regulation

of epithelial differentiation and maintenance, develop-

ment and organ function through cellular signaling.

Expression of GTP-locked Rab8 induces cilial exten-

sion, whereas depletion of Rab8 inhibits cilia forma-

tion and trafficking, which often results in renal and

retinal defects (Nachury et al. 2010). Numerous studies

have linked Rab GTPases to the transport of mem-

brane-bound ciliary proteins to the primary cilium;

however, the distinct mechanisms are not well defined.

Many questions about ciliary trafficking have been

brought to the forefront of basic cell biology research,

such as: How many ciliary trafficking routes exist?

How are they modulated for specific cell types? What

are the signaling pathways that drive ciliary transport?

What are the mechanisms that switch cellular transport

to direct ciliary trafficking of molecules with multiple

localization patterns? How do vesicular transport path-

ways cooperate with other trafficking pathways such as

intraflagellar transport (IFT)?

In the context of cilial function, Rab8 trafficking is

best characterized in the rhodopsin transport model.

Rhodopsin requires several small GTPases, including

Rab8, to shuttle from the Golgi to an elaborate primary

cilium, known as the rod outer segment, of photore-

ceptor cells; a Rab8 GDP-locked mutant (Rab8-T22N)

inhibits docking and fusion of rhodopsin-containing

exocytic vesicles in transgenic frogs which results in

dramatic retinal degeneration (Wandinger-Ness and

Deretic 2008). Rab8 in concert with the Rab6, Rab11,

and Arf4 GTPases is responsible for rhodopsin trans-

port from the Golgi to the rod outer segment, in concert

with regulatory proteins (e.g., ASAP, the Arf4 GAP)

and effector proteins (e.g., FIP3) (Mazelova et al.

2009a). Mutations in the extreme C-terminus of rho-

dopsin impair interactions between rhodopsin and the

GTPase trafficking complex, which leads to autosomal

dominant retinitis pigmentosa. The altered interaction

results in aberrant trafficking of rhodopsin to the rod

outer segment, and explains at the molecular level the

cause of retinitis pigmentosa. These careful studies

were the first to reveal GTPase-mediated vesicular

hand-offs in ciliary transport, beginning with interac-

tion of a cargo protein with Rab6 and Rab11 in the

Golgi. The cargo recruits and binds Arf4 via a specific

targeting sequence to facilitate vesicle budding and

Arf4 cooperates with Rab11. ASAP1, the Arf4 GAP,

recognizes membrane curvature and is recruited to

bind Arf4 to promote GTP hydrolysis and removal of

Arf4 from the ciliary-targeted vesicle. FIP3, the Rab/

Arf effector also bridges Arf and Rab GTPases via

interactions with Rab11 and ASAP1 (Fig. 2a). The

described pathway of rhodopsin transport prompted

further questions about mechanism conservation

between different cell types, the identification of

other trafficking molecules that participate in the

GTPase vesicle ciliary relay, and the composition of

the coat complex of the ciliary transport vesicles.

Further studies in renal epithelial cells provided

clues to conservation of Rab-mediated ciliary traffick-

ing. In normal cultured renal cells, Rab8 can be found

at the perinuclear Golgi region, whereas in cultured

R 1554 Rab8

ADPKD cells, Rab8 is mislocalized to disperse vesi-

cles (Wandinger-Ness and Deretic 2008). Polycystin-1,

a protein that when mutant causes ADPKD, uses the

same GTPase transport mechanism as rhodopsin to

traffic to primary cilia of renal epithelial cells (Ward

et al. 2011). Thus, the trafficking mechanism consisting

of Rab GTPases (Rab6, Rab11, Rab8), Arf4 GTPase,

and Arf GAP ASAP1 is conserved between retinal

photoreceptor cells and renal epithelial cells (Ward

et al. 2011). In contrast, fibrocystin, a protein mutant

in patients with autosomal recessive PKD, also utilizes

Rab8 to traffic to the primary cilium, but the fibrocystin

C-terminal sequence does not bind Rab6 and Rab11

(nor Rab17, Rab23, and IFT20), suggesting that Rab8

may serve as the common GTPase between different

vesicular transport mechanisms leading to ciliary deliv-

ery (Follit et al. 2010).

Until recently, the mechanism of Rab8 recruitment

and activation along the cilial trafficking route was an

enigma. For example, in the sequential transport of

cargo that traffics with Rab6, then Rab11, and finally

with Rab8 vesicles, how is Rab8 recruited to the

exocytic vesicle? Identification of the interaction

between Rabin8, a Rab8 GEF, and a complex of

Bardet-Biedl proteins (known as the BBSome) at the

centriole provided insight into the GTPase coordination

and localized GTPase activation required for ciliary

formation. The BBSome-Rabin8 interaction was identi-

fied at the ciliary base within pericentriolar recycling

endosomes, offering the first clue for spatial regulation

(Wandinger-Ness and Deretic 2008). Here BBSome

components assist in recruitment of Rab8 to the

pericentriolar recycling endosome for nucleotide

exchange and activation, presumably by Rabin8. Fur-

ther studies revealed that transport protein particle II

complex (TRAPPII) components (C3, C9 and C10)

and Rab11 are required for vesicular trafficking of

Rabin8 to the centrosome, where Rabin8 subsequently

recruits and activates Rab8 (Fig. 2a) (Knodler et al.

2010; Westlake et al. 2011). Thus, Rabin8 serves both

as a Rab11 effector and as a Rab8 activator and both

Rab8 and Rab11 are required for ciliogenesis. Together,

the data highlight the involvement of a spatially and

temporally regulatedGTPase cascade in cilial formation

and ciliary membrane protein targeting.

The dissection of a specific Golgi to cilia pathway

is revealing key players in a ciliary targeting, but

there are numerous examples of Rab8 involvement

in cilial targeting that do not seem to follow the

above-mentioned GTPase sequence and the unifying

mechanisms remain elusive. CEP290 mutations are

linked to several inherited cystic diseases including

Senior-L€oken syndrome, nephronophthisis, Joubert

syndrome, Meckel-Gr€uber syndrome, and BBS.

CEP290 protein satellites the base of the primary cil-

ium, interacts with pericentriolar material 1 (PCM-1),

and CEP290 knockdown significantly inhibits Rab8

localization to the primary cilium (Kim et al. 2008);

however, the mechanism that mediates CEP290/PCM-1

Rab8 recruitment to the primary cilium remains

undefined. An alternate Rab8-mediated ciliary traffick-

ing mechanism has been suggested in Caenorhabitis

elegans worms in the context of the transmembrane

olfactory receptor ODR-10 (Kaplan et al. 2010). Rab8

associates with AP-1 in the clathrin-dependent delivery

of ODR-10 to dendritic sensory cilia. Overexpression

of GTP-locked Rab8 or mutations in the clathrin heavy

chain perturbed ODR-10 trafficking and caused ODR-

10 to localize to all plasma membrane compartments.

Therefore, AP-1 and Rab8 likely cooperate to direct

ODR-10 to the dendritic cilium. The authors suggested

that a default secretory pathway directs proteins to all

plasma membrane regions, and that this default path-

way can be re-routed by AP-1 and Rab8 activation. The

level of Rab8 activity may serve as the determining

factor in protein destination. If so, then active Rab8

may serve as the switch between a general secretory

and cilial-directed transport pathway. The authors’

leading model predicts that AP-1 functions at the

trans-Golgi network in a clathrin-dependent manner,

where the cargo is packaged into budding vesicles.

Upon leaving the Golgi, the vesicle uncoats, fuses

with Rab8 positive vesicles, and targets to the cilium.

It is interesting to speculate that Rab8 vesicles that

traffic ODR-10 from neuronal soma to the dendritic

cilium may resemble secretory granules in exocrine

cells and contain components of the pericentriolar

recycling endosomes described in epithelial cells.

At site of the cilium, Rab8 serves as an evolution-

arily conserved regulator of ciliary trafficking in sev-

eral cell types. In turn, the exocyst complex, first

identified in Saccharomyces cerevisiae, also serves as

a conserved trafficking regulator and as an effector of

several GTPases. The exocyst complex is comprised of

several subunits and regulates polarized secretion via

docking of intracellular vesicles to the plasma mem-

brane. The exocyst also localizes to the primary cilium

of renal cells, and Sec6 and Sec8 exocyst components

Rab8 1555 R

R

are overexpressed or diminished in ADPKD cells. The

exocyst complexes with small GTPases including

RalA, Rho1, and Rab8, and regulates function via

interactions with GAPs and other molecules such as

aPKCs and the Arp2/3 complex (Hertzog and Chavrier

2011). Rab8a-Sec6/8 interactions are thought to con-

trol vesicle docking and fusion with the basolateral

plasma membrane, thus linking the exocyst with Rab

GTPase trafficking. In photoreceptor cells, Rab8 coop-

erates with phosphatidylinositol (4,5)-bisphosphate,

moesin, Rac1 and actin to tether and fuse vesicles to

the base of the modified photoreceptor cell cilium. The

Sec6/8 complex likely serves as a Rab8 effector during

GTPase-mediated vesicular trafficking, as Sec8

colocalizes with Rab8 at fusion sites of vesicles

transporting rhodopsin, and, like Rab8, the exocyst

localizes to the primary cilia of renal epithelial cells

(Fig. 2a) (Nachury et al. 2010). In the case of retinal

cells, the Sec6/8 complex coordinates with syntaxin 3

and SNAP-25, whose interactions are regulated by

omega-3 docosahexaenoic acid, to regulate rhodopsin

delivery (Mazelova et al. 2009b).

Rab8, Rab11, Rabin8 and exocyst components play

dual roles in renal epithelial cells. Once renal cells are

polarized, Rab8, Rab11 and Rabin proteins participate

in delivery of trans-membrane protein cargo to the

primary cilium. However, during polarization and

lumen formation, Rab11, Rabin 8 and Rab8 mimic

a yeast trafficking pathway and traffic cargo, such as

podocalyxin, from the trans-Golgi to the forming pre-

apical membrane (Fig. 2b). Rab11 recruits Rabin8,

which recruits and activates Rab8. Rab11 (and poten-

tially Rab8) also recruits Sec15a, an exocyst compo-

nent, which binds Sec10 at the plasmamembrane. Rab8

recruitment to the transport vesicle enhances active

Cdc42 localization, likely driven by Rab8 activity on

Tube, the Cdc42 GEF. Cdc42 and Par6 recruit aPKC,

which then recruits Par3-Sec8 and Sec10. Thus, similar

Rab8 trafficking mechanisms are utilized throughout

the cellular polarization process, but the role that

Rab8 plays in the regulation and switch between differ-

ent target destinations remains to be explored.

One important question in the field of ciliary traf-

ficking is how the intraflagellar transport (IFT) system,

which transports non-membranous particles to and

within the primary cilium, interfaces with the Rab8

GTPase-mediated vesicular trafficking pathway. The

IFT protein Elipsa provided one of the first links to

IFT-vesicular cross talk. Elipsa localizes to primary

cilia and interacts with Rab8 via the Rab8 effector,

Rabaptin5 (Omori et al. 2008). Elipsa also directly

interacts with IFT20, which has been shown to localize

to the Golgi and within primary cilia, and when

knocked down, decreases the amount of polycystin-2

delivered to primary cilia (Follit et al. 2010). Com-

bined, these data suggest that IFT plays a role in the

transport of membrane-bound proteins to the primary

cilium. Taken together, one can speculate that mem-

brane-bound proteins interact in a complex associated

with GTPase-mediated vesicular transport, which in

turn interacts via effector proteins, such as Rabaptin5,

with IFT components. Of note, IFT molecules have

been linked to the formation of the immune synapse in

T-lymphocytes (Nachury et al. 2010), and immune

synapse formation shares similarities with cilial traf-

ficking, namely, the use of IFT20, IFT57, IFT88, Kif3a

motor protein. In contrast, IFT transport was not

affected when ODR-10 transport was perturbed by

Rab8 and clathrin manipulation (Kaplan et al. 2010).

Further studies are necessary to dissect out the

overlapping and independent roles of IFT and Rab8-

mediated vesicular transport pathways to cilia.

Though advances have been made in the context of

Rab8 and cilial trafficking, numerous questions remain

about coat complexes used by cilia-destined vesicles in

various cell types. Since Rab8, along with FIP-2, facil-

itates AP-1-mediated cargo export from the Golgi and

may be associated with AP-1 in zymogen granule

transport, could AP-1 (an Arf4 effector) also serve to

generate the exocytic vesicle coat in mammalian epi-

thelial cells? Alternatively, going back to the Rab

GTPase-mediated transport in epithelial cells (Fig. 2),

note that Arf4 facilitates vesicle budding. Arf1 and

Arf4 regulate COPI recruitment and TRAPPII compo-

nents bind COPI (Angers and Merz 2011). Thus, COPI

may serve as the initial vesicle coat for some ciliary

targeted vesicles. The coat-like BBSome plays a role in

Rab8 recruitment and activation at the pericentrosomal

region and the complex shares similarities with the

COPI and clathrin coat complexes (Nachury et al.

2010). Therefore, after initial budding, does the vesicle

coat morph to resemble components of the BBSome,

or can the BBSome itself become the late-stage Rab8-

positive vesicle coat prior to fusion with the ciliary

membrane? Further questions arise about how Rab8,

a potentially central ciliary component, may play a role

in recruitment of the motor proteins and tethering

components for ciliary transport vesicles. Recently,

R 1556 Rab8

helical SNARE [Soluble NSF Attachment Protein

(SNAP) Receptor] tethering proteins have been incor-

porated into the rhodopsin trafficking model

(Mazelova et al. 2009b). Syntaxin 3 and SNAP-25

regulate rhodopsin delivery and localize to the base

of the cilium, where Rab8 recruitment and activation is

predicted to occur. SNARE proteins can function as

Rab effectors or GEFs and future studies may reveal

that SNARE proteins, in addition or as an alternative to

the BBSome and Rabin8, may play a role in Rab

activation at the primary cilium.

Summary

Vesicular transport can be simplified into three major

steps: budding from the donor membrane, transloca-

tion of the trafficking vesicle along the cytoskeleton,

and docking and fusion with the acceptor membrane

compartment (Wandinger-Ness and Deretic 2008).

Rab GTPases facilitate this process by serving as

molecular scaffolds and interacting with cargo, pro-

teins that promote vesicle budding and coat proteins,

and through recruitment of motor protein complexes

and tethering molecules. Rab8 is involved in several

transport pathways and interfaces with endocytic path-

ways, regulates exocytosis, and coordinates regulation

of the cytoskeleton and trafficking of diverse cargo to

multiple subcellular destinations. Rab effectors, motor

proteins and specific GEFs, GAPs and kinases mediate

regulation of Rab8. Defects in Rab8-mediated traffick-

ing have profound effects on cell morphogenesis, cyto-

skeletal organization, and cellular polarity. Rab8

serves to coordinate vesicle recycling and delivery of

newly synthesized vesicle components to target mem-

branes and functions as a nexus between vesicular

endocytic and exocytic pathways. There are a number

of unanswered questions in the field of vesicle traffick-

ing: How do cells temporally and spatially regulate the

sorting decisions for GTPases? What are the molecular

switch mechanisms that cause a shift of shuttling cargo

from one target to another?What signals cause Rab8 to

associate with different effectors and how are these

signals processed? Further investigation of Rab8 in

diverse cell types and polarization states is predicted

to reveal the unifying and diverse mechanisms of

GTPase trafficking and lead to specific signaling tar-

gets for small molecule therapies for tumor invasion,

cyst formation, and neurologic disease.

Acknowledgments We acknowledge fellowships from NCRR

INBRE 5P20RR016480 and NIGMS 1K12GM088021 to HW

and research support from NIDDK DK50141 to AWN.

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Rab8a

▶Rab8

Rab-RP4 (Drosophila melanogaster)

▶RAB18

Rac GTPases

Diamantis Konstantinidis and Theodosia A. Kalfa

Cancer and Blood Diseases Institute, Cincinnati

Children’s Hospital Medical Center and University of

Cincinnati College of Medicine, Cincinnati, OH, USA

Historical Background and Taxonomy

Rac GTPases comprise one of the eight subfamilies of

the Rho (Ras homology) GTPases family, itself a sub-

group of the Ras superfamily of small G proteins

(Burridge and Wennerberg 2004). They were first

identified as a substrate for the bacterial C3-like trans-

ferases that block Rho by ADP-ribosylation, although

the C3-like transferases act on Rac rather inefficiently.

More effective are the large clostridial cytotoxins

(with prototypes the Clostridium difficile toxin A and

B) which glycosylate Rac at Thr35, inhibiting its func-

tions by preventing effector coupling (Aktories et al.

2000). Rac GTPases are preferred targets for bacteria

since they act as molecular switches in a multitude of

R 1558 Rac GTPases

signaling processes, regulating many fundamental cel-

lular functions, including actin cytoskeleton, cell adhe-

sion, motility and migration, vesicular transport

pathways and cytokinesis, ▶ reactive oxygen species

(ROS) production via NADPH oxidase, as well as cell

proliferation and survival (Hall 1998).

The Rac GTPase subfamily consists of four mem-

bers; Rac1, Rac2, Rac3, and RhoG. They all have high

homology, with the first three sharing over 90% amino

acid sequence identity between them (Heasman and

Ridley 2008). Despite this sequence similarity, studies

using knockout mice indicate that many of their func-

tions are nonredundant (Wang and Zheng 2007).

Homologues of Rac GTPases have been found in all

eukaryotic cells studied, but are totally absent in pro-

karyotes. In mammalian tissues, Rac1 is ubiquitously

expressed while expression of Rac2 is restricted to

hematopoietic cells. Rac3 is abundant in the brain but

has also been identified in a variety of tissues including

heart, placenta, and pancreas. RhoG (Ras homology

growth-related) is widely expressed in a variety of

organs but reaches a particularly high level in lung

and placenta. Deletion of the Rac1 gene in mouse

germline produces an early embryonic lethal pheno-

type and thus studies of Rac1 function have utilized

tissue-specific conditional knockout. In contrast, Rac2,

Rac3, and RhoG knockout mice are viable, fertile, and

do not exhibit obvious developmental defects. Never-

theless, they do exhibit cell-type-specific functional

defects and in the case of Rac3-null mice, a mild neu-

rological phenotype (Wang and Zheng 2007; Heasman

and Ridley 2008).

Activation of Rac GTPases and DownstreamSignaling

Rac GTPases are classical Rho GTPases and as such

act as molecular switches cycling between a GTP-

bound active form and a GDP-bound inactive form.

In their active form, they recognize target proteins,

“turning on” signaling pathways, until they hydrolyze

GTP to GDP, “switching off” (Etienne-Manneville and

Hall 2002). The activation of Rac GTPases is mediated

and regulated by guanine nucleotide exchange factors

(GEFs), which exchange GDP for GTP on the Rac

GTPase enzyme. GEFs exhibit a varied degree of

specificity with some being able to activate a large

number of different GTPases, while others specialize

in activating only one particular isoform. GEFs are

antagonized by GTPase-activating proteins (GAPs),

which increase intrinsic GTPase activity resulting in

GTP hydrolysis and the return of the GTPase to the

inactive GDP-bound state. Another level of GTPase

activity regulation is achieved through guanosine

nucleotide dissociation inhibitors (GDIs), which

inhibit nucleotide exchange and sequester small

GTPases away from the membrane. Following the

effect of specific stimuli such as soluble molecules,

cell surface-bound ligands, or mechanical stress on cell

surface receptors, GEFs and GAPs are mobilized

accordingly to regulate the activation state of the Rac

GTPases. Upon adopting an active conformation, Rac

GTPases can bind to a wide variety of effectors and

lead to initiation of the corresponding downstream

signaling pathways (Bishop and Hall 2000; Schwartz

2004) (Fig. 1).

Rac GTPases bind and activate the p21-Activating

Kinases (PAK1, PAK2, PAK3), serine/threonine

kinases, which drive cytoskeletal remodeling

(lamellipodia and membrane ruffling), cell adhesion

and proliferation, and gene transcription. While PAK

can activate c-Jun NH2-Terminal Kinase (JNK), Rac1

activates JNK mostly independently of PAK

(Westwick et al. 1997), and mainly through Mixed

Lineage Kinases (MLKs). Pathways downstream

PAK and MLK stimulate AP1-dependent gene

expression. AP1 can upregulate the expression of

genes that control cell cycle progression, such as

cyclin D1 and c-myc, proteins that when

overexpressed are associated with cell transformation

and cancer (Bosco et al. 2009). Through PAK, Rac

GTPases phosphorylate and activate ▶LIMK, which

in turn phosphorylates and inhibits cofilin, an actin

filament severing protein, hence inducing actin poly-

merization into lamellipodia and membrane ruffling.

Additionally, Rac stimulates the Wiskott–Aldrich

syndrome protein (WASP)-family verprolin-

homologous protein (WAVE) complex, which in

turn activates actin-related proteins 2/3 complex

(Arp2/3) that nucleates unbranched actin filaments

(Heasman and Ridley 2008). Rac regulation of cell

contractility includes PAK-mediated phosphorylation

of myosin light chain kinase (▶MLCK) and hence its

inactivation causing decreased phosphorylation of the

myosin regulatory light chain (MRLC) and reducing

actomyosin assembly and contraction (Bishop and

Hall 2000). PAK can also inhibit the microtubule

IQGAP

PIP5KWASP WAVE

LIMKP

Pcofilin

MLC-kinase

Raf-1

P

P

PAK

PI3K

GTP

Rac

Rac

GDP

GEF

O2.–

NF-κB

JNK

MEK1

Op18

ERK

p38

Arp2/3ERMproteins

Actinpolymerization,cross-linking,

and cytokinesis

Actinpolymerization

Association of F-actinto plasma membrane

Decreased MRLCphosphorylationand decreased

actomyosin contraction

Microtubulestability

AP1-dependentgene expression

NF-κB-dependentgene expression

MLK

GTPNox

Rac

p67phox

NADPH

NADP+ + H+

p41phox

GAP

Rac GTPases, Fig. 1 Rac GTPases regulate their multiple

functions within the cell via a variety of effectors, which initiate

separate or interacting signaling cascades. Following the effect

of specific stimuli GEFs and GAPs are mobilized accordingly to

regulate the activation state of the Rac GTPases and direct Rac

GTP to the appropriate effector

Rac GTPases 1559 R

R

destabilizing activity of OP18/stathmin by phosphor-

ylation. Other functions of Rac GTPases mediated by

PAK are interactions with the myosin heavy chain,

also leading to decreased actomyosin filaments, with

filamin A to promote membrane ruffling, as well as

with components of the paxillin-GIT/PKL-P1X com-

plex to regulate cell adhesion and motility (Schwartz

2004). Independently of PAK, Rac binds to the

actin-binding protein IQGAP (named GAP because

of some homology with Ras GAP, but actually a Rac

effector) which oligomerizes and cross-links F-actin

in vitro and has been shown to arrange actin filaments

into the cytokinetic contractile ring in yeast

(Bishop and Hall 2000). Rac GTPases bind and

activate Phosphatidylinositol-4-Phosphate 5-Kinase

(PIP5K) leading to production of phosphatidy-

linositol (4,5)-bisphosphate (PIP2) and activation of

the ERM (ezrin, radixin, moesin) complex of proteins.

ERM proteins have actin-binding domains as well as

domains that bind to cytosolic domains of plasma

membrane integral proteins and mediate the associa-

tion of F-actin to plasma membrane. (Schwartz 2004).

Phosphatidylinositol 3-Kinase (PI3K) is also a Rac

effector with multiple actions. It stimulates WASP

and Arp2/3, thus inducing actin polymerization and

produces 30-phosphorylated lipids that bind to and

stimulate Rac GEFs, creating a positive feedback

loop that maintains cell migration. In addition, it

also activates AKT Ser/Thr kinase to support cell

survival (Schwartz 2004; Bosco et al. 2009). Rac-

GTP binds to the p67phox component of NADPH

oxidase, activating the enzyme to produce superox-

ide. Superoxide and other reactive oxygen species

(ROS) have multiple roles and effects to cells and

Fibronectin

α β /α β

SCF SDF-1a

R 1560 Rac GTPases

tissues, including signaling and stimulation of NFkB-dependent gene expression (Schwartz 2004; Hordijk

2006).

CXCR4

4 1 5 1

Survival

pAKTpERK

Rac1

c-kit

Rac2Actin cytoskeleton

Adhesion & Migration

Proliferation

Rac GTPases, Fig. 2 Rac1 and Rac2 GTPases mediate prolif-

eration and survival, and regulate the actin cytoskeleton, adhe-

sion, and migration of hematopoietic stem cells and myeloid and

erythroid progenitors in the bone marrow microenvironment, in

response to cell surface receptors triggered by cytokines (SCF),

chemokines (SDF-1a), and extracellular matrix (fibronectin).

Their deficiency results in massive mobilization of HSC/P and

increased homing in the spleen, while it also disturbs the actin

cytoskeleton of neutrophils and mature erythrocytes

Rac GTPases in Hematopoiesis

Rac1 and Rac2 GTPases play distinct and overlapping

roles in hematopoietic and mature blood cells, regu-

lating homing, engraftment, actin cytoskeleton orga-

nization, ROS production, cell survival, and

proliferation (Gu et al. 2003; Mulloy et al. 2010).

Hematopoietic stem cells and progenitors (HSC/P)

deficient of Rac1 demonstrate decreased proliferation

when stimulated with stem cell factor (SCF), associ-

ated with nondetectable cyclin D1 levels and with

decreased Extracellular Signal–Regulated Kinase

(ERK) (p42/p44) phosphorylation. In contrast, loss

of Rac2 activity leads to a pro-apoptotic phenotype

in HSC/P as well as in mast cells, with reduced AKT

activation in the presence of SCF. Rac1�/�;Rac2�/�

HSC/Ps display decreased adhesion to fibronectin,

despite normal expression of a4b1 and a5b1 integrinson their surface and decreased migration in response

to stromal-derived factor-1 (SDF-1), although they

have significantly increased expression of CXCR4,

the SDF-1 receptor (Gu et al. 2003) Since Rac

GTPases are key components of the signaling path-

ways downstream of the SCF-ligand c-kit, the chemo-

kine receptor CXCR4, and the b1 integrin-receptors

for fibronectin, all significant mechanisms of interac-

tion of HSC/Ps with the bone marrow microenviron-

ment (Fig. 2), it is not surprising that combined Rac1

and Rac2 deficiency results in massive mobilization

of progenitor colony-forming unit cells (CFU-C) into

the peripheral circulation and results in increased

homing of CFU-C in the spleen (Cancelas et al.

2005; Mulloy et al. 2010).

In neutrophils, Rac1 affects cell spreading and

adhesion, while Rac2 regulates directed migration

and superoxide production (Gu et al. 2003). Rac2-

deficient mice exhibit a phagocyte immunodeficiency

syndrome. Interestingly, after the description of this

phenotype, the case of a patient with leukocytosis and

neutrophilia but multiple, recurrent, life-threatening

infections in infancy was described. A dramatic

decrease of neutrophil infiltration (absence of pus) in

areas of infections was noted. The neutrophils of the

patient exhibited decreased chemotaxis and

superoxide generation in response to fMLP (N-for-myl-methionyl-leucyl phenylalanine), as well as

reduced rolling on the L-selectin ligand GlyCAM-1;

the latter a defect that had been observed in Rac2�/�

mouse neutrophils. After LAD (leucocyte adhesion

disorder) was ruled out with normal presence of

CD11b, CD11c, and CD18, the patient was found to

have a p.Asp57Asn (D57N) mutation of Rac2 (Wil-

liams et al. 2000). This is a highly conserved position

in Rac GTPases as well as in the Ras superfamily as

a whole, since it is located in the GTP-binding pocket

of the GTPase. The mutation creates a dominant neg-

ative protein that is not only dysfunctional but also

antagonizes Rac1 and Rac3 for GTP.

Studies in gene-targeted mice demonstrated that

Rac1 and Rac2 play an overlapping but essential role

in organizing the erythrocyte cytoskeleton. Mice with

combined deficiency of Rac1 and Rac2 GTPases in

their hematopoietic cells develop hemolytic anemia,

as evidenced by concurrent reticulocytosis. Rac1�/�;Rac2�/� red blood cells exhibit a disorganized mem-

brane cytoskeleton with increased actin-to-spectrin

ratio, F-actin aggregates and meshwork gaps, irregular

clamping of band 3, decreased content of the proteins

adducin and dematin, and decreased cellular

Rac GTPases 1561 R

deformability (Kalfa et al. 2006). These mice develop

successful stress-erythropoiesis in the spleen while

homeostatic erythropoiesis in the bone marrow is sig-

nificantly compromised, implying different signaling

pathways for homeostatic and stress erythropoiesis

(Kalfa et al. 2010). Rac GTPases were also shown to

play a role in enucleation by using constitutively active

and dominant negative mutants of Rac1 and Rac2;

both inhibited enucleation in cultured mouse fetal

liver erythroblasts indicating that either inhibition or

excessive activation of Rac GTPases inhibits enucle-

ation via disruption of the contractile actin ring in

enucleating erythroblasts (Ji et al. 2008).

Combined Rac1 and Rac2 deficiency has also

been shown to impair T and B cell development, pro-

liferation, survival, adhesion, and migration, while

Rac1 deficiency compromises platelet aggregation,

lamellipodia formation, granule secretion, and clot

retraction (Mulloy et al. 2010).

R

Rac GTPases in Cancer

Rac GTPases have been implicated in cellular trans-

formation, oncogenesis, cancer invasiveness, and

metastasis. Rac1 can be induced by oncogenes like

Ras and collaborates with p53 loss of function to

promote transformation in primary fibroblasts.

Although no Rac mutations have been reported in

tumors, overexpression or increased activity of Rac1

have been found in breast, lung, and colon cancer

(Bosco et al. 2009). Activated Rac3 was detected in

the malignant precursor B-lymphoblasts in p190-BCR/

ABL transgenic mice (Cho et al. 2005) while Rac1 and

Rac2 gene targeting was found to significantly delay or

abrogate disease development in a p210-BCR/ABL

mouse model of chronic myelogenous leukemia

(CML) (Thomas et al. 2007). These data suggest

that targeting modulation of Rac GTPases activity

may provide clinical benefit for patients with CML,

Ph-positive ALL, or other cancers.

Summary

The Rac subfamily of Rho GTPases consists of four

members: Rac1, Rac2, Rac3, and RhoG. Although

they exhibit high-sequence similarity, a great number

of their functions are nonredundant. Via proteins that

activate them (guanosine exchange factors, GEFs) or

deactivate them (GTPase-activating proteins, GAPs),

they receive signals from the cell surface after soluble

ligand-receptor binding, interaction with the extracellu-

lar matrix or mechanical stress on cell surface receptors

and propagate them through the appropriate down-

stream signaling pathways. They regulate many funda-

mental cellular functions, including actin cytoskeleton,

cell adhesion, motility and migration, vesicular trans-

port pathways and cytokinesis, ROS production via

NADPH oxidase, gene transcription, and cell prolifera-

tion and survival. They have been implicated in many

physiological and pathological processes, including

hematopoiesis and cancer, and their role continues to

be investigated using gene-targeted mouse models.

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Johnson JF, Joiner CH,Williams DA, Zheng Y. Rac GTPases

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R 1562 RAC3

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RAC3

▶ Steroid Receptor Coactivator Family

Rad

▶Ras-Related Associated with Diabetes

Raf1

▶RAF-1 (C-RAF)

Raf-1

▶RAF-1 (C-RAF)

RAF-1 (C-RAF)

Andrea Varga and Manuela Baccarini

Department of Microbiology and Immunobiology,

Center for Molecular Biology, University of Vienna,

Max F. Perutz Laboratories, Vienna, Austria

Synonyms

C-Raf; c-Raf-1; Murine leukemia viral (v-raf-1)

oncogene homolog 1 (3611-MSV); Murine sarcoma

3611 oncogene 1; Raf1; Raf-1; v-Raf; v-Raf-1

leukemia viral oncogene 1

Historical Background

Raf-1, also known as C-Raf-1 or C-Raf, was identified

about 30 years ago as the oncogene (v-raf ) in the

murine sarcoma virus 3611 (3611-MSV) and, in

parallel, in the naturally occurring avian retrovirus

Mill Hill 2 (MH2). The gene was named after its

enhancing effect on fibrosarcoma induction in

newborn mice: Rapidly accelerated fibrosarcoma, or

Raf. The sequences of the oncogenes, v-raf (derived

from 3611-MSV) and v-mil (derived fromMH2), were

found to encode a serine/threonine protein kinase

containing the catalytic, but not the N-terminal regu-

latory domain of the enzyme. This deletion rendered

the protein constitutively active and was responsible

for its transforming effect, making Raf the first

oncogenic serine/threonine kinase discovered.

A pseudogene (c-raf-2) and two paralogues of

c-raf-1, named a-raf and b-raf, were subsequently

identified (Wellbrock et al. 2004; Niault and Baccarini

2010). About 20 years ago, Raf-1 was reported to be

phosphorylated in response to growth factor stimula-

tion and was identified as the activator of the ▶MEK/

ERK pathway, the first mitogen-activated protein

kinase (MAPK) module discovered that acts down-

stream of receptor tyrosine kinases; and finally, the

finding that Raf-1 could be recruited to the membrane

and stimulated by active Ras, already then recognized

as a human oncogene, made the picture complete and

led to the “textbook” description of the pathway as it is

known today. Briefly, pathway activation involves the

growth factor–induced dimerization and tyrosine

SIGNAL

SOS

Ras

Raf

MEK

ERK

RTK

RAF-1 (C-RAF), Fig. 1 Outline of the ERK pathway. The Grb2-SOS complex is recruited via the binding of Grb2 to tyrosine

phosphorylated residues in the cytoplasmic domain of activated

Receptor Tyrosine Kinases (RTK). This brings Sos in the prox-

imity of Ras, which is activated by the exchange of GDP for

GTP. GTP-bound Ras recruits Raf to the membrane, where it is

activated by phosphorylation (see Fig. 2 for more details on

Raf-1 activation). From here, the signal is passed, in the form

of phosphorylation, from Raf to MEK to ERK (solid arrows).

ERK, in turn, quenches pathway activity at different levels by

phosphorylating SOS, B-Raf, Raf-1, and MEK on negative reg-

ulatory residues (broken arrows)

RAF-1 (C-RAF) 1563 R

R

phosphorylation of cell surface receptors, which in

turn triggers the binding of a complex containing

a scaffolding protein, Grb2, and a nucleotide exchange

factor for Ras, SOS. The interaction with SOS cata-

lyzes the exchange of GDP for GTP on Ras; with the

help of scaffolding proteins (e.g. KSR), active, GTP-

bound Ras can now induce the formation of mem-

brane-associated Raf/MEK/ERK complexes. Each

protein in the cascade can activate its downstream

target via phosphorylation, i.e., Raf can phosphorylate

MEK and MEK can phosphorylate ERK. Active ERK

can induce the expression of several genes involved in

cell proliferation, differentiation, and survival. In addi-

tion, active ERK feeds back on SOS, Raf, and MEK,

providing a mechanism for signal attenuation [Fig. 1;

reviewed in (Niault and Baccarini 2010)]. Adaptor

proteins other than KSR have been reported to direct

pathway components to distinct subcellular compart-

ments, with potentially different signaling outcomes

(Kolch 2005; McKay and Morrison 2007), and inhib-

itors of the pathway can regulate it at different levels

(Kolch 2005).

The study of the essential functions of Raf-1 in

conventional and conditional knockout mice

[(Galabova-Kovacs et al. 2006) and references therein]

has revealed that most of Raf-1’s essential functions

are not linked to the activation of the MEK/ERK path-

way and to proliferation, but rather to pathways that

counteract apoptosis and promote migration and dif-

ferentiation. These new roles of Raf-1 are based on its

interaction with three other kinases: the Rho-

dependent kinase Rok-a, also known as ▶ROCK2

(Ehrenreiter et al. 2005; Piazzolla et al. 2005;

Ehrenreiter et al. 2009), involved in cytoskeletal

rearrangements; the mammalian Sterile-20-like

kinase-2, MST2 (O’Neill et al. 2004), homolog of

Drosophila’s Hippo; and the apoptosis signal-

regulating kinase 1, ASK1 (Yamaguchi et al. 2004),

upstream regulator of the p38 and JNK pathways.

Structure of Raf and Activation of Its Kinase

Function

Structure: The structure of Raf consists of three con-

served regions [Fig. 2a; see also (Baccarini 2005) and

references therein]. The regulatory/autoinhibitory

domain of Raf is composed of two Conserved Regions,

CR1 and CR2. CR1 contains the RBD – Ras BindingDomain, which is required for membrane recruitment of

the protein after activation by Ras; and the CRD –

Cysteine-Rich Domain, which, besides being

a secondary Ras binding site, is responsible for Raf-1

autoinhibition. CR2 is rich in Ser/Thr residues, whose

phosphorylation can inactivate protein function (i.e.,

negative regulatory residues such as S259, whose

dephosphorylation is prerequisite for Ras binding and

Raf activation). CR3 is responsible for the catalytic

activity and contains residues (S338, Y341) whose

phosphorylation is involved in growth factor-induced

kinase activation. The three-dimensional structure of

the RBD [NMR structure, PDB 1RFA, (Emerson et al.

1995)], CRD [NMR, PDB 1FAR, (Mott et al. 1996)],

and of the kinase domain [X-ray, PDB 3OMV,

(Hatzivassiliou et al. 2010)] is known. The RBD domain

fold resembles that of ubiquitin, while the CRD domain

is an atypical C1 domain, which binds to phosphati-

dylserine and needs Zn2+ ions to preserve its folded

RAF-1 (C-RAF), Fig. 2 Domain Structure of Raf-1 and mecha-nism of activation. (a) Schematic representation of the domain

structure of Raf-1. CR1, encompassing the RBD and the CRD,

and CR2, containing some of the phosphorylation sites which

restrain Raf-1 activity, comprise the regulatory domain; CR3

consists essentially of the kinase domain and contains the posi-

tive regulatory sites whose phosphorylation stimulates Raf-1

activity. (b) three-dimensional structure of the RBD, with its

ubiquitin fold, of the Zn2+-bound CRD, and of the Raf kinase

domain. The inactive structure of the Raf-1 kinase domain

R 1564 RAF-1 (C-RAF)

RAF-1 (C-RAF) 1565 R

R

structure (Fig. 2b, left panel). The structure of the kinase

domain consists of a smaller (N-terminal) and a larger

(C-terminal) lobe. The latter contains the activation

segment (the region between the DFG. . .APE motif,

from D486 to E515), including the P-loop and b-strand9 (b9). The P-loop is responsible for the correct posi-

tioning of both the adenosine and the gamma-phosphate

of ATP for catalysis. The inactive conformation of the

enzyme (“DFG-out”, yellow) is stabilized by hydropho-

bic interactions between the P-loop and the activation

segment (Fig. 2b, right panel). Kinase activation is

mediated by phosphorylation of serine/threonine resi-

dues in the activation segment (T491 and S494), which

results in a conformational change from the “DFG-out”

to the “DFG-in” conformation (red). In the “DFG-in”

conformation, the b9 strand of the N-lobe interacts withtheb6 strand of theC-lobe of the kinase, closing the cleftbetween the two lobes and switching the enzyme to its

active state. The b9 strand contains the V482 residue

which corresponds to the B-Raf residue frequently

mutated to E in human melanoma [V600E; activating

B-Raf mutation (Wellbrock et al. 2004)]. Interestingly,

corresponding mutations in Raf-1, which would have

the same activating effect, have not been reported.

Activation–Inactivation: In the inactive state of Raf,

the N-terminal, regulatory domain of the protein binds

to its kinase domain and inhibits its activity [Fig. 2c;

see also (Wellbrock et al. 2004; Niault and Baccarini

2010)]. This conformation is stabilized by the binding

of 14-3-3 proteins, which recognize two phosphory-

lated Raf-1 residues: S259 on the N-terminal and S621

on the C-terminal part of the protein. The current

model of Raf-1 activation postulates that this binding

must be disrupted to enable Raf-1 activation. This

process is accomplished by protein phosphatases 1

and 2A, which dephosphorylate residue S259. 14-3-3

��

RAF-1 (C-RAF), Fig. 2 (continued) (PDB: 3OMV,

(Hatzivassiliou et al. 2010); ribbon representation, in green) issuperimposed on the active structure of the B-Raf kinase domain

(PDB: 2FB8, (King et al. 2006); cartoon representation, inwhite). The aminoacid numbering corresponds to the human

Raf-1 protein. The inactive “DFG-out” conformation is shown

in yellow, the active “DFG-in” conformation in red. Note the

interaction of the b9 strand of the N-lobe (red) with the b6 strandof the C-lobe in the “DFG-in” conformation. The start of the

DFG (D486) and the position of the V492 residue corresponding

to the V600 in B-Raf frequently mutated in melanoma are

indicated. (c) Mechanism of Raf-1 activation. In quiescent

cells, intramolecular inhibition, stabilized by 14-3-3 binding to

proteins remain bound to the phosphorylated S621 and

maintain a productive conformation of the kinase

domain. After S259 dephosphorylation, Raf-1 can be

recruited to the membrane by binding to activated Ras,

primarily via the RBD. Ras binding is followed by

disruption of the inhibitory interaction between the

regulatory and the kinase domain of the protein. The

activation process is completed by the phosphorylation

of the activating residues in the CR3 region (T491,

S494), which stabilizes the active, “DFG-in” confor-

mation. Inactivation of Raf-1 occurs via the phosphor-

ylation on its negative regulatory residues by ERK,

which is followed by the dephosphorylation of activat-

ing residues by PP2A. Finally, PKA/▶ PKB

rephosphorylates the residue S259, making the

rebinding of 14-3-3 possible (Wellbrock et al. 2004;

Niault and Baccarini 2010).

Raf-1-Containing Complexes and Their Biological

Functions

Raf-1 can also be activated by (Ras-dependent)

homodimerization or by heterodimerization with

other Rafs, particularly B-Raf. As part of this complex,

Raf-1 can stimulate the MEK/ERK pathway and there-

fore regulate cell proliferation and several other bio-

logical functions (Fig. 3, pathway 1). In most cells and

tissues, however, Raf-1 is not essential for MEK/ERK

activation and proliferation. Instead, Raf-1 is required

to promote survival, either through the inhibition of

proapoptotic kinases such as MST2 and ASK1 (Fig. 3,

pathways 2 and 3) or by restraining the cytoskeleton-

based kinase Rok-a, which regulates the trafficking of

the death receptor Fas (Fig. 3, pathway 4; and right

panel). Interaction with, and inhibition of Rok-a is alsothe molecular basis of Raf-1’s role in cell migration, in

keratinocyte differentiation, and in Ras-driven

the phosphorylated S259 and S621 sites, prevents Raf-1 activa-

tion. The transition to the active state is mediated by the dephos-

phorylation of 259 and Ras binding, which recruits Raf-1 to the

membrane, where phosphorylation of activating residues occurs.

The Raf-1 signal is quenched by the phosphorylation of negative

regulatory sites (in grey) mediated by active ERK, which results

in kinase desensitization, followed by dephosphorylation of both

positive and negative regulatory sites (resensitized state).

Finally, rephosphorylation of S259 restores the close, inactive

conformation of Raf-1. The residues phosphorylated at each step

are shown at their approximate localization in the molecule. The

kinases responsible for phosphorylation of the positive (PAK,

Src) or negative (ERK, PKA, PKB) regulatory sites are shown

coiled-coil

RB

D

CR

DPH

N

PH

c

PHc CRDCRD

RhoRas

PHN

RBD

RBD

coile

d-co

il

Raf-1

Raf-1

Raf-1

SIGNAL

plasmamembrane

MST2

B-Raf

MEK

ERK

proliferation survival

Raf-1

Raf-1

Raf-1 Rok-α

Raf-1 Rok-α

KINASE

KIN

AS

E

CR

DR

BD

migrationsurvival

differentiation block

(4)

(3)(2)

(1)

ASK1

Rok-α

RTK

KINASEKINASE

RAF-1 (C-RAF), Fig. 3 Interactions of Raf-1 with differentpartners and their biological consequences. Left, As part of

a Raf dimer, Raf-1 functions as a MEK/ERK activator and

stimulates many cellular functions, in particular proliferation

(1). Most of the essential Raf-1 functions rely on protein/protein

interaction and are independent of Raf-1 kinase activity. Binding

of Raf-1 to the MST2 (2) and ASK-1 (3) kinases promotes

survival by reducing the strength of the downstream

proapoptotic signal. Interaction with the cytoskeleton-based

kinase Rok-a (4) promotes migration and survival and restrains

differentiation. Right, molecular basis of Rok-a inhibition by

Raf-1. Raf-1 and Rok-a share similar autoinhibitory domains,

which, in quiescent cells, interact with the kinase domains and

restrain their activity. When the kinases are activated by the

respective upstream GTPases, autoinhibition is relieved, and

the regulatory domain of Raf-1 is free to bind to the kinase

domain of Rok-a and modulate its kinase activity

R 1566 RAF-1 (C-RAF)

epidermal tumorigenesis (Niault and Baccarini 2010;

Wimmer and Baccarini 2010; Kern et al. 2011).

The following section describes the individual Raf-

1-containing complexes.

The Raf-1/B-Raf complex (Fig. 3, pathway 1): All

three Raf isoforms, ▶A-Raf, B-Raf, and Raf-1, can

mediate MEK/ERK activation. Conditional mutagene-

sis has revealed that B-Raf is the essential MEK activa-

tor in various cells and tissues (Galabova-Kovacs et al.

2006; Niault and Baccarini 2010). A-Raf and Raf-1 can

heterodimerize with B-Raf, yielding a MEK kinase

more potent than the individual monomeric forms.

Whether A-Raf or Raf-1 are preferred B-Raf partners

in the context of the heterodimer is yet unknown; how-

ever, both A-Raf and Raf-1 must be ablated to reduce

MEK/ERK phosphorylation in fibroblasts (Mercer et al.

2005), consistent with an interchangeable role of these

two kinases as dimer subunits. The heterodimerization

of Raf has been in the limelight since the discovery that

B-Raf inhibitors currently in the process of being

approved for the treatment of melanoma patients acti-

vate the Raf/MEK/ERK pathway, instead of stopping it

(see below; and Fig. 4).

Raf-1 and MST2 (Fig. 3, pathway 2): In mammalian

cells, MST2 is activated by stress signals and causes

apoptosis acting upstream of the mammalian Hippo

signaling pathway (Pan 2010). MST2 was identified

as a Raf-1 interacting partner using mass spectrometry

(O’Neill et al. 2004). In quiescent cells, MST2 is

phosphorylated on two negative regulatory sites in its

N-terminal and C-terminal region (Romano et al.

2010). This diphosphorylated form can interact with

Raf-1 (region between amino acids 150–303) and this

inhibits MST2 activation by preventing

homodimerization. This interaction is disrupted by

proapoptotic stimuli, enabling MST2 activation by

the tumor suppressor RASSF1.

Raf-1 and ASK1 (Fig. 3, pathway 3): ASK1 is a Ser/Thr kinase which acts upstream of JNK and p38 and

promotes apoptosis induced by stress or death recep-

tors. Cardiac-specific ablation of Raf-1 induces apo-

ptosis in the cardiac muscle in vivo and leads to

a transient increase in ASK1, JNK, and p38 activity

during postnatal heart development, without affecting

theMEK/ERK pathway (Yamaguchi et al. 2004). Con-

comitant ablation of ASK1 rescues the phenotype of

Raf-1 conditional knockout mice, indicating a causal

role of ASK1 in the abnormalities observed in Raf-1

knockout hearts and suggesting that Raf-1 limits ASK1

activity in cardiomyocytes. How exactly Raf-1 inhibits

RAF-1 (C-RAF), Fig. 4 Paradoxical activation of MEK/ERKpathway by B-Raf inhibitors. Upper panel, in wild type (WT)

cells, Raf dimer formation and MEK/ERK activation are stimu-

lated by extracellular signals acting through RTKs and Ras. In

melanoma cells harboring constitutively active B-RafV600E,

MEK/ERK activation is Ras and Raf-1-independent; while mel-

anoma cells harboring mutated N-Ras activate MEK/ERK by

stimulating the formation of B-Raf-Raf-1 dimers. Lower panel,B-Raf inhibitors will block B-RafV600E kinase activity, reduc-

ing ERK activation and inducing a proliferation block and sub-

sequent tumor regression. However, in cells harboring NRAS

mutations, inhibitors can induce Raf dimerization and ERK

activation. This is due to the fact that dimers containing only

one functional kinase subunit are active as MEK kinases. Such

dimers may arise if the inhibitors used have fast off-rates, or if

low concentrations of inhibitors are used. In both situations, the

conformational change induced by the inhibitors would suffice

to stimulate dimerization, but not to completely inhibit the

resulting dimeric kinase. This mechanism may be linked to the

development of drug-related tumors observed in melanoma

patients treated with B-Raf inhibitors

RAF-1 (C-RAF) 1567 R

R

ASK1 has not yet been established, but it is known that

this function of Raf-1 is independent of the MEK/ERK

pathway, although the phosphorylation of the activat-

ing Raf-1 regulatory residues S338 and S339 is

a prerequisite, at least in endothelial cells (Alavi et al.

2007). Raf-1 physically interacts with the N-terminal

autoinhibitory domain of ASK1; it is therefore possible

that by doing so it will promote and/or stabilize an

inactive ASK1 conformation (Chen et al. 2001).

Raf-1 and Rok-a (Fig. 3, pathway 4): In mammalian

cells, the Rho effector Rok-a is responsible for cyto-

skeletal rearrangements essential for cell adhesion and

motility. Conditional gene ablation studies have

revealed that Rok-a is hyperactive and mislocalized

in the absence of Raf-1 (Ehrenreiter et al. 2005;

Piazzolla et al. 2005). Importantly, chemical or genetic

inhibition of Rok-a rescues all phenotypes of Raf-1

knockout mouse embryonic fibroblasts, defining

Rok-a hyperactivity as the rate-limiting factor in this

context (Piazzolla et al. 2005). In addition, both the

cellular phenotypes and Rok-a hyperactivity could be

rescued by complementation with Raf-1 mutants

devoid of kinase activity, as well as by mutants featur-

ing the isolated Raf-1 regulatory domain, indicating

that the physical presence of at least part of Raf-1 is

necessary for the inhibition of Rok-a activity

(Ehrenreiter et al. 2005; Piazzolla et al. 2005). The

molecular basis of Raf-1 interaction with Rok-a is

understood in some detail. Both Raf-1 and Rok-a are

modular kinases featuring a similar domain structure.

In quiescent cells, the activity of Raf-1 and Rok-a is

restrained by intramolecular inhibition. The negative

RAF-1 (C-RAF), Fig. 5 Raf-1-mediated Rok-a inhibition isessential for the establishmentand maintenance of Ras-induced epidermal tumors.Activated Ras stimulates the

interaction between Raf-1 and

Rok-a. The resultingattenuation of Rok-a leads to

decreased expression of the

epidermal differentiation

cluster genes (EDC) and

reduces the phosphorylation of

cofilin (via ▶LIMK). Since

phosphocofilin, in turn,

inhibits the pro-proliferative

STAT3/myc pathway, Raf-1-

mediated Rok-a attenuation

supports Ras-driven

tumorigenesis

R 1568 RAF-1 (C-RAF)

regulatory domain (cysteine-rich region, CRD) of each

protein can interact with its own kinase domain and

inhibit its activity, likely by preventing substrate bind-

ing. Upon mitogenic stimulation, binding to activated

small G-proteins (Ras for Raf-1 and Rho for Rok-a)relieves autoinhibition and, at the same time, makes

the interaction between the regulatory domain (CRD)

of Raf and the kinase domain of Rok possible (Niault

et al. 2009). In this situation, the autoinhibitory domain

of Raf-1, much like an ill-fitting lego brick, can restrain

the activity of the Rok-a kinase domain without

blocking it completely (Fig. 3, right panel). This mech-

anism of inhibition in trans is the first example of

kinase regulation mediated by physical interaction

rather than phosphorylation on negative regulatory

residues.

Raf-1 and Cancer

A wealth of reports has implicated Raf isoforms, par-

ticularly B-Raf, in different aspects of tumor develop-

ment (Niault and Baccarini 2010; Maurer et al. 2011).

The next chapters will focus on two recently described

functions of Raf-1 in melanoma and squamous cell

carcinoma, both of which relay on Raf-1’s ability to

form physical complexes with other kinases.

Raf-1-B-Raf interaction and melanoma: MEK/

ERK signaling is particularly important in melanoma.

Somatic mutations occur in B-Raf and N-Ras in�50%

and �15% of cutaneous melanomas, respectively

(www.sanger.ac.uk/genetics/CGP/cosmic/). The most

frequent mutation in B-Raf is the V600E mutation,

which causes constitutive activation of the kinase and

thus of the MEK/ERK pathway. After this discovery,

tremendous efforts were made to find inhibitors

targeting the mutated form of B-Raf. Surprisingly,

these inhibitors could inactivate the enzyme in vitro,

but activated the RAF/MEK/ERK pathway in cells not

harboring the V600E mutation. The molecular basis of

the Raf-inhibitor paradox is the ability of Raf enzymes

to heterodimerize and form a potent MEK kinase, even

when only one dimer subunit is enzymatically active

[Fig. 4; reviewed in (Cichowski and Janne 2010;

RAF-1 (C-RAF) 1569 R

R

Wimmer and Baccarini 2010)]. Selective inhibitors

cause a conformational change in the structure of

B-Raf, which promotes the dimerization of this

“inhibited” form of B-Raf with Raf-1 or A-Raf. This

leads to the stabilization of an active MEK kinase and

to the stimulation of the MEK/ERK pathway.

Inhibitor-induced stabilization of Raf hetero- or

homodimers is predicted to be particularly dangerous

in cells containing mutations that stimulate dimer

formation, such as melanoma cells with an N-Ras

mutation, as indicated by a recent animal study

(Heidorn et al. 2010), and also in any other tumor-

prone cell. This mechanism might be the reason for

the appearance of keratoacanthomas and squamous

cell carcinomas in about 30% of patients treated with

Raf inhibitors in clinical studies (Arkenau et al. 2011).

Raf-1-Rok-a interaction and Ras-driven epidermal

carcinogenesis: To date, Raf-1 is the only Ras effectorthat has been shown to be essential for the mainte-

nance of Ras-driven tumors. This has been achieved

by conditional gene ablation experiments conducted

in mice with epidermis-restricted Raf-1 ablation.

Besides showing defects in wound healing,

keratinocyte adhesion and migration (Ehrenreiter

et al. 2005), these animals are refractory to epidermal

tumors caused by Ras activation; more importantly,

Raf-1 ablation causes the complete regression of

established tumors. Thus, Ras-driven tumors are

addicted to endogenous Raf-1, constituting a prime

example of non-oncogene addiction.Mechanistically,

Ras drives the formation of a complex between Raf-1

and Rok-a, ultimately resulting in Rok-a inhibition.

In the absence of Raf-1, hyperactive Rok-a drives

keratinocyte differentiation and tumor regression via

a pathway involving phosphorylated cofilin, the inhi-

bition of STAT3 phosphorylation, and of Myc expres-

sion (Fig. 5). Thus, inhibiting Raf-1-Rok-a complex

formation, either by silencing the Raf-1 gene or by

using small molecule inhibitors which can disrupt the

complex, may be a viable strategy for the (co-)therapy

of Ras-driven epidermal tumors (Ehrenreiter et al.

2009).

Summary

Born as the first serine/threonine kinase oncogene and

intensively studied as the link between Ras and the

mitogenic MEK/ERK pathway, Raf-1 is coming of

age as a versatile signal transducer with multiple

partners impinging on cell motility, differentiation,

and survival. The basis for this is its modular struc-

ture, featuring a kinase domain kept in check by an

autoinhibitory domain. This autoinhibition is relieved

by intricate regulatory mechanisms involving dephos-

phorylation of negative regulatory sites, Ras binding,

and phosphorylation of activating sites. Once

autoinhibition is relieved, Raf-1 can function as

a MEK kinase, in the context of homodimers or of

Raf heterodimers, but it can also exert kinase-

independent functions by binding to, and directly

regulating, serine/threonine kinases operating in dis-

tinct pathways. These functions in pathway cross-talk

are the essential ones, as revealed by conventional and

conditional gene ablation studies. One obvious

unresolved question in this context is how extracellu-

lar cues direct Raf-1 (or, for that matter, other signal

transducers) to the appropriate signaling complex in

order to implement the correct biological response.

One of the kinase-independent functions of Raf-1, the

inhibition of the cytoskeleton-based kinase Rok-a, isessential for the development and maintenance of

Ras-driven epidermal tumors. Will other Raf-1 inter-

actions prove similarly essential in tumorigenesis,

possibly in the context of other cell types/tissues?

And if yes, will it be possible to design inhibitors for

molecule-based therapy? The key to these questions

lies in the further investigation of Raf-1’s role in

tumor models in vivo and in obtaining structural

information on the complexes between Raf-1 and its

interacting proteins.

Acknowledgements The authors wish to thank all the members

of the Baccarini group for helpful discussions. Dr. Andrea Varga

is supported by a FEBS long term fellowship. Work in the

Baccarini lab is supported by funds of the Austrian National

Research Fund (FWF), the Austrian Society for the Advance-

ment of Research (FFG), the Obermann Foundation, and the

European Community.

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RAFA1

▶A-RAF

Ramp

Debbie L. Hay1, Patrick M. Sexton2 and

David R. Poyner3

1School of Biological Sciences, University of

Auckland, Auckland, New Zealand2Monash Institute of Pharmaceutical Sciences,

Monash University, Melbourne, VIC, Australia3School of Life and Health Sciences, Aston University,

Birmingham, UK

Historical Background

The receptor activity-modifying protein (RAMP) fam-

ily was first reported in 1998 during attempts to iden-

tify the cell surface receptor for a neuropeptide known

as calcitonin gene-related peptide (CGRP) (McLatchie

et al. 1998). Formerly, a protein known as the calcito-

nin receptor-like receptor (CLR) was thought to be the

receptor for CGRP but no study had convincingly

shown that this was the case. McLatchie and col-

leagues were able to show that CLR needs RAMP1

for a CGRP receptor to be formed. RAMP1 assists

CLR in reaching the cell surface. Thus, RAMP1 and

CLR together at the cell surface form the receptor for

CGRP, which binds and activates this protein complex,

Ramp 1571 R

R

leading to downstream signaling events such as an

accumulation of intracellular cAMP. In this same

study, two other related proteins were found, named

RAMP2 and RAMP3. Each of these proteins could

also assist CLR in reaching the cell surface but remark-

ably, CGRP was less effective at activating these pro-

tein complexes. Instead, a peptide similar to CGRP,

called adrenomedullin (AM), preferentially activated

them. Thus, RAMPs can be considered as “pharmaco-

logical switches” by virtue of their ability to change the

peptide hormone for which CLR has a preference.

Overview

The receptor complexes that are formed by RAMPs

interacting with CLR are shown in Fig. 1. Since

RAMPs were first identified, a great deal has been

learned about their functions. It is now broadly under-

stood how they change peptide recognition but also

that they have other functions and interact with other

receptors.

Pharmacological Importance of RAMPs

At first it was thought that the way RAMPs could

change peptide preference was by changing the con-

formation of the receptor protein, CLR, but it is now

known that RAMPs also play their own role in binding

CGRP and AM and also small molecule drugs (Sexton

et al. 2009). For instance, RAMP1 and CLR both

participate in the binding of the drugs Olcegepant and

Telcagepant. This makes the drugs extremely selective

for the CGRP receptor; they have only low affinity for

the two AM receptors or for the RAMP1-based AMY1

receptor. RAMPs also change the pharmacology of

another receptor, the calcitonin receptor (CTR). This

is the closest protein relative to CLR and can also

interact with all three RAMPs. In this case, CTR is

a receptor for the peptide hormone, calcitonin, when it

is expressed alone in cells. However, when CTR is

expressed with RAMPs, the resulting RAMP/CTR

complexes have a preference for a peptide hormone

known as amylin (Fig. 1) (Poyner et al. 2002).

RAMPs and Cellular Signaling

CLR and CTR are proteins known as G protein-

coupled receptors (GPCRs). As their name suggests,

these proteins interact with G proteins (guanine nucle-

otide binding proteins) to initiate intracellular signal-

ing. There are several types of G proteins that initiate

different downstream signaling events and many

receptors can interact with more than one G protein

to regulate cellular function. For instance, Gs

G proteins activate ▶ adenylate cyclase to cause

increases in cAMP, while Gq G proteins activate phos-

pholipase C to hydrolyze membrane inositol phos-

phates to form inositol trisphosphate (▶ IP3Receptors) and diacyl glycerol (DAG); these in turn

mobilize intracellular calcium and activate various

kinase proteins. Both CLR and CTR can couple to Gs

and signal via adenylate cyclase/cAMP pathway. In

the case of the RAMP/CTR complexes, that is the

amylin receptors, the amount of amylin binding can

be influenced by G protein type in a RAMP-dependent

manner (Morfis et al. 2008), and conversely RAMP

interaction with the CTR can change the receptor pref-

erence for different G proteins. For example, the

RAMP1/CTR (AMY1) and RAMP3/CTR (AMY3)

receptors have greater relative preference for Gs over

Gq G proteins than the CTR when expressed alone

(Morfis et al. 2008), and this may change to overall

cellular response to receptor activation.

The activity of a GPCRmust be carefully controlled

to make sure that cells are kept responsive when they

need to be. In many cases, a process called desensiti-

zation occurs after a GPCR has been activated, which

reduces the amount of signal that the receptor can

generate. Following this, GPCRs are often removed

from the cell surface membrane; the receptors are

internalized. The GPCR may then be returned to the

cell surface and, thus, recycled or it can be degraded

and not returned. RAMPs can also modulate these

internalization and recycling processes. RAMP3 has

a specific sequence of amino acids, a PDF-like domain,

in its intracellular domain. This allows it to interact

with different regulatory proteins to RAMP1 and

RAMP2, such as N-ethylmaleimide-sensitive factor

(Bomberger et al. 2005). Therefore, in model systems,

RAMP3/CLR (AM2 receptor) complexes can be

recycled unlike AM1 and CGRP receptors.

RAMP Interactions with Other Receptors

There is accumulating research which shows that

RAMPs may have much broader roles and control

aspects of the activity of many receptors. For example,

the calcium sensing receptor which is unrelated to

CLR or CTR requires RAMP1 or RAMP3 for it to

reach the cell surface and therefore signal (Bouschet

et al. 2005). The VPAC1 receptor can associate with

RAMP2 and this leads to enhanced, agonist-mediated

CGRP

ECD

TM

ICD

CT AMY1 AMY2 AMY3

AM2AM1

CLR

CLR CLR

CT

R

CT

R

CT

R

CT

R

RA

MP

1

RA

MP

1

RA

MP

2

RA

MP

2

RA

MP

3R

AM

P3

Ramp, Fig. 1 Receptor complexes that are formed when CLR

and RAMPs or CTR and RAMPs associate. CLR is the seven

transmembrane (TM) protein in white and CTR is the seven TM

protein in gray. RAMP1 is shown in red, RAMP2 in blue, andRAMP3 in yellow. RAMP1 with CLR is known as the CGRP

receptor whereas RAMP2 or RAMP3 with CLR are known as

AM1 and AM2 receptors, respectively. CTR is the receptor for

calcitonin (CT) but with RAMP1 it is an AMY1 (amylin subtype

1) receptor. AMY2 and AMY3 receptors are formed when

RAMP2 and 3 associate with CTR. ECD extracellular domain,

ICD intracellular domain

R 1572 Ramp

phosphoinositide breakdown but there is no effect on

cAMP production (Christopoulos et al. 2003). The

secretin receptor is another example of a RAMP-

interacting GPCR but in this case the reason why

these proteins associate has not yet been revealed.

The Structure of RAMPs

Sequence analysis shows that the RAMPs all have

a single transmembrane region, with a small cytoplas-

mic tail of around 10 amino acids and a much larger

N-terminus of around 100 amino acids (Figs. 1 and 2).

All RAMPs have four cysteines that take part in for-

mation of two disulfide bonds; RAMPs 1 and 3 have an

extra cysteine pair which form a third disulfide.

A crystal structure is available for the majority of

RAMP1, both by itself and in combination with CLR

(Kusano et al. 2008; Koth et al. 2010). The extracellu-

lar domain is a trihelical structure. Residues at the base

of helix 2 and the C-terminus of helix 3, among others,

appear to be important for making contact with CLR;

the region connecting these two helices may be partic-

ularly important for peptide binding (Fig. 3).

RAMP Expression

RAMPs appear to be widely expressed in mammals,

although there are actually very few studies where the

RAMP3 -------------METGALRRPQLLPLLLLLCG---------------GCPRAGGCNETGRAMP2 MASLRVERAGGPRLPRTRVGRPAAVRLLLLLGAVLNPHEALAQPLPTTGTPGSEGGTVKNRAMP1 -------------MARALCRLPRRGLWLLLAHH----------------LFMTTACQEAN

RAMP3 MLERL-PLCGKAFADMMGKVDVWKWCNLSEFIVYYESFTNCTEMEANVVGCYWPNPLAQGRAMP2 YETAV-QFCWNHYKDQMDPIEK-DWCDWAMISRPYSTLRDCLEHFAELFDLGFPNPLAERRAMP1 YGALLRELCLTQFQVDMEAVGETLWCDWGRTIRSYRELADCTWHMAEKLGCFWPNAEVDR

RAMP3 FITGIHRQFFSNCTVDRVHLEDPPDEVLIPLIVIPVVLTVAMAGLVVWRSKRTDTLLRAMP2 IIFETHQIHFANCSLVQPTFSDPPEDVLLAMIIAPICLIPFLITLVVWRSKDSEAQARAMP1 FFLAVHGRYFRSCPISGRAVRDPPGSILYPFIVVPITVTLLVTALVVWQSKRTEGIV

TM domain

Ramp, Fig. 2 Amino acid

sequences of human RAMPs.

Comparison of the sequences

of human RAMPs 1, 2, and 3.

Signal peptides are shown in

italics, potential glycosylation

sites are in bold. Cysteines

involved in disulfides are

shaded. TM transmembrane

Ramp, Fig. 3 Structure of the extracellular domain of RAMP1.

Structure based on 2YX8 in the protein structure database.

Helices are shown in red. Residues at the base of helix 2

and the C-terminus of helix 3 likely to be involved in CLR

recognition are in gray; the region between helices 2 and 3

which may be important for ligand binding is in green

Ramp 1573 R

R

proteins themselves have been measured, rather than

mRNA. This has been because there have been few

reliable antibodies. This has meant that it has been

difficult to properly colocalize RAMPs and receptors.

A recent study has reported the co-expression of

RAMP1 and CLR in neurons of the human and rodent

trigeminal ganglion, which is involved in pain trans-

mission (Eftekhari et al. 2010). More studies of this

nature are needed to confirm the physiological signif-

icance of the AM and amylin receptor subtypes and the

interactions of RAMPs with other receptors.

Several animal models of RAMP under or

overexpression have been generated. Mice which

selectively overexpress human RAMP1 in neurons

are sensitized to CGRP, whereas those which

overexpress RAMP2 in the smooth muscle were sen-

sitized to the effects of AM (Tam et al. 2006; Zhang

et al. 2007). These types of observation help to confirm

that these are valid components of AM and CGRP

receptors in vivo. Mice that genetically lack RAMP2

(RAMP2 knockout mice) have severe defects and

show that RAMP2 is essential for the blood and lym-

phatic vascular systems to develop properly in the

embryo (Fritz-Six et al. 2008). Interestingly, RAMP3

knockout mice do not have any obvious phenotype,

suggesting that RAMP3, and the AM2 receptors which

it forms with CLR, has different functions to the AM1

receptor (Dackor et al. 2007).

Evolutionary Considerations

There is some evidence that a form of CGRP first

evolved in insects. In Drosophila melanogaster, the

protein CG17415 shows homology to CLR. It is acti-

vated by the diuretic hormone DH31 and this response

can be amplified when it is co-expressed with human

RAMP1 or RAMP2 (Johnson et al. 2005). However,

no ortholog of a RAMP has yet been identified in

Drosophila. RAMPs are certainly present in bony

fish, where they are found with homologues of CLR

and AM. The functions of RAMPs have been best

studied in the pufferfish, Takifugu obscurus. This

expresses five forms of AM, three forms of CLR, and

five RAMPs. The expanded AM/RAMP/CLR family

appears to be involved in fluid homeostasis (Nag et al.

2006). The evolutionary history of RAMPs between

insects and fish remains obscure.

R 1574 Ran

Summary

This entry has provided a snapshot of what RAMPs are

and what they are currently understood to do. Most of

this research has been performed in isolated cellular

systems because these have been the models that have

been available. It is now important to move toward

whole organism studies so it can be fully appreciated

how broad the functions of these proteins may be in

physiology and disease.

References

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WS. Novel function for receptor activity-modifying proteins

(RAMPs) in post-endocytic receptor trafficking. J Biol

Chem. 2005;280:9297–307.

Bouschet T, Martin S, Henley JM. Receptor-activity-modifying

proteins are required for forward trafficking of the calcium-

sensing receptor to the plasma membrane. J Cell Sci.

2005;118:4709–20.

Christopoulos A, Christopoulos G, Morfis M, Udawela M,

Laburthe M, Couvineau A, Kuwasako K, Tilakaratne N,

Sexton PM. Novel receptor partners and function of receptor

activity-modifying proteins. J Biol Chem. 2003;278:3293–7.

Dackor R, Fritz-Six K, Smithies O, Caron K. Receptor activity-

modifying proteins 2 and 3 have distinct physiological func-

tions from embryogenesis to old age. J Biol Chem.

2007;282:18094–9.

Eftekhari S, Salvatore CA, Calamari A, Kane SA, Tajti J,

Edvinsson L. Differential distribution of calcitonin gene-

related peptide and calcitonin gene-related peptide receptor

components (calcitonin receptor-like receptor and receptor

activity-modifying protein 1) in the human trigeminal gan-

glion. Neuroscience. 2010;169:683–96.

Fritz-Six KL, Dunworth WP, Li M, Caron KM. Adrenomedullin

signaling is necessary for murine lymphatic vascular devel-

opment. J Clin Invest. 2008;118:40–50.

Johnson EC, Shafer OT, Trigg JS, Park J, Schooley DA, Dow JA,

Taghert PH. A novel diuretic hormone receptor in Drosoph-ila: evidence for conservation of CGRP signaling. J Exp Biol.2005;208:1239–46.

Koth CM, Abdul-Manan N, Lepre CA, Connolly PJ, Yoo S,

Mohanty AK, Lippke JA, Zwahlen J, Coll JT, Doran JD,

Garcia-Guzman M, Moore JM. Refolding and characteriza-

tion of a soluble ectodomain complex of the calcitonin

gene-related peptide receptor. Biochemistry. 2010;

49:1862–72.

Kusano S, Kukimoto-Niino M, Akasaka R, ToyamaM, Terada T,

Shirouzu M, Shindo T, Yokoyama S. Crystal structure of the

human receptor activity-modifying protein 1 extracellular

domain. Protein Sci. 2008;17:1907–14.

McLatchie LM, FraserNJ,MainMJ,WiseA, Brown J, Thompson

N, Solari R, Lee MG, Foord SM. RAMPs regulate the trans-

port and ligand specificity of the calcitonin-receptor-like

receptor. Nature. 1998;393:333–9.

Morfis M, Tilakaratne N, Furness SG, Christopoulos G, Werry

TD, Christopoulos A, Sexton PM. Receptor activity modify-

ing proteins differentially modulate the G protein-coupling

efficiency of amylin receptors. Endocrinology.

2008;149:5423–31.

Nag K, Kato A, Nakada T, Hoshijima K, Mistry AC, Takei Y,

Hirose S. Molecular and functional characterization of

adrenomedullin receptors in pufferfish. Am J Physiol Regul

Integr Comp Physiol. 2006;290:R467–78.

PoynerDR, Sexton PM,Marshall I, SmithDM,QuirionR,BornW,

Muff R, Fischer JA, Foord SM. International Union of

Pharmacology. XXXII. The mammalian calcitonin gene-

related peptides, adrenomedullin, amylin, and calcitonin recep-

tors. Pharmacol Rev. 2002;54:233–46.

Sexton PM, Poyner DR, Simms J, Christopoulos A, Hay DL.

Modulating receptor function through RAMPs: can they

represent drug targets in themselves? Drug Discov Today.

2009;14:413–9.

Tam CW, Husmann K, Clark NC, Clark JE, Lazar Z, Ittner LM,

G€otz J, Douglas G, Grant AD, Sugden D, Poston L, Poston R,McFadzean I, Marber MS, Fischer JA, Born W, Brain SD.

Enhanced vascular responses to adrenomedullin in mice

overexpressing receptor-activity-modifying protein 2. Circ

Res. 2006;98:262–70.

Zhang Z, Winborn CS, Marquez de Prado B, Russo AF. Sensi-

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glion. J Neurosci. 2007;27:2693–703.

Ran

Carlo Petosa

Institut de Biologie Structurale Jean-Pierre Ebel,

UMR 5075 (CEA/CNRS/Universite Joseph Fourier),

Grenoble, France

Synonyms

Gsp1; Spi1; TC4

Historical Background

Ran is an abundant member of the Ras superfamily

of small GTPases that is highly conserved across

eukaryotes. Originally cloned from a human teratocar-

cinoma cell line as one of four novel genes with

sequence homology to the GTP-binding domain of Ras

(Drivas et al. 1990), the gene was initially named TC4(teratocarcinoma clone 4) and found to encode a protein

of 216 amino acid residues. TC4wasmarkedly different

Ran 1575 R

from other members of the Ras superfamily in two

respects: it lacked the sites required for post-

translational lipid modification and it was primarily

localized to the nucleus. Accordingly, it was renamed

Ran for Ras-related nuclear protein. Ran was subse-

quently purified as an essential cofactor for nuclear

protein import (Moore and Blobel 1993) and over the

following years was extensively characterized for its

role in regulating nucleocytoplasmic transport. Ran

was later discovered to be critical for mitotic spindle

assembly (Carazo-Salas et al. 1999; Ohba et al. 1999)

and for the post-mitotic assembly of the nuclear enve-

lope (Hetzer et al. 2000; Zhang and Clarke 2000) and of

nuclear pore complexes (Walther et al. 2003). Ran has

more recently been implicated in diverse processes,

including centrosome duplication (Budhu and Wang

2005), apoptosis (Wong et al. 2009), injury response

signaling in neurons (Yudin and Fainzilber 2009), and

ciliary trafficking (Dishinger et al. 2010).

R

Regulation of Guanine Nucleotide State andSubcellular RanGTP Distribution

Like other GTPases, Ran undergoes cycles of GTP

exchange and subsequent hydrolysis to GDP. The

rates of nucleotide exchange and hydrolysis by Ran

are intrinsically low, and in vivo these reactions

require accessory factors to proceed at physiological

rates. GTP hydrolysis is stimulated by a GTPase-

activating protein, RanGAP, and further enhanced by

the RanGTP-binding proteins RanBP1 and RanBP2,

whereas the replacement of GDP with GTP is acceler-

ated by a guanine nucleotide exchange factor,

RanGEF, which is called Regulator of Chromosome

Condensation 1 (RCC1) in vertebrates (Binding part-

ners of Ran are summarized in Table 1). These proteins

act together to define an enzymatic cycle whereby Ran

hydrolyzes GTP to GDP, releases the GDP, and

accepts a new molecule of GTP (Fig. 1). Importantly,

the accessory factors that modulate this cycle are dis-

tributed asymmetrically in the cell. RanGEF has a high

affinity for chromatin and is restricted to the nucleus

during interphase, whereas RanGAP, RanBP1, and

RanBP2 localize to the cytosol or to the cytosolic

face of the nuclear envelope. This gives rise to an

asymmetric distribution of Ran, with the GTP- and

GDP-bound forms prevailing in the nucleus and cyto-

sol, respectively.

The function of Ran depends on its conformation,

which in turn is determined by the state of the guanine

nucleotide bound to it (Fig. 2). As with other GTPases,

the conformational changes in Ran involve two

regions, called switch I and II, that are sensitive to

the presence of the g-phosphate of GTP (Wittinghofer

and Vetter 2011). In addition, Ran possesses a 40-

residue C-terminal extension which also displays

a nucleotide-dependent conformation. In the GDP-

bound state, the C-terminal extension folds intimately

against the core domain of Ran; in the GTP-bound

state, this region detaches from the core and becomes

highly solvent accessible. Thus, the GDP- and GTP-

bound conformations of Ran are strikingly different,

allowing Ran-interacting factors to discriminate

between RanGDP and RanGTP with high selectivity.

Point mutations in Ran that either block GTPase activ-

ity or prevent RanGTP generation by inhibiting

RanGEF-mediated nucleotide exchange have been

instrumental in elucidating the role of Ran in diverse

cellular pathways. These mutations are listed in

Table 1.

Role in Nucleocytoplasmic Transport

Ran regulates nuclear transport during interphase by

acting as a molecular switch for the karyopherin-bfamily of nuclear transport receptors, also known as

importins and exportins (Fried and Kutay 2003).

Karyopherins are responsible for delivering various

classes of macromolecular cargo through the nuclear

pore complex (NPC). Representative members include

Importin b, which cooperates with the adaptor protein

Importin a to deliver proteins bearing a basic nuclear

localization signal (NLS) from the cytosol to the

nucleus; Transportin 1, which mediates the nuclear

import of ribosomal proteins and heterogeneous

nuclear ribonucleoproteins (hnRNPs); CRM1/

Exportin 1, which mediates the nuclear export of pro-

teins bearing a leucine-rich nuclear export signal

(NES); and Xpot, which exports tRNA. Additional

family members are listed in Table 1.

Crystal structures determined for a number of trans-

port receptors, both in isolation and in complex with

binding partners, have greatly elucidated how these

proteins function (Cook and Conti 2010; Stewart

2007). Karyopherins are superhelical structures

made of tandem HEAT repeats that interact with the

Ran, Table 1 Ran point mutants and binding partners

Protein Function

Ran G19V, L43E, Q69L Ran point mutants locked in the GTP-bound form due to lack of GTPase activity

Ran T24N Ran point mutant that binds to RanGEF and inhibits its exchange activity, thereby preventing RanGTP

generation

RanGAP GTPase-activating protein for Ran. Localizes to the cytoplasm and nuclear pores during interphase and

to mitotic spindle during mitosis

RanGEF/RCC1 Guanine nucleotide exchange factor for Ran. Associates with chromatin throughout the cell cycle

RanBP1 RanGTP-binding protein that localizes to the cytoplasm of nondividing cells. Acts as a cofactor for

RanGAP, enhancing the rate of GTP hydrolysis on Ran

RanBP2/Nup358 Nucleoporin that localizes to the cytosolic face of the nuclear pore complex (NPC). Acts as a cofactor

for RanGAP, enhancing the rate of GTP hydrolysis on Ran

RanBP3 Cofactor for CRM1-mediated nuclear export

RanBPM/RanBP9 Centrosomal protein that interacts with the GTP-bound form of Ran and is required for correct

nucleation of microtubules

RanBP10 Tubulin-binding protein and cytoplasmic guanine nucleotide exchange factor for Ran

NTF2/p10 Import carrier for RanGDP

Mog1 Stimulates release of GTP from Ran. In combination with RanBP1 promotes GDP release and the

selective binding of GTP to Ran

Dis3 Exoribonuclease subunit of the RNA-processing exosome complex that enhances the nucleotide-

releasing activity of RCC1

Karyopherin-b family members

Importin b/Importin b1 Associates with Importin a to mediate the nuclear import of proteins with a basic nuclear localization

signal (NLS). Associates with Snurportin1 to mediate the import of UsnRNPs

Transportin 1/Importin b2 Nuclear import receptor for diverse RNA-binding proteins

Transports the Kif17 motor protein and retinitis pigmentosa 2 protein to the primary cilium

Transportin SR Nuclear import receptor for serine/arginine-rich (SR) proteins

Transportin SR2/

Transportin 3

Nuclear import receptor for SR proteins, stem-loop binding protein (SLBP), HIV integrase

Importin 4 Nuclear import receptor for histones, ribosomal proteins, vitamin D receptor, transition protein 2

Importin 5/Importin b3 Nuclear import receptor for histones, ribosomal proteins, recombinase protein RAG-2

Importin 7 Nuclear import receptor for ribosomal proteins, glucocorticoid receptor, HIV reverse transcriptase

complex. Heterodimerizes with importin beta to import histone H1

Importin 8 Nuclear import receptor for Smad4 and signal recognition particle protein 19 (SRP19)

Importin 9 Nuclear import receptor for core histones and ribosomal proteins

Importin 11 Nuclear import receptor for ribosomal protein L12 and for the class III ubiquitin conjugating enzymes

UbcM2, UbcH6, and UBE2E2

Importin 13 Nuclear import receptor for Ubc9, Rbm8, Mago-Y14, Pax6

Export receptor for translation initiation factor eIF1A

CAS Export receptor for Importin aCRM1/Exportin1 Export receptor for proteins bearing a leucine-rich nuclear export signal (NES). Cooperates with Ran

to recruit nucleophosmin to centrosomes

Exportin-t/Xpot Export receptor for tRNAs

Exportin 4 Export receptor for eukaryotic translation initiation factor 5A (eIF5A) and Smad3. Import receptor for

transcription factors Sox2 and SRY

Exportin 5 Export receptor for microRNA precursors and 60S ribosomal subunit

Exportin 6 Export receptor for profilin/actin complexes

Exportin 7 Export receptor for p50RhoGAP, 14-3-3s

R 1576 Ran

GTP-bound form of Ran. RanGTP binds to the N-

terminal half of these proteins, while the cargo gener-

ally binds to the C-terminal half (Fig. 3a). RanGTP and

cargo bind to importins in a mutually exclusive

manner, whereas they bind to exportins cooperatively.

This difference in binding mode, combined with the

asymmetric distribution of RanGTP across the nuclear

envelope, ensures the directionality of nuclear

H2O

PO4RanGDP

RanGAP

RanBP1

RanGEF(RCC1)

GTP

GDP

Nucleus

nucleotideexchange

RanGTP

NPC

GTP

hydrolysis

Cytosol

Ran, Fig. 1 Regulation of Ran’s guanine nucleotide state. The

guanine nucleotide exchange factor RanGEF localizes to the

nucleus, while the GTPase-activating protein RanGAP and the

RanGTP-binding protein RanBP1 localize to the cytosol. This

gives rise to an asymmetric subcellular distribution of RanGTP,

which is at high concentration in the nucleus and at low concen-

tration in the cytosol. NPC nuclear pore complex

switch II

switch II

C-terminalextension

C-terminalextension

switch I

switch I

RanGDP

RanGTP

β

βγ

α

α

Ran, Fig. 2 Structure of Ran in the GDP- and GTP-bound

states. The conformations of the switch I and II regions and of

the C-terminal extension depend on the bound nucleotide. GDP

and GTP are shown as stick models, with the a, b, and gphosphate groups labeled. Structures shown are those of pdb

entries 1BYU (RanGDP) and 1IBR (RanGTP)

Ran 1577 R

R

transport. Thus, importins bind their cargo in the cyto-

sol, translocate through the NPC, and release the cargo

in the nucleus upon encountering RanGTP (Fig. 3b). In

contrast, exportins associate with their cargo in the

nucleus together with RanGTP, forming a ternary

complex that traverses the NPC and subsequently dis-

sociates in the cytosol. In the cytosol, the binding

of RanBP1 releases RanGTP from importins and

exportins, and rebinding is prevented by RanGAP-

mediated hydrolysis of Ran to the GDP-bound state.

Nuclear transport factor 2 (NTF2) recycles RanGDP to

the nucleus, where RanGEF mediates conversion to

the GTP-bound form. RanGTP, thus, acts as a posi-

tional cue that defines the nuclear compartment and

directs the disassembly and assembly of import and

export complexes, respectively.

Role in Mitotic Spindle Organization

In addition to regulating nucleocytoplasmic transport

during interphase, Ran plays an important role during

mitosis, regulating several aspects of mitotic spindle

assembly (Clarke and Zhang 2008; Gruss and Vernos

2004). These include microtubule nucleation, micro-

tubule stability, production of antiparallel microtubule

arrays, and the focusing of spindle poles. Many of

these functions for Ran were discovered and charac-

terized in studies using Xenopus laevis egg extracts.

The importance of Ran during mitosis has been con-

firmed in mammalian somatic cells, where, for exam-

ple, Ran regulates microtubule attachment to

IMPORTIN

Mutually exclusive binding

b

a

Import Cargo

Cargo

Import

NTF2

NTF2

NTF2

NTF2

RanGTP RanGTP

Cytosol

HEAT repeats HEAT repeats

Cooperative binding

Export Cargo

EXPORTIN

Export

RanGDP

RanGTP

RanGTPRanGTP

RanGDPC

C

C

C

CNCN

RanGDPR R

RRnucleotideexchange

GTPhydrolysis

GTP hydrolysis

nucleotideexchange

R

R

R

R

R

R

R R

RCC

C

C

C

C

R

NTF2

NTF2

NTF2

NTF2

Importin Import cargo

Export cargoExportin

Nucleus

Cargo

Ran, Fig. 3 Role of Ran in nucleocytoplasmic transport. (a)Mode of interaction with importins and exportins. Whereas

RanGTP and cargo associate with importins in a mutually exclu-

sive manner, they associate with exportins cooperatively.

(b) Generic import (left) and export (right) pathways mediated

by karyopherin b members. Nucleotide exchange in the nucleus

is mediated by RanGEF/RCC1, while GTP hydrolysis in the

cytosol is promoted by RanGAP and RanBP1 or RanBP2

R 1578 Ran

kinetochores, is required for microtubule nucleation by

both centrosomes and kinetochores, and regulates cen-

trosome cohesion during spindle pole formation

(Arnaoutov and Dasso 2005; Roscioli et al. 2010).

The molecular mechanism by which RanGTP

affects mitotic spindle assembly is closely related to

that by which it regulates nucleocytoplasmic transport.

After nuclear envelope breakdown at the onset of

mitosis, RanGTP is concentrated near chromosomes

by chromatin-bound RanGEF, while RanGAP and

RanBP1 promote GTP hydrolysis by Ran distal to

chromatin. This gives rise to a concentration gradient

of RanGTP that is centered on chromosomes (Fig. 4).

RanGTP influences the organization of the mitotic

apparatus by activating spindle assembly factors

(SAFs), which mediate microtubule stabilization and

spindle assembly. One example of a RanGTP-

activated SAF is TPX2 (Targeting Protein for Xklp2).TPX2 promotes spindle formation by targeting the

kinesin-like motor protein Xklp2 to microtubule

minus ends and by activating Aurora A kinase, which

is involved in centrosome function. Another SAF acti-

vated by RanGTP is NuMA (Nuclear Mitotic Appara-tus protein). NuMA associates with the motor protein

centrosome centrosomeImpα

Impα

Impβ

Impβ

SAF

SAF

RanGTPgradient

RanGDP

RanGTPRanGEF(RCC1)

R

R

R

Ran, Fig. 4 Role of Ran in

mitotic spindle assembly. The

binding of RanGTP to

Importin b in the vicinity of

chromosomes causes the

release of spindle assembly

factors such as TPX2 and

NuMA. The gradient of

RanGTP established by

chromatin-bound RanGEF is

indicated by the magenta

shading. SAF spindle

assembly factor, Impaimportin a, Impb importin b

Ran 1579 R

dynein and its motility-activating complex dynactin,

and translocates along microtubules to the spindle

poles where it organizes and tethers microtubules to

spindle poles. TPX2 and NuMA are inhibited by the

Importin a/b heterodimer, which bind to the NLS

motifs of these SAFs. Near chromatin, RanGTP binds

and displaces Importin b, releasing and activating the

SAFs. RanGTP, thus, acts as a positional marker that

ensures the correct spatial regulation of spindle assem-

bly in the vicinity of chromosomes.

R

Role in Nuclear Envelope Assembly

In higher eukaryotes, the nuclear envelope is

reconstituted around the segregated DNA at the end of

mitosis. This process occurs in three steps: first, mem-

brane vesicles are recruited to the vicinity of chromatin;

next, these vesicles fuse into a continuous nuclear mem-

brane; and finally, nucleoporins assemble to formNPCs

that insert into the nuclear envelope. Although the

mechanisms underlying these events are not fully under-

stood, Ran clearly plays an important role. RanGTP

stimulates membrane fusion and nuclear pore assembly,

while Importin b negatively regulates these events.

More specifically, artificial beads coated with Ran and

added to X. laevis egg extracts or other cell-free systems

accumulate membrane vesicles that fuse into

a continuous lipid layer, incorporate nucleoporins, and

form NPCs in the absence of chromatin (Zhang and

Clarke 2000). Both the generation of RanGTP by

RanGEF/RCC1 and GTP hydrolysis by Ran are

required for membrane fusion to occur (Hetzer et al.

2000). The generation of RanGTP is also required to

release specific nucleoporins from Importin b, to targetthese proteins to chromatin and to allow the association

of NPC subcomplexes (Walther et al. 2003).

Additional Functions of Ran

1. Centrosome duplication. Centrosomes, the major

microtubule organizing center of mammalian

cells, are duplicated once and only once during the

G1/S transition of the cell cycle. Ran and the

nuclear export receptor CRM1 help orchestrate

this event through their effect on nucleophosmin

(NPM), which has been implicated as a licensing

factor that regulates centrosome synthesis (Budhu

and Wang 2005). A fraction of Ran and CRM1

localizes to centrosomes, where they recruit NPM

through the latter’s leucine-rich NES motif to form

a centrosomal CRM1/Ran/NPM complex. Mutation

of the NES motif or inactivation of CRM1 leads to

dissociation of NPM from centrosomes and to the

premature initiation of centrosome duplication.

Viral oncoproteins that cause abnormal centrosome

duplication (e.g., adenovirus E1A and human pap-

illomavirus E7 proteins) also interact physically

with Ran and disrupt its centrosomal regulatory

functions (Lavia et al. 2003).

2. Apoptosis. Apoptosis triggered by DNA damage

leads to the redistribution of Ran from the nucleus

to the cytosol and to an overall reduction in

R 1580 Ran

RanGTP levels (Wong et al. 2009). This redistribu-

tion has been linked to the action of Mst1, a kinase

localized primarily to the cytosol but which accu-

mulates in the nucleus during apoptosis following

caspase-mediated cleavage of its nuclear export

signal. Nuclear Mst1 phosphorylates serine residue

S14 on histone H2B. This leads to RanGEF becom-

ing more tightly bound to chromosomes and to

inhibition of its guanine nucleotide exchange activ-

ity toward Ran. The resulting dissipation of nuclear

RanGTP blocks entry into the nucleus of▶NF-kB,a transcription factor with an important role in res-

cuing cells from apoptosis. Thus, dissipation of the

RanGTP gradient due to histone phosphorylation

prevents the initiation of an anti-apoptotic program.

3. Ciliary trafficking. The primary cilium is

a microtubule-based organelle that projects from

the cell surface and transduces environmental stim-

uli into intracellular signals. Entry of proteins into

cilia appears to be regulated at the base of the cilia at

a region known as the transition zone, where

a structure analogous to the NPC has been proposed

to exist. Studies of the microtubule motor Kif17

revealed a role for Ran in regulating protein entry

into primary cilia which is strikingly similar to

Ran’s role in nucleocytoplasmic transport

(Dishinger et al. 2010). Targeting of Kif17 to pri-

mary cilia depends on a short carboxy-terminal

sequence that contains several basic residues and

shares similarities with NLSs. This “ciliary locali-

zation signal” (CLS) mediates binding to the

nuclear import receptor Transportin 1. The Kif17/

Transportin 1 complex is then targeted to the

intraciliary compartment, where high RanGTP con-

centrations release the motor protein from the

importin. Ciliary import of Kif17 thus mirrors the

nuclear import of an importin/cargo complex and its

dissociation by nuclear RanGTP.

4. Neuronal processes. Ran appears to play an impor-

tant role in neuron development, as RNAi knock-

down of Ran revealed defects in neuron

development in both drosophila and mouse neu-

rons, while the Ran-binding protein RanBPM has

been implicated in cytoplasmic signaling in neuro-

nal processes and in the regulation of neuronal

outgrowth (Yudin and Fainzilber 2009). A role for

Ran has also been identified in the regulation of

retrograde injury signaling in peripheral sensory

neurons. In axons, RanGTP forms part of

a multimeric complex that includes the nuclear

export receptor CAS, Importin a, and the microtu-

bule-associated motor protein dynein. Nerve injury

causes an increase in the cytoplasmic levels of

RanBP1, RanGAP, and Importin b. RanBP1 and

RanGAP induce the release of RanGTP and CAS

from the Importin a/dynein complex and promote

the hydrolysis of RanGTP to RanGDP. This allows

the newly translated Importin b to associate with

Importin a/dynein, thereby creating a retrograde

injury-signaling complex capable of binding (via

Importin a) NLS-bearing signaling cargos. As

with the ciliary trafficking example above, these

findings show that Ran can act as a regulator of

importin-dependent transport and signaling at sites

that are distant from the nucleus.

Summary

Ran regulates several fundamental processes through-

out the cell cycle, including nucleocytoplasmic trans-

port during interphase, the organization of the mitotic

apparatus after nuclear envelope breakdown, the

reassembly of the nuclear envelope after mitosis, and

duplication of the centrosome. Ran also plays a role in

more specialized processes, such as ciliary trafficking,

the apoptotic response to a variety of conditions, and

neuronal development and injury signaling. In many of

these processes, Ran functions as a spatial marker

describing where the chromatin or nucleus is.

A commonly observed feature is the role played by

transport receptors of the karyopherin b family, which

sequester different activators and inhibit their function

until relieved by RanGTP. A challenge for the future

will be to understand these Ran-regulated processes in

detail, defining all the players involved and the molec-

ular mechanisms by which these events are precisely

orchestrated during the cell cycle.

References

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ment in somatic cells. Cell Cycle. 2005;4:1161–5.

Budhu AS, Wang XW. Loading and unloading: orchestrating

centrosome duplication and spindle assembly by Ran/Crm1.

Cell Cycle. 2005;4:1510–14.

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JW, Truong YN, Margolis B, Martens JR, Verhey KJ. Ciliary

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Ran/M1

▶TLR4, Toll-Like Receptor 4

RANK and RANKL

Tomoki Nakashima1,2 and Hiroshi Takayanagi1,2,3

1Department of Cell Signaling, Graduate School of

Medical and Dental Sciences, Tokyo Medical and

Dental University, Bunkyo-ku, Tokyo, Japan2Japan Science and Technology Agency (JST),

Explorative Research for Advanced Technology

(ERATO) Program, Takayanagi Osteonetwork

Project, Bunkyo-ku, Tokyo, Japan3Global Center of Excellence (GCOE) Program,

International Research Center for Molecular Science

in Tooth and Bone Diseases, Bunkyo-ku, Tokyo, Japan

RANK and RANKL Family Members

RANK; receptor activator of nuclear factor-kB,TNFRSF11A; tumor necrosis factor receptor super-

family, member 11a, CD265. RANKL; receptor

activator of nuclear factor-kB ligand, TNFSF11;

tumor necrosis factor (ligand) superfamily,member 11,

OPGL; osteoprotegerin ligand, ODF; osteoclast

differentiation factor, TRANCE; TNF-related

activation-induced cytokine, CD254.

Historical Background

In the late 1980s, an in vitro coculture system for

osteoclast formation was established. This system

was shown to require cell-to-cell contact between

calvarial cells and bone marrow cells for osteoclast

differentiation (Suda et al. 1999). Based on this

finding, it was proposed that osteoclastogenesis-

supporting mesenchymal lineage cells express an

osteoclast differentiation factor (ODF) in the form of

a membrane-associated protein (Suda et al. 1999). In

the late 1990s, the potential inhibitor of osteoclas-

togenesis osteoprotegerin (OPG) was cloned. OPG is

a decoy receptor that associates with a transmembrane

protein of the tumor necrosis factor (TNF)

superfamily, OPGL, which turned out to be the

long-sought ODF (Takayanagi 2007; Theill et al.

2002). Interestingly, immunologists cloned the

same molecule as a stimulator of dendritic cells

expressed by T cells, and named it receptor activator

R 1582 RANK and RANKL

of nuclear factor-kB ligand (RANKL), or TNF-related

activation-induced cytokine (TRANCE) (Lorenzo

et al. 2008). The receptor for RANKL is RANK,

a type I transmembrane protein, which assembles into

a functional trimer upon ligand binding, which is

similar to other members of the TNF receptor family

(Nakashima and Takayanagi 2009). The RANK and

RANKL system currently provides a paradigm that

enables the molecular understanding of the linkage

among bone metabolism, the organization of lymphoid

tissues, the regulation of body temperature, mammary

gland development, and tumorigenesis.

The Role of RANKL in the Bone and theImmune Systems

Mice with a disruption of Rank or Rankl exhibit severeosteopetrosis accompanied by a defect in tooth erup-

tion owing to a complete lack of osteoclasts. These

genetic findings clearly demonstrate that RANK and

RANKL are essential for osteoclastogenesis in vivo. In

contrast, mice lacking Opg exhibit severe osteoporosis

due to both an increased number and enhanced activity

of osteoclasts (Takayanagi 2007; Theill et al. 2002). In

humans, mutations in RANK, RANKL, and OPG have

been identified in patients with bone disorders, includ-

ing familial expansile osteolysis, autosomal recessive

osteopetrosis, and juvenile Paget’s disease of bone

(Nakashima and Takayanagi 2009).

RANKL functions as a membrane-anchored mole-

cule and is released from the cell surface as a soluble

molecule following proteolytic cleavage by matrix

metalloproteinases (MMPs) (Nakashima et al. 2000).

Both the soluble and membrane-bound forms of

RANKL function as agonistic ligands for RANK. How-

ever, previous reports have suggested that membrane-

bound RANKL is more efficient than soluble RANKL

(Nakashima and Takayanagi 2009). In addition, previ-

ous studies have indicated that RANKL serves as both

a chemotactic and survival factor for osteoclasts, and

that RANKL is mainly expressed in cells of mesenchy-

mal lineage such as osteoblasts, bone marrow stromal

cells, and synovial cells. RANKL expression can be

upregulated by certain osteoclastogenic factors such as

vitamin D3, prostaglandin E2, parathyroid hormone,

interleukin (IL)-1, IL-6, IL-11, IL-17, and TNF-a(Nakashima et al. 2000; Theill et al. 2002). However,

the major source of RANKL in vivo remains unclear,

since RANKL is expressed by several different cell

types in both the bone and bone marrow, including

osteoblasts, osteocytes, bone marrow stromal cells, and

lymphocytes. A recent report demonstrated that osteo-

cytes embedded within the bone matrix both express

a much higher amount of RANKL and have a much

greater capacity to support osteoclastogenesis than oste-

oblasts or bone marrow stromal cells. Furthermore, the

crucial role of RANKL expressed by osteocytes was

confirmed by the severe osteopetrotic phenotype

observed in mice specifically lacking RANKL in osteo-

cytes. These results clearly indicate that the osteocytes

are the major source of RANKL in bone remodeling

in vivo (Nakashima et al. 2011).

Intriguingly, in addition to the defect in osteoclasts,

both RANK- and RANKL-deficient mice are defective

in the development and organization of secondary

lymphoid tissue (Takayanagi 2007; Theill

et al. 2002). However, RANKL-deficient mice also

have a reduced thymus size and impaired thymocyte

differentiation. Although the mRNA of RANK is pre-

sent in the thymus of RANKL-deficient mice,

RANK-deficient mice do not display any obvious

defects in thymocytes. This phenotypic difference in

the thymus is the only evident distinction between

RANK- and RANKL-deficient mice (Lorenzo

et al. 2008; Takayanagi 2007; Theill et al. 2002).

This observation suggests that RANKL has the poten-

tial to act on another receptor during the course of

thymocyte development, a subject which remains to

be investigated further. Severe immunodeficiency is

not observed in RANKL-deficient mice, nor are there

any obvious adverse effects in the immune system due

to the administration of anti-RANKL antibody in

humans (McClung et al. 2006). The loss of RANKL

in T cells seems to be compensated by CD40L in mice

(Lorenzo et al. 2008). These observations initially

suggested that the immunological function of

RANKL is of lesser importance, but recent studies

have revealed a crucial role for RANKL in the immune

system. RANKL has been shown to play a critical role

in a pathological model of inflammatory bowel disease

by stimulating dendritic cells (Nakashima and

Takayanagi 2009; Takayanagi 2007), suggesting that

RANKL is distinctly involved in the activation of

dendritic cells under certain autoimmune conditions.

On the other hand, keratinocytes express RANKL in

response to ultraviolet stimulation of the skin, which

appears to activate Langerhans cells and trigger the

RANK and RANKL 1583 R

expansion of regulatory T (Treg) cells in draining

lymph nodes (Nakashima and Takayanagi 2009).

Vitamin D3, which is produced in the skin in

response to sun exposure, has long been known to

have immunosuppressive functions and to induce

RANKL on osteoclastogenesis-supporting mesenchy-

mal cells in bone. Thus, the suggested role for

RANK/RANKL might be the missing link which

mediates sunlight-induced immunosuppression. In

addition, recent reports suggest that RANK is

a key molecule in the development of autoimmune

regulator (Aire)-expressing medullary thymic

epithelial cells (mTECs), and cooperation between

RANK and CD40 also promotes mTEC development,

thereby establishing self-tolerance (Nakashima and

Takayanagi 2009). Although the functions of

RANKL/RANK in the immune system need to be

elucidated in greater detail, the discovery and subse-

quent functional analysis of RANKL has become the

driving force behind advances in the understanding of

the osteoimmune axis.

R

The Intracellular Signal Transduction ofRANKL

RANK is a transmembrane molecule expressed on

osteoclast precursor cells and mature osteoclasts. The

ligation of RANK with RANKL results in the commit-

ment of monocyte/macrophage precursor cells to the

osteoclast lineage and the activation of mature osteo-

clasts. RANK lacks intrinsic enzymatic activity in its

intracellular domain and transduces signals by

recruiting adaptor molecules such as the TNF

receptor-associated factor (TRAF) family proteins

(Takayanagi 2007). Genetic approaches coupled with

intensive molecular analyses have identified▶TRAF6

as the main adaptor molecule that links RANK to both

osteoclastogenesis and lymph node development

(Nakashima and Takayanagi 2009; Takayanagi 2007).

By an as yet unknown mechanism, RANKL binding to

RANK induces the trimerization of RANK and

TRAF6, which leads to the activation of nuclear

factor-kB (▶NF-kB) and certain mitogen-activated

kinases (MAPKs), including Jun N-terminal kinase

(JNK) and p38. It has not yet been determined how

RANK alone, among the TRAF6-binding receptors, is

able to stimulate osteoclastogenesis so potently. Addi-

tional RANK-specific adaptor molecules may exist

which link RANK signaling to other pathways. For

example, the molecular scaffold Grb2-associated bind-

ing protein 2 (▶Gab2) and four-and-a-half LIM

domain 2 (FHL2) have been shown to be associated

with RANK and to exert an important regulatory role

in its signal transduction. On the other hand, recent

investigation has revealed that the deubiquitinating

enzyme CYLD negatively regulates RANK signaling

by inhibiting TRAF6 ubiquitination and the activation

of downstream signaling events (Nakashima and

Takayanagi 2009). The control of the RANK signaling

cascade during osteoclastogenesis is summarized in

Fig. 1.

The essential role of NF-kB in osteoclastogenesis

has been demonstrated genetically (Takayanagi 2007).

NF-kB p50 and p52 double-deficient mice develop

severe osteopetrosis because of a defect in osteoclas-

togenesis. The upstream kinase complex that mediates

the phosphorylation and degradation of inhibitor of

NF-kB (IkB) comprising the catalytic subunits IkBkinase a (IKKa), IKKb, and the non-catalytic subunit

IKKg (also known as NEMO) are also important for

RANK signaling and osteoclastogenesis. In mice,

IKKb is required for RANKL-induced osteoclas-

togenesis both in vitro and in vivo, whereas IKKaappears to be required only in vitro, not in vivo.

Importantly, patients with X-linked osteopetrosis,

lymphedema, anhidrotic ectodermal dysplasia, and

immunodeficiency (OL-EDA-ID syndrome) bear

a X420W point mutation in IKKg (Nakashima and

Takayanagi 2009).

The activator protein 1 (AP-1) transcription factor

complex is also essential for osteoclastogenesis

(Wagner and Eferl 2005). RANK activates AP-1

through an induction of c-Fos. Induction of c-Fos is

dependent on the activation of calcium/calmodulin-

dependent protein kinase type IV (CaMKIV) and cyclic

AMP-responsive element-binding protein (▶CREB)

(Sato et al. 2006), but there are several reports that

suggest NF-kB is involved in the c-Fos induction

(Nakashima and Takayanagi 2009). In addition, c-Fos

expression is induced by treatment with macrophage

colony-stimulating factor (M-CSF). Recent investiga-

tion has revealed that peroxisome proliferator-activated

receptor-g (PPAR-g) plays an unexpected role in

osteoclastogenesis by directly regulating c-Fos expres-

sion (Nakashima and Takayanagi 2009). Thus, it

appears that the induction of c-Fos is not regulated by

a single pathway.

RANK and RANKL, Fig. 1 Signaling cascades duringosteoclastogenesis. Osteoclastogenesis is cooperatively inducedby M-CSF, RANKL, and its costimulatory factor, immunoglob-

ulin-like receptor. (a) Precursor cell stage; the binding ofM-CSF

to its receptor, c-Fms, activates the proliferation, survival, and

cytoskeletal reorganization of osteoclast precursor cells of the

monocyte/macrophage lineage and induces RANK expression.

The costimulatory receptors appear to be stimulated at early

stages. Proximal RANK signals; RANKL binding to RANK

results in the recruitment of TRAF6 and, at the same time, the

phosphorylation of the ITAM in DAP12 and FcRg, which are

adaptor proteins associating with distinct immunoglobulin-like

receptors. (b) Initial induction of NFATc1; NFATc1, a master

transcription factor for osteoclastogenesis, is initially induced by

the TRAF6-activated NF-kB and NFATc2 that are present in the

cell before RANKL stimulation. RANK and ITAM signals

cooperate to phosphorylate PLCg and activate calcium signal-

ing, which is critical for the activation of NFATc1. The tyrosine

kinases Btk and Tec are activated by RANK and are important

for the phosphorylation of PLCg, thus linking the two pathways.(c) Disinhibition of NFATc1; NFATc1 activity is negatively

regulated by other transcription factors such as IRF-8, MafB,

and Bcl6. The expression of such negative regulators was

observed to be repressed in osteoclastogenesis. Blimp1, which

is induced by RANKL through NFATc1 during osteoclas-

togenesis, functions as a transcriptional repressor of

anti-osteoclastogenic genes. (d) Autoamplification of NFATc1;

calcium signal-mediated persistent activation of NFATc1, as

well as cooperation with AP-1, is a prerequisite for the robust

induction of NFATc1. AP-1 activation is mediated by the induc-

tion and activation of c-Fos by CaMKIV-stimulated CREB and

c-Fms. The NFATc1 promoter is epigenetically activated

through histone acetylation and NFATc1 binds to an

NFAT-binding site on its own promoter. (e) Induction of osteo-

clast-specific genes; NFATc1 works together with other tran-

scription factors, such as AP-1, PU.1, CREB, and MITF, to

induce various osteoclast-specific genes

R 1584 RANK and RANKL

Importantly, RANKL specifically and potently

induces nuclear factor of activated T cells, cytoplas-

mic 1 (▶NFATc1), the master regulator of osteoclast

differentiation, and this induction is dependent on both

the TRAF6-NF-kB and c-Fos pathways (Takayanagi

et al. 2002). The NFAT family of transcription factors

was originally discovered in T cells, but its members

are involved in the regulation of a variety of biological

systems (Crabtree and Olson 2002). The activation of

NFAT is mediated by a specific phosphatase,

RANK and RANKL 1585 R

R

calcineurin, which is activated by calcium-calmodulin

signaling. The essential and sufficient role of the

Nfatc1 gene in osteoclastogenesis has been shown

both in vitro and in vivo. TheNfatc1 promoter contains

NFAT binding sites and NFATc1 specifically

autoregulates its own promoter during osteoclas-

togenesis, thus enabling the robust induction of

NFATc1 (Takayanagi 2007). AP-1 containing c-Fos,

together with continuous activation of calcium

signaling, is crucial for this autoamplification

(Takayanagi et al. 2002). NFATc1 regulates

a number of osteoclast-specific genes in cooperation

with other transcription factors such as AP-1, PU.1,

and MITF (Takayanagi 2007). Osteoclasts mature into

multinuclear giant cells by the fusion of numerous

mononuclear osteoclasts. The expression of fusion-

mediating molecules such as the d2 isoform of

the vacuolar ATPase Vo domain (Atp6v0d2) and

the dendritic cell-specific transmembrane protein

(DC-STAMP) is directly regulated by NFATc1

(Nakashima and Takayanagi 2009). A previous study

indicated that CREB, activated by CaMKIV, also

cooperates with NFATc1 in the activation of

osteoclast-specific genes (Fig. 1). On the other hand,

NFATc1 activity is negatively regulated during

osteoclastogenesis by other transcription factors, such

as interferon regulatory factor-8 (IRF-8), B cell lym-

phoma 6 (Bcl6), and v-maf musculoaponeurotic

fibrosarcoma oncogene family protein B (MafB)

(Miyauchi et al. 2010; Nishikawa et al. 2010; Zhao

et al. 2009). The expression of such negative regulators

was observed to be repressed through osteoclas-

togenesis (Fig. 1). This repression is consistent with

the notion that high NFATc1 activity is a prerequisite

for efficient osteoclastogenesis, but the mechanism by

which the expression of these anti-osteoclastogenic

regulators is repressed during RANKL-induced

osteoclastogenesis has remained obscure. Recent data

indicate that B lymphocyte-induced maturation pro-

tein-1 (Blimp1), which is induced by RANKL through

NFATc1 during osteoclastogenesis, functions as

a transcriptional repressor of anti-osteoclastogenic

genes such as IRF-8, Bcl6, and MafB (Nishikawa

et al. 2010). Therefore, NFATc1 choreographs the

determination of cell fate in the osteoclast lineage by

inducing the repression of negative regulators as well

as through its effect on positive regulators. However,

compared with the wealth of information on RANK

signaling in osteoclasts, it is as yet unclear whether

RANK uses the same signaling mechanisms in the

immune system and other systems.

Phospholipase Cg (PLCg), which mediates Ca2+

release from intracellular stores, is crucial for the acti-

vation of the key transcription factor NFATc1 via

calcineurin (Takayanagi et al. 2002). However, despite

the evident importance of the calcium-NFAT pathway,

it had long been unclear how RANKL activates cal-

cium signals. RANK belongs to the TNF receptor

family, which has yet to be directly connected to cal-

cium signaling. The activation of PLCg by RANK

requires the protein tyrosine kinase Syk, along with

immunoreceptor tyrosine-based activation motif

(ITAM)-bearing molecules, such as DNAX-activating

protein (DAP12) and the Fc receptor common gamma

chain (FcRg) (Koga et al. 2004). In the osteoclast

lineage, the immunoglobulin-like receptors (IgLR)

associated with DAP12 include triggering receptor

expressed in myeloid cells 2 (TREM-2) and signal-

regulatory protein b1 (SIRPb1) while those associatedwith FcRg include osteoclast-associated receptor

(OSCAR) and paired immunoglobulin-like receptor

A (PIR-A). As ITAM signals are essential for

osteoclastogenesis, but by themselves cannot induce

osteoclastogenesis, these signals are most accurately

described as costimulatory signals for RANK. The

binding of M-CSF to its receptor c-Fms also

generates a signaling complex comprised of phosphor-

ylated DAP12 and the nonreceptor tyrosine kinase Syk

(Nakashima and Takayanagi 2009). In addition,

mutation of TREM-2 or DAP12 in humans leads to

Nasu-Hakola disease, which is characterized by bone

cysts (Koga et al. 2004; Takayanagi 2007). Thus,

RANKL and M-CSF signals appear to converge on

the ITAM signaling pathway (Fig. 1).

It is also conceivable that RANK activates an as yet

unknown pathway that specifically synergizes with or

upregulates ITAM signaling. Tec family tyrosine

kinases such as Btk and Tec are activated by RANK

and are involved in the phosphorylation of PLCg,which leads to the release of calcium from endoplas-

mic reticulum (ER) through the generation of IP3

(Shinohara et al. 2008). An osteopetrotic phenotype

in Tec and Btk double-deficient mice revealed these

two kinases play an essential role in the regulation of

osteoclastogenesis. Tec and Btk had already been

reported to play a key role in proximal BCR signaling,

but this study established their crucial role in linking

the RANK and ITAM signaling pathways (Fig. 1).

R 1586 RANK and RANKL

This study also identified an osteoclastogenic signaling

complex, composed of Tec kinases and scaffold pro-

teins, which affords a new paradigm for understanding

the signal transduction mechanisms involved in osteo-

clast differentiation.

Bone Destruction with Arthritis as a RANKLDisease

In rheumatoid arthritis (RA), a long-standing question

is how abnormal T cell activation (characterized by the

infiltration of CD4+ T cells) mechanistically induces

bone damage. The identification of osteoclast-like

giant cells at the interface between synovium and

bone in rheumatoid joints dates back to the early

1980s (Takayanagi 2009). These pathological findings

led us to hypothesize that osteoclasts play an important

role in the bone resorption that occurs in arthritis and

that the osteoclasts are formed in the synovium

(Takayanagi 2009). Can osteoclasts be generated

from synovial cells alone? This question was answered

in the affirmative by generating osteoclasts in synovial

cell culture without adding any other cells, thus dem-

onstrating that rheumatoid synovial cells contain

both osteoclast precursor and osteoclastogenesis-

supporting cells (Takayanagi 2009). Further studies

indicated that synovial fibroblasts express membrane-

bound factor(s) that stimulate osteoclastogenesis and

induce the differentiation of synovial macrophages

into osteoclasts, but it was not until RANKL was

cloned that the membrane-bound factor in synovial

cells was brought to light (Takayanagi et al. 2000a).

Importantly, inflammatory cytokines such as IL-1,

IL-6, and TNF-a, which are abundant in both the

synovial fluid and synovium of RA patients, have

a potent capacity to induce RANKL on synovial

fibroblasts/osteoblasts and to accelerate RANKL

signaling, thus directly contributing to the bone

destruction process. Several groups have demonstrated

the high expression of RANKL in the synovium of

RA patients (Takayanagi 2009). RANKL was shown

to be expressed by synovial cells and T cells, both of

which are found in the inflamed synovium

(Takayanagi 2007, 2009). As RANKL is expressed in

activated T cells, T cells may have the capacity to

induce osteoclast differentiation by directly acting on

osteoclast precursor cells under pathological condi-

tions (Kong et al. 1999). However, ▶ interferon-g

(▶ IFN-g), which is produced by T cells, potently

suppresses RANKL signaling through a rapid degra-

dation of TRAF6 (Takayanagi et al. 2000b). To fully

understand the effects of T cells on osteoclastogenesis,

it is absolutely necessary to elucidate the specific

effects of the various cytokines which T cells produce.

It has been shown that IL-17-producing Th17 cells are

the exclusive osteoclastogenic T cell subset among the

known Th subsets (Takayanagi 2007, 2009). Since

even Th17 cells stimulate osteoclastogenesis mainly

through RANKL induction on synovilal fibroblasts, it

is as yet still unclear how T cell RANKL contributes to

bone destruction in the face of synovial fibroblasts

expressing RANKL to a higher extent.

Nevertheless, a series of reports has established that

the bone damage associated with inflammation is the

fundamental pathological condition caused by an

abnormal expression of RANKL. Additionally, osteo-

clast-deficient mice and osteopetorosis patients are

protected from bone erosion in arthritis (Kadono

et al. 2009; Takayanagi 2009). In the absence of oste-

oclasts, bone destruction did not occur, despite

a similar level of inflammation, indicating that

RANKL and osteoclasts are indispensable for the

bone loss associated with inflammation. Blocking

RANKL by OPG treatment significantly prevented

bone destruction in adjuvant arthritis (Kong

et al. 1999). Consistent with this, anti-RANKL and

anti-osteoclast therapies have been shown in clinical

trials as well as in the treatment of an animal model

of arthritis to be beneficial for the inhibition of

bone loss without affecting the immune system

(Takayanagi 2007).

RANK and RANKL in Mammary GlandDevelopment and Tumorigenesis

During pregnancy, increased ductal side branching

and the development of lobuloalveolar structures

are the result of an expansion and proliferation of

ductal and alveolar epithelium (Hennighausen and

Robinson 2005). Previously, genetic findings demon-

strated that mice with a disruption of Rank or Rankl fail

to develop mammary glands during pregnancy,

resulting in the death of newborns (Fata et al. 2000).

These mice exhibit normal mammary development

and normal ductal elongation and side-branching of

the mammary epithelial tree into the mammary fat

RANK and RANKL 1587 R

R

pad during puberty. However, their mammary epithe-

lium fails to proliferate and form lobuloalveolar struc-

tures during pregnancy (Fata et al. 2000). The

mammary gland defect in female RANKL-deficient

mice can be reversed by recombinant RANKL treat-

ment. These data clearly indicate that RANKL is an

essential regulator of alveolar epithelial cell prolifera-

tion. Although RANK is constitutively expressed on

mammary epithelial cells, the expression of RANKL is

absent in virgin glands, but gradually increases during

pregnancy. RANKL expression in mammary epithelial

cells is induced by pregnancy hormones such as pro-

lactin, progesterone, and PTHrP (Fata et al. 2000).

A previous report showed that kinase-dead IKKamutant mice display a severe lactation defect due to

the impaired proliferation of mammary epithelial cells

(Cao et al. 2001). The phenotype can be rescued by

mammary-specific overexpression of cyclin D1.

These data suggest that IKKa activity in response to

RANKL is required for NF-kB activation and cyclin

D1 induction in mammary epithelial cells during

pregnancy. However, it is reported that cyclin D1 is

normally expressed in mammary epithelial cells of

RANK-deficient mice, but in these animals there is

a defect in nuclear translocation of the basic helix-

loop-helix transcriptional regulator, inhibitor of DNA

binding 2 (Id2) (Kim et al. 2006). Genetic deletion of

Id2 results in a similar phenotype having a lactation

defect. Id2 regulates the proliferation of mammary

epithelial cells through a suppression of the cell cycle

inhibitor p21 in response to RANKL. Thus,

RANK/RANKL plays an essential role in mammary

gland development, but further study is required to

completely elucidate the signaling pathways and

transcriptional regulators.

The mammary gland in the period from puberty to

menopause develops through tightly choreographed

stages of cell proliferation (Hennighausen and

Robinson 2005). Steroid hormones such as estrogen

and progesterone have a prominent role in both the

healthy and diseased states of breast tissue. Reproduc-

tive history is the strongest risk factor for breast cancer,

and increased risk of breast cancer is correlated with

a greater number of ovarian hormone-dependent

reproductive cycles (Beral et al. 2005). There is also

an increased risk of breast cancer associated with preg-

nancy in the short term. Although a proliferative role

for the steroid hormones in this gland is well accepted,

it is still unclear how the mammary gland translates

hormonal signals into cell proliferation. Recent studies

have implied that RANK/RANKL functions in mam-

mary stem cell (MaSC) biology. MaSC is defined as

a cell that can both self-renew and propagate the full

spectrum of cell types that make up the mammary

gland (Shackleton et al. 2006). The MaSC activity

that was increased in mice treated with steroid hor-

mones and pregnancy led to a dramatically increased

number of MaSC in mice (Asselin-Labat et al. 2010;

Joshi et al. 2010). In contrast, ovariectomy or aroma-

tase inhibitor treatment markedly reduced the MaSC

number and outgrowth potential in vivo (Asselin-

Labat et al. 2010). In aged mice, MaSCs also display

stasis upon cessation of the reproductive cycle (Joshi

et al. 2010). MaSCs carry no known receptors for

estrogen or progesterone, but these stem cells are

highly responsive to steroid hormone signaling. Stud-

ies have shown that neutralization of RANKL in preg-

nant mice reduces the capacity of the MaSC-enriched

basal cell population to form colonies. These data

suggest that RANKL, a known progesterone target,

may act as a crucial molecule that links progesterone-

responsive mammary cells to MaSCs.

Hormone replacement therapy (HRT) is associated

with an increased risk of breast cancer (Beral

et al. 2005). In particular, progesterones or their syn-

thetic derivatives (progestins) such as medroxypro-

gesterone acetate (MPA) markedly increase the risk

of an abnormal mammogram and breast cancer.

Recently, it was revealed that MPA treatment triggers

the induction of RANKL expression in progesterone

receptor (PR)-positive luminal mammary epithelial

cells, resulting in autocrine or/and paracrine stimula-

tion of RANK signaling in the mammary epithelium

(Schramek et al. 2010). Importantly, specific deletion

of RANK in mammary epithelium cells prevents both

the onset and progression of MPA-driven mammary

cancer and impairs self-renewal of breast cancer stem

cells. Tumorigenesis in an MPA-driven tumor model

as well as in a spontaneous tumor model was also

inhibited by a neutralizing antibody to RANKL

(Gonzalez-Suarez et al. 2010). In contrast, mammary-

specific overexpression of RANK results in the accel-

eration of preneoplasias of the mammary glands and an

increase in mammary tumor formation after either

multiparity or treatment with a carcinogen and proges-

tin (Gonzalez-Suarez et al. 2010). These findings show

that the RANK/RANKL system is crucial for

tumorigenesis.

R 1588 RANK and RANKL

Bone is the most common site for the distal spread

of breast and prostate cancer (Mundy 2002). Bone

metastases result in serious morbidity, including

skeletal-related events such as pain, fractures, and

hypercalcemia, increasing the mortality risk. There-

fore, the clinical priority is to prevent metastases and

bone loss owing to excessive osteoclastic bone

resorption. Indeed, many clinical trials have evalu-

ated the potential activity of anti-osteoclastic agents

in cancer having bone metastases. The representative

anti-osteoclastic agents include bisphosphonates and

a RANKL neutralizing antibody (Denosumab)

(Fornier 2010). A recent clinical study showed that

Denosumab is superior to bisphosphonates such as

Zoledronic acid for the delay or prevention of

skeletal-related events in patients with advanced

breast cancer with bone metastases (Stopeck et al.

2010). Interestingly, RANK is highly expressed in

several human breast cancer cell lines and primary

human breast tumors (Jones et al. 2006). Functionally,

it has been shown that RANKL can stimulate the

directed migration of mammary epithelial cells as

well as prostate cancer and melanoma cells toward

a source of RANKL. Furthermore, in an in vivo

metastasis model, OPG reduced the tumor burden in

bones and ameliorated clinical paralysis, but did not

affect the frequency of the spread of metastases into

other tissues (Jones et al. 2006). A recent clinical

study reported that the level of RANK expression in

primary breast cancer positively correlates with the

development of bone metastases and may be

a predictive marker of bone metastasis risk (Santini

et al. 2011). Similar to breast cancer, prostate cancer

metastasizes to bone through RANK signaling.

Previous reports showed that IKKa activation

by RANKL inhibits the expression of Maspin, a

metastasis suppressor in prostate epithelial cells.

Maspin expression reduced metastatic activity,

whereas Maspin ablation restored this activity (Luo

et al. 2007). These findings suggest that

RANK-expressing tumor cells might sense RANKL

as a chemoattractant and migrate in a coordinated

fashion to a source of RANKL produced in the bone.

Summary

Bone-related diseases such as osteoporosis and RA

afflict a great number of patients. Women taking

progesterone derivatives for contraception or HRT

have been shown epidemiologically to have an

increased risk of breast cancer. These diseases are

presenting a tremendous burden to the health care

costs. Genetic approaches have established that the

RANKL/RANK system is the central regulator of

osteoclastogenesis, lymph node organogenesis, mam-

mary gland development, and thymic epithelial cell

development. In addition, recent data have revealed

an entirely novel and unexpected function for

RANKL/RANK in female thermoregulation and the

central fever response (Hanada et al. 2009). RANKL

has attracted the attention of scientists and pharmaceu-

tical companies, since it plays a pivotal role in the

pathogenesis of osteoporosis, RA, tumorigenesis, and

metastasis. Novel drugs specifically targeting RANK/

RANKL and their signaling pathways provide

a potential means to revolutionize the treatment of

various diseases associated with this pathway (Kearns

et al. 2008).

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RANTES

▶CCL5

R 1590 Rap GEF Family

Rap GEF Family

Hoa B. Nguyen and Lawrence A. Quilliam

Department of Biochemistry and Molecular Biology,

Indiana University School of Medicine, Indianapolis,

IN, USA

Synonyms

Guanine nucleotide exchange factor

List of Discussed GEFs

RapGEF1: C3G

RapGEF2: PDZ-GEF1, RA-GEF, CnRasGEF,

KIAA0313, nRapGEP

RapGEF3: Epac, cAMP-GEF I

RapGEF4: Epac2, cAMP-GEF II

RapGEF5: MR-GEF, Repac, GFR, KIAA0277

RapGEF6: PDZ-GEF2, RA-GEF2

RasGRP2: CalDAG-GEF I

RasGRP3: GRP3, CalDAG-GEF III, KIAA0846

NSP2: BCAR3 (human), AND34 (mouse)

NSP3: Chat, SHEP

SmgGDS: RAP1GDS1

Historical Background

GTP binding proteins are extensively used in nature to

regulate biological processes. Most of these proteins

act as molecular switches that transition between inac-

tive GDP-bound and active GTP-bound conforma-

tions. The largest family of GDP/GTP switches is the

Ras superfamily of small-molecular-weight GTP bind-

ing proteins that constitute approximately 150 mem-

bers in mammalian cells. This superfamily can be

subdivided into Ras, Rho, Rab, ▶Ran, and ARF fam-

ilies that regulate a myriad of cellular functions (Takai

et al. 2001). True Ras proteins play major roles in

coupling cell surface receptors to intracellular signal-

ing pathways that control proliferation, differentiation,

and survival. Due to their mutational activation in over

20% of human cancers, much research has focused on

H-, K-, and N-Ras. In the shadow of these oncoproteins

there are additionally over 30 Ras-related proteins that

include the Rap proteins, Rap1A, 1B, Rap2A, 2B, and

2C. These proteins were initially discovered due to

sequence similarity to Ras, their ability to revert the

actions of Ras, and their abundance in leukocytes

(Bokoch 1993). More recently Rap1 has been shown

to play multiple roles that include inside-out signaling

to control the affinity of integrins for extracellular

matrix and consequently cell adhesion and migration.

Rap1 also localizes to adherens junctions where it

influences cell–cell adhesion and plays a number of

additional roles in cell signaling, often overlapping

with those of Ras (Raaijmakers and Bos 2009).

So how do Ras/Rap proteins act as molecular

switches and how does the extracellular environment

control their activity? Ras proteins normally exist in

an inactive or resting GDP-bound state. To acquire an

active GTP-bound confirmation they must exchange

nucleotide. An intrinsic GTPase activity then hydro-

lyzes the terminal phosphate off the bound GTP

enabling return to the inactive confirmation. The

intrinsic rates of nucleotide exchange and GTP hydro-

lysis are in the order of minutes to an hour, not practi-

cal for rapid response to the extracellular environment.

This means that additional factors must exist to both

accelerate and tightly regulate these processes. Gua-

nine nucleotide exchange factors (GEFs) promote the

release of GDP whereas GTPase activating proteins

(GAPs) have evolved to assist in the rapid hydrolysis

of GTP (Quilliam et al. 2002). Whereas inhibition of

GAPs and activation of GEFs both tip the balance in

favor of Ras-GTP accumulation, nature most fre-

quently uses GEFs to activate Ras proteins and enable

biological responses. This article will focus on the

nature and regulation of Rap family GEFs.

The baker’s yeast CDC25 protein was the first Ras

family GEF to be identified. Consequently the catalytic

domain is referred to as a CDC25 homology domain.

This region is conserved in approximately 35mammalian

GEFs,most of which also contain an REM (Ras exchange

motif) thatwas required for aminimalCDC25 fragment to

function in vitro (Quilliam et al. 2002; Raaijmakers

and Bos 2009), see Fig. 1. Structural studies on the Ras

GEF Sos1 and Rap GEF Epac demonstrated that

the REM, which is located various distances N-terminal

to the CDC25 homology region, contributes to the

stabilization of the core catalytic domain.

Although there are approximately equal numbers of

Ras subfamily members and GEFs, there is not

a simple monogamous pairing of GEFs to Ras proteins.

RapGEF1/C3G

RapGEF3/Epac

RapGEF4/Epac2

RapGEF5/MR-GEF

RapGEF2/PDZ-GEF1

RapGEF6/PDZ-GEF2

Phospholipase Cε

RasGRP2 and 3

SHEP1 or BCAR3

SmgGDS ARM ARM ARM ARM ARM ARM ARM ARM ARM

CDC25PxxP

PxxP

PxxP

PxxP

SH2

REM

REM

REM

REM

REM

REM

REM

REM

CDC25

CDC25

CDC25

CDC25

CDC25

CDC25

CDC25

CDC25

EF

EFPH

EF C1

C2 RA RAY

Y

X

cAMP cAMP

cAMP

cAMP

cAMP

PDZ

PDZ

RA

RA

RA

RA

RA

PY

PY PBM

PBM

DEP

DEP

DEP

PLC

cAMP

Rap GEF Family, Fig. 1 Schematic representation of Rap GEFfunctional domains. The CDC25 homology domain (core

catalytic unit) and associated Ras exchange motif (REM) are

shown in red; protein kinase C-conserved region 1 (C1) and C2,

Ca2+ binding motif are shades of light green; Armadillo (ARM)

repeats are light blue; cAMP binding domains are pink; cAMP

represents similar sequences not thought to bind cyclic nucleo-

tide; DEP (Disheveled, Egl-10 and Pleckstrin) domains are

turquoise; Ca2+-binding EF hands are gray, PDZ (PSD-95, Dgl

and ZO-1) domains are shown in green with PDZ-binding motifs

(PDM), proline-rich SH3 bindingmotifs (PxxP), protein tyrosine

kinase phosphorylation site (Y), and WW domain-binding PY

site is shown in light blue. Pleckstrin homology (PH) domain is

brown, Ras associated (RA) domains are shown in yellow,

Src-homology (SH) 2 domain is orange, and the X and

Y domains that form the catalytic domain of phospholipase

C are dark blue

Rap GEF Family 1591 R

R

Interestingly more GEFs have evolved to regulate Rap

than any other group. Rap GEFs contain multiple reg-

ulatory domains suggesting that Rap proteins are under

the control of diverse extracellular stimuli. Regulation

includes protein–protein or protein–lipid interactions,

binding of second messengers, and/or posttranslational

modifications. The need for so many GEFs may be to

activate Rap in different tissues or different locations

within a given cell, at key points in development, or in

response to different hormones/growth factors, etc.,

that use unique signaling mechanisms. Recent knock-

out studies in mice have supported the notion that

Rap1A and 1B play fundamental roles in mammalian

development and function, and likewise, creation of

mice lacking various Rap GEFs (discussed below)

support the notion that individual exchange factors

play equally critical functions.

RapGEF1/C3G

One of the earliest mammalian Ras family GEFs to be

isolated was the Crk SH3-binding guanine nucleotide

exchange factor (C3G) (3, 4). In addition to the CDC25

homology and REM domains, C3G contains central

proline-rich sequences that bind to the N-terminal

SH3 domain of the Crk adapter protein, a more

N-terminal proline-rich motif that associated with

▶ p130Cas, c-Abl, and Hck SH3s, and a central tyro-

sine residue (Y504) that is phosphorylated by Src

family tyrosine kinases (Fig. 1). C3G is perhaps the

most ubiquitously expressed Rap GEF. It is activated

by numerous cell surface molecules that include

integrins, T and B cell receptors, E-cadherin, and var-

ious growth factors and G-protein-coupled receptors.

C3G was initially found to activate Rap1 but has

since been reported to also promote nucleotide

exchange on several other small GTPases: Rap2,

R-Ras, and TC10. Interestingly, TC10 is a member

of the Rho family of GTPases that are typically regu-

lated by a distinctly different family of GEFs. TC10

plays a role in insulin-stimulated glucose uptake and

in 2010 a Korean study linked mutations within C3G

to type II diabetes (Hong et al. 2009). Two C3G

knockout mice were created and demonstrated

embryonic lethality, exemplifying its important

R 1592 Rap GEF Family

biological role. C3G is frequently localized through

its interaction with the Crk and CrkL SH2/SH3-

containing adapter proteins. For example, the Abl

tyrosine kinase can recruit a CrkL-C3G complex to

the immune synapse.

RapGEF3 and 4/Epac1 and 2

The second messenger cyclic adenosine

monophosphate (cAMP) exerts many effects on cell

biology. While these were classically known to be

mediated by the cAMP-dependent protein kinase/pro-

tein kinase A (PKA) and olfactory cyclic nucleotide–

gated ion channels, two cAMP-activated Rap GEFs

were discovered in 1998 that helped explain the

PKA-independent actions of cAMP (Quilliam et al.

2002; Raaijmakers and Bos 2009; Gloerich and Bos

2010). They are called Epacs (exchange proteins acti-

vated by cAMP) 1 and 2 or cAMP-GEFs. While the

former name is most popular (and used herein) their

official gene names are RapGEFs 3 and 4.

In addition to the REM/CDC25 exchange region

that acts on Rap1 and 2, Epac proteins have either

one (Epac1) or two (Epac2) cyclic nucleotide–binding

domains (CNBD), a Disheveled, Egl-10, Pleckstrin

(DEP) domain, and an RA domain (Fig. 1). Structural

studies on Epac2 indicate that the N-terminus folds

over the C-terminus and hinders Epacs binding to its

substrate Rap. This autoinhibition is relieved by the

binding of cAMP to the CNBDs (Gloerich and Bos

2010). Epacs’ CNBDs lack a glutamate residue found

in PKA and cAMP-gated ion channels that typically

interacts with the 2-OH group of the ribose of

cAMP. Consequently, Epacs can bind bulky cAMP

analogs such as 8-(4-chloro-phenylthio)-2’-O-methyladenosine-cAMP (8CPT) where the 2-OH

group has been replaced with a O-Me to selectively

activate Epac proteins versus other cAMP effectors.

These compounds have proven to be very useful tools

to implicate Epac in biological events and may have

clinical potential (Gloerich and Bos 2010).

Epacs, like most other GEFs and GAPs, regulate

Rap proteins in a spatial and temporal manner. For

instance, localization of Epac1 and 2 is regulated by

their distinct RA domains. The RA domain of Epac2

specifically binds K- and N-Ras (with weaker affinity

for H-Ras), enabling Ras-GTP to translocate Epac2,

but not Epac1, from the cytosol to the plasma

membrane. Consequently, a pool of plasma mem-

brane-bound Rap1 can become activated upon concur-

rent cAMP and Ras signaling (Li et al. 2006).

Meanwhile, the RA domain of Epac1 interacts with

Ran, a small G-protein best known for its role in

regulating nuclear transport. Ran and its binding

partner RanBP2 anchor Epac1 to the nuclear pore,

allowing localized Rap1 activation at the nuclear enve-

lope upon cAMP elevation (Liu et al. 2010). In a

different scenario, Epac1 can be targeted to the plasma

membrane via its DEP domain. This is necessary for

Rap to regulate integrin-mediated adhesion at the

membrane. However, in Rat1a fibroblasts, peripheral

Rap1 activation by Epac1 is counteracted by high

RapGAP activity, resulting in predominantly

perinuclear Rap-GTP (Gloerich and Bos 2010). Fur-

thermore, in both interphase and mitotic cells, Epac1 is

targeted to microtubules by tubulin or the microtubule-

associated protein 1 and may play a role in microtubule

polymerization. Other reports show Epacs localizing to

centrosomes, mitochondria, macrophage phagosomes,

the apical epithelial membrane, and regulating the

DNA damage–responsive kinase, DNA-PK, in the

nucleus. Temporal expression also contributes to

Epac action: As monocytes differentiate into macro-

phages, their Epac protein levels increase threefold and

play a role in chemokine secretion.

Epacs regulate a variety of physiological processes

that include secretion of insulin from pancreatic beta

cells, permeabilization of vascular endothelium,

transmigration of leukocytes, and regulation of car-

diac calcium channels. Consequently, Epac activity

has been associated with diabetes, vascular inflamma-

tion, and heart disease. Interestingly, PKA is also

involved in these processes demonstrating the close

partnership of Epac and PKA in mediating cAMP

action.

Using Epac2 knockout mice, Seino’s lab estab-

lished that cAMP potentiates glucose-induced exocy-

tosis via Epac2 rather than PKA. They later

demonstrated that Epac2 is the direct target of the

antidiabetic sulfonylurea drugs that promote insulin

secretion (Zhang et al. 2009). A number of studies

have implicated Epacs in inflammation both through

regulation of leukocytes and vascular permeability

(Borland et al. 2009). In cardiac myocytes, Epacs and

another Rap GEF, phospholipase Ce both play critical

synergistic roles in calcium-induced calcium release

downstream of b-adrenergic receptors.

Rap GEF Family 1593 R

R

RapGEF2 and 6/PDZ-GEFI and II

Similar in structure to Epacs are PDZ-GEF-I and II,

also called RA-GEF-1 and 2, or official names

RapGEFs 2 and 6. PDZ-GEF-I has also been described

as CNrasGEF or nRapGEP. Like Epacs, PDZ-GEFs

have a REM-CDC25 GEF module, an RA domain, and

a region sharing homology with CNBDs (Quilliam

et al. 2002; Raaijmakers and Bos 2009) (Fig. 1). How-

ever, most reports indicate that cAMP does not bind to

this latter region with high affinity. PDZ-GEFs have

a PSD-95/DlgA/ZO-1 (PDZ) domain that can bind

the b1 adrenergic receptor, potentially linking G-pro-

tein-coupled receptors to Ras activation. At its C-ter-

minus, PDZ-GEFs have a proline-rich region and

a PDZ-binding motif, which interact with the PDZ

domains of cell junctional proteins, MAGI-1 and �2

(Sakurai et al. 2006). This links PDZ-GEF with

▶ b-catenin and contact-induced activation of Rap1

(Sakurai et al. 2006). In addition, two PY motifs at

its C-terminus are responsible for binding to the

WW domain of the ubiquitin protein ligase Nedd4.

This interaction regulates PDZ-GEF protein turnover

rate via proteasomal degradation.

PDZ-GEF1 and 2 functions have been studied

through gene knockouts in both Drosophila and

mice. In Drosophila, PDZ-GEF (Gef26) regulates

DE-cadherin to control stem cell adhesion to its

niche. Functional mutation of this GEF leads to loss

of cell polarity, impaired adherens junctions, and thus

reduction in stem cell number (Wang et al. 2006). Two

labs found that PDZ-GEF knockout is embryonic

lethal in mice. Kataoka’s lab found that vascular devel-

opment is impaired in PDZ-GEF1 knockout mice at

around E7.5 with embryonic lethality occurring by

E9.5, while Hou and colleagues found PDZ-GEF2�/�

mouse embryos surviving to ~E11.5. Mice died of

vasculature defects in the yolk sac and the allantois.

Further analysis revealed dysregulation in B-Raf/ERK

signaling, and a reduction in Scl/Gata transcription

factor expression (Satyanarayana et al. 2010). These

molecular signaling events underline the reduction of

definitive hematopoietic CD41 cells. Hematopoietic

progenitors isolated from these mouse embryos also

lacked the potential to form erythroid or granulocyte/

macrophage colonies, indicating that PDZ-GEF-2

plays a role in hematopoiesis development and progen-

itor functions. In addition, deletion of PDZ-GEF-2 late

in embryogenesis resulted in defective fetal liver

erythropoiesis. However, such deletion in the adult

bone marrow, or specific deletion in B-cells, T-cells,

hematopoietic stem cells, or endothelial cells had no

impact on hematopoiesis (Satyanarayana et al. 2010).

This reiterates the importance of temporal control and

the role of various GEFs at different stages of

development.

Conditional knockout of PDZ-GEF showed other

crucial functions of this GEF in neural migration and in

splenocyte responses. Mice with dorsal telencephalon-

specific PDZ-GEF-1 knockout develop heterotopic

cortical mass and commissural fiber defect. Mean-

while, PDZ-GEF-2 and Rap1 mediate TNFa-inducedM-Ras activation in order to activate the integrin,

lymphocyte function-associated antigen 1 (LFA-1),

and subsequently, cell aggregation in response to

inflammation (Yoshikawa et al. 2007).

Others also reported PDZ-GEF2 interacts through

its PDZ domain with junctional adhesion molecule-A

(JAM-A), which also interacts with Afadin/AF6 in

human colonic epithelial cells. JAM-A and AF6 both

act upstream of a signaling pathway that specifically

activates Rap1A but not Rap1B in order to regulate b1integrin and mediate cell migration (Severson et al.

2009). This is a rare report of differential signaling to

Rap1A versus Rap1B.

RapGEF5/MR-GEF

MR-GEF was characterized by numerous groups, as

a Rap-specific GEF (acting on Rap1 and 2) (Quilliam

et al. 2002; Raaijmakers and Bos 2009). Due to sharing

highest homology to Epacs it was referred to as Repac

or alternatively as MR-GEF (M-Ras regulated) due to

the presence of an RA domain that selectively bound to

M-Ras-GTP. M-Ras over-expression inhibited Rap1

activation but based on the experiences of us and

others it is likely that M-Ras is specifically targeting

the GEF to activate a plasma membrane pool of Rap1

at the expense of the activity of bulk GTPase (Li et al.

2006). An apparent splice variant that swaps the first

70 amino acid residues for an alternative 208 residue

sequence (NP_036426) places a DEP domain at the

extreme N-terminus that may play a role in membrane

localization. A DEP domain is also found in Epacs

a similar distance from the REM domain (Fig. 1).

However, unlike Epacs, MR-GEF does not contain an

intervening cAMP binding motif.

R 1594 Rap GEF Family

MR-GEF expression was induced by exposure to

anthrax and expression is also turned on in developing

rodent GABAergic neurons. Interestingly, MR-GEF

expression is also altered in individuals with bipolar

disorder. Correlation of the percentage of MR-GEF

expressing neurons and 2D neuronal density between

cortical layers II and IV in bipolar disorder support

a growing body of evidence for its contribution to

defects in cortical organization and communication in

this disease (Bithell et al. 2010).

RasGRPs

In addition to RapGEFs 1–6, several other Rap1

exchange factors exist, if not implicitly acknowledged

by their official gene names. The four RasGRP

(Ras guanyl releasing proteins) or CalDAG-GEF

gene products contain an N-terminal REM and

CDC25 homology regions that are followed by two

tandem Ca2+-binding EF hands similar to those found

in calmodulin (Quilliam et al. 2002; Raaijmakers and

Bos 2009). Farther C-terminal is a C1 domain similar

to the diacylglycerol (DAG)/phorbol ester binding

domain found in classical and atypical protein kinases

C – hence the CalDAG moniker. Both RasGRP2/

CalDAG-GEF1 and RasGRP3 act as GEFs for Rap

proteins. GRP2 is specific for Rap1/2 and activates it

in a Ca2+-dependent manner (although an N-terminally

myristoylated splice variant was reported to also act on

N- and K-Ras but to be inhibited by Ca2+ elevation). In

contrast, RasGRP3 has the broadest substrate specific-

ity of all Ras GEFs, acting on true Ras, R-Ras, and Rap

subfamilies (Quilliam et al. 2002; Raaijmakers and

Bos 2009). RasGRPs 1 and 4 are Ras-specific.

RasGRPs are highly abundant in brain: RasGRP2

is most highly expressed in the basal ganglia whereas

RasGRP3 is found primarily in glial cells of the cere-

bral and cerebellar white matter. Both GRPs are also

highly expressed in cells of hematopoietic origin.

GRP2/Cal-DAG1 is particularly abundant in platelets

(that are also replete with Rap1b) where they play

a major role in coupling chemoattractant receptors

to aIIbb3 integrin activation during “inside-out” sig-

naling as well as in thromboxane A2 release. Leuko-

cyte adhesion deficiency (LAD) syndrome III,

characterized by an inability of leukocytes to adhere

and migrate during inflammatory and host defense

reactions was initially attributed to mutations in

RasGrp2 but recent studies suggest that kindlin-3

rather than GEF mutation is the true culprit behind

this rare disease.

Phospholipase C«

Like other phospholipases C (PLCs) PLCe contains

X and Y regions that make up the phospholipase

catalytic domain. This enzyme cleaves phosphatidyl

inositol 4,5 bisphosphate (PIP2) into the second mes-

sengers inositol 3,4,5 trisphosphate (IP3) and DAG.

PLCe similarly possesses, a PH domain, C2 domain,

and EF hands that bind to phospholipids and Ca2+

(see Fig. 1). However, unlike other PLC isozymes,

PLCe is also a Rap GEF. PLCe has both an N-terminal

REM/CDC25 Rap GEF module and tandem

RA domains located at its C-terminus (Fig. 1). The

GEF function of PLCe helps maintain persistent

Rap1-GTP levels following G-protein-coupled recep-

tor stimulation (Suh et al. 2008). The C-terminal RA

domain (RA2) interacts with activated Ras and Rap1

enabling PLCe recruitment to either the plasma mem-

brane or the perinuclear area, respectively, following

growth factor stimulation. Meanwhile, the other RA

domain confers protein stability and possibly also

autoinhibition.

Significant crosstalk between PLCe and other

RapGEFs has been reported and further scenarios can

readily be imagined. For example, Ca2+ and DAG

generated by PLCe activity might promote the activa-

tion of GRP2 or 3. Additionally, upon adrenaline or

prostaglandin E2 stimulation, G-protein-coupled

receptors elevate cAMP levels and activate Epac.

This results in Rap2B activation that in turn associates

with RA2 of PLCe and induces PIP2 hydrolysis. PLCecan also be activated by other ligands such as

lysophosphatidic acid or sphingosine 1-phosphate

that couple to Ga12 and Ga13. In addition, Ga12 and

Ga13 can activate various RhoGEFs. GTP-loaded

RhoA can bind directly to the phospholipase Y domain

and stimulate PLCe activity.PLCe knock-down or mutation is embryonic lethal

in Caenorhabditis elegans due to its role in epidermal

morphogenesis while PLCe�/� mice exhibit multiple

cardiac defects. These include ventricular dilation,

aortic and pulmonary valve defects, and stenosis due

to the thickening of valve leaflets. Additionally, car-

diac myocytes from PLCe null mice have a decrease

Rap GEF Family 1595 R

in contractile response to acute b-adrenergic stimula-

tion. Human kidney development also requires PLCeand truncating mutations in PLCe were found in

nearly 30% of children having the nephrotic syn-

drome, diffuse mesangial sclerosis (Suh et al. 2008).

However, loss of PLCe can be advantageous to mice,

resulting in reduced susceptibility to carcinogen

induced skin tumor formation (Suh et al. 2008). This

is likely the result of PLCe mediating both direct

agonist-dependent proliferation and an indirect

inflammatory response. PLCe both mediates PDGF-,

EGF-, and Rho-dependent cell growth and inhibits

EGF receptor down-regulation via PIP2-derived sec-

ond messengers. Furthermore, PLCe can transduce

mitogenic signals through its Rap1 GEF activity. On

the other hand, PLCe-null mice have a reduction in

phorbol ester-induce edema, granulocyte infiltration,

and expression of the proinflammatory cytokine,

interleukin-1a.

R

Additional Potential Rap GEFs

Dock4 is a member of the Dock180/CZH family of

Rho GEFs and, in partnership with ELMO, is known to

regulate the activity of Rac1. However Dock4 was

reported to effectively activate Rap1 in a mouse oste-

osarcoma cell line (Yajnik et al. 2003). A dominant

inhibitory mutant of Rap1 blocked the biological con-

sequences of Dock4 expression and a constitutively

active Rap1-63E mutant mimicked Dock4-induced

adherens junction formation; however, there is no

direct demonstration of RapGEF activity in vitro.

Since Rap1 is intimately associated with Rac and

adherens junction regulation the effect of Dock4

reported here may have been indirect.

NSP1-3 (novel SH2-containing proteins) have

a C-terminal CDC25 homology domain, an N-terminal

SH2 domain, and a central proline-rich region that may

interact with SH3 domains (Quilliam et al. 2002)

(Fig. 1). They have various alternate names such as

BCAR3, AND-34, SHEP1, and Chat and have been

reported to associate with Rap1.While exchange activ-

ity was attributed to NSP2/BCAR3/AND34 in one

study, this was likely indirect due to its association

with Crk/Src to influence Rap1 and Rac GEF activity.

NSPs were identified in breast cancer (BCAR3 ¼breast cancer antiestrogen resistance gene 3) or

associated with receptor tyrosine kinases (EGFR for

NSP1, EphB2 for NSP3/SHEP1/Chat). All 3 NSPs

associate with the scaffold protein p130 Cas, implicat-

ing them in adhesion. Surprisingly the CDC25 homol-

ogy domain is responsible for this interaction. Thus

although NSPs likely play a pivotal role in coupling

adhesion receptors and tyrosine kinases to actin cyto-

skeletal organization their CDC25 domains act as

adapter modules rather than as Rap GEFs.

SmgGDS (small-molecular-weight G-protein gua-

nine nucleotide dissociation stimulator) is relegated

to last place in this article but was the first mammalian

Rap (or Ras family) GEF to be discovered in 1990,

reviewed in Quilliam et al. (2002). It is comprised of

ARM/armadillo repeats (similar to, e.g., catenins or

importins) rather than having a CDC25 homology

domain (Fig. 1) and selectivity is based on the pres-

ence of positively charged residues in C-terminal tail

of Ras proteins: target substrates include Rap1, K-

Ras, Rac1, RhoA, and Ral that each have multiple

lysine residues in their C-terminal hypervariable

regions. SmgGDS (gene name RAP1GDS1) expres-

sion is critical during development and appears to

possess weak GEF activity and to promote the malig-

nant phenotype of certain cancer cells. However,

whether it acts as a true GEF in vivo has always

been in question. A recent study suggests that two

splice variants of smgGDS increase the activity of

polybasic-region-containing GTPases such as Rap1

by facilitating their posttranslational modification by

lipids and subsequent transit to the plasma membrane

(Berg et al. 2010).

Summary

Multiple Rap GEFs have been characterized at both the

molecular level and frequently also in mouse models

of development/disease. However there is much still to

be learned from animal models. The number of GEFs

and variety of regulatory domains suggest that Rap

plays key roles in cell signaling that need to be acti-

vated in unique spatiotemporal scenarios. While Rap1

plays many roles in cell biology and likely represents

a poor drug target, targeting Rap GEFs might result in

specific regulation of Rap1 in certain diseases. The

8CPT-cAMP molecule represents a specific activator

of Epacs that may impact signaling events in vascular

disease and diabetes, but to date no Rap GEF inhibitors

have been developed.

R 1596 Rapamycin and FKBP12 Target-1 Protein (RAFT1)

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Bithell A, Hsu T, Kandanearatchi A, Landau S, Everall IP,

TsuangMT, et al. Expression of the Rap1 guanine nucleotide

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Rapamycin and FKBP12 Target-1 Protein(RAFT1)

▶mTOR

RARa

▶Retinoic Acid Receptors (RARA, RARB, and

RARC)

RARb

▶Retinoic Acid Receptors (RARA, RARB, and

RARC)

RARg

▶Retinoic Acid Receptors (RARA, RARB, and

RARC)

RAS (H-, K-, N-RAS)

Michael S. Samuel

Centre for Cancer Biology, SA Pathology, Adelaide,

SA, Australia

Synonyms

c-H-ras; c-K-ras; c-N-ras; Ha-ras; Ki-ras; Ras onco-

genes; v-H-ras; v-K-ras; v-N-ras

RAS (H-, K-, N-RAS) 1597 R

Historical Background

Evidence that viral genes were translated in virus-

transformed cells was first reported in the early 1970s

(Green et al. 1971). Following on from these observa-

tions, it was discovered that certain 21 kDa proteins

encoded by viral genes and possessing guanine nucleo-

tide binding properties (Scolnick et al. 1979) were

essential for the maintenance of transformation in cells

infected with the Kirsten and Harvey sarcoma viruses

(Shih et al. 1979). Intriguingly, sequences containing

a very high degree of homology to genes encoding these

transforming proteins were found in normal rat, mouse,

and human genomes, suggesting a physiological role,

unrelated to disease, for these proteins. Mutant versions

of the oncogenes associated with the Kirsten sarcoma

virus (K-ras) and theHarvey sarcoma virus (H-ras) were

found in cancer cell lines of various tissue origins (Der

et al. 1982). The majority of these contained point

mutations that resulted in the replacement of the guano-

sine residue at position 12. A third Ras family member

termed “N-ras,” also containing activating point muta-

tions, was subsequently identified in leukemia and neu-

roblastoma cell lines (Hall et al. 1983). Indeed, it was

soon realized that the Ras family of proto-oncogenes are

among the most frequently mutated genes in human

cancers. The field of Ras research is now a large and

complex enterprise owing to the discovery of this pro-

tein family’s fundamental roles in cell and cancer biol-

ogy, development, and disease.

R

Structure and Function of Ras Proteins

The Ras proteins are 21 kDa small G proteins with

intrinsic GTP binding and GTPase properties (Sweet

et al. 1984). The three-dimensional structures of Ras in

the GTP- and GDP-bound forms determined by crys-

tallography were published in 1990 and revealed that

Ras consists of a six stranded b-sheet and five a-helicesforming a hydrophobic core and linked together by ten

loops. Five of these loops mediate the high-affinity

interactions between Ras and GTP and act by stabiliz-

ing the g-phosphate of the bound GTP (Brunger et al.

1990). The p-loop, which contains the guanosine-12

often mutated in cancers, directly binds the

g-phosphate of GTP. The structures of GTP-bound

and GDP-bound Ras differ in two regions called switch

I and switch II (Fig. 1), which are crucial for the

interaction of Ras with both its upstream regulators

and downstream effector partners that mediate its func-

tion and intracellular localization.

In the GTP-bound state, Ras proteins are in an

active conformation and can participate in high-

affinity interactions with Ras effector proteins. These

proteins are usually enzymes that transduce down-

stream signaling cascades. However, the intrinsic

GTPase activity of Ras proteins results in the hydroly-

sis of GTP to GDP, with which Ras proteins associate

only weakly. Following hydrolysis and loss of the

g-phosphate, the GDP molecule is released and the

Ras protein returns to an inactive state. This GTPase

cycle is facilitated by the Ras GTPase-activating pro-

teins (Ras GAPs) and the Ras guanine nucleotide-

exchange factors (Ras GEFs) (Fig. 2). The Ras GAP

proteins promote the GTPase activity of Ras and facil-

itate its transition from the active to the inactive state,

resulting in the switching off of Ras signaling. The Ras

GEF proteins, on the other hand, bind inactive Ras-

GDP and promote the exchange of GDP for GTP,

triggering the activation of Ras. Ras GEF proteins

contain many protein-protein interaction domains that

mediate their activation. One way in which Ras GEFs

are activated is in response to ligand binding to recep-

tors with which they are associated. Similarly, GAPs

Ras proteins are often large and complex and contain

a variety of signaling motifs that enable them to asso-

ciate with the many interacting partners that regulate

their activity. Mutant versions of Ras proteins often

found in cancers exhibit attenuated affinity for GTP,

but maintain an active conformation enabling them to

interact with and activate effector proteins and signal

constitutively downstream in the absence of upstream

activating signals (Karnoub and Weinberg 2008).

Regulation of Ras Activity

In addition to the action of Ras GEFs and GAPs, the

activity of Ras is regulated by a variety of other mech-

anisms. These include posttranslational modifications

such as the addition of fatty acid side chains and

proteolytic processing, which determine its localiza-

tion. Palmitoylation of Ras at the C terminus facilitates

its association with the cell membrane (Sefton et al.

1982) and this particular localization is essential for its

function. A C-terminal CAAX motif within Ras is the

target of a prenylation reaction by farnesyl transferase,

RAS (H-, K-, N-RAS), Fig. 1 Linear representation of thedomain structure of the Ras proteins. The a-helices are denoteda1–a6 and the six strands making up the b-sheet are denoted

b1–b6. The P-loop between b1 and a1 stabilizes the g-phosphate

of GTP and is a region often mutated in oncogenic versions of

the protein. The Switch 1 and Switch 2 regions are important for

protein-protein interactions and the CAAX region is a target of

posttranslational modifications

LigandGrowthfactor

GPCR RTK

RasGDP

RasGTP

SHP2

Ras effectors

Cel

l sur

viva

l

Cyt

oske

leta

lre

gula

tion

Cel

l pro

lifer

atio

n

Ves

cicl

etr

affic

king

Ca2+

sig

nalin

g

PI3K TIAM1 RAF RALGDS PLCε

GRB2

SOS1

GAPNF1

Integrins

ECM

RAS (H-, K-, N-RAS), Fig. 2 Signaling through Ras. This sche-matic illustration of the Ras signaling pathway highlights the

modes of Ras activation and the effector pathways downstream

of Ras that regulate cellular processes. Binding of growth factor

to receptor tyrosine kinases (RTK) activates proteins such as

GRB2 and SHP2, which activates SOS1, a Ras GEF, resulting in

the accumulation of GTP-bound active Ras. GPCR and Integrin

signaling can also indirectly activate Ras. NF1 and other GAPs

in turn bind active Ras and catalyze its conversion to the GDP-

bound inactive form, turning off Ras signaling. Downstream,

Ras-GTP signals through a variety of effector pathways to reg-

ulate many cellular processes

R 1598 RAS (H-, K-, N-RAS)

which is followed by cleavage of the AAX sequence,

leaving a C-terminal cytosine residue. This cytosine

residue is then carboxy-methylated as an essential step

for full Ras function. There are slight variations in the

specific modifications to K-Ras, H-Ras, and N-Ras

proteins, but the final outcome of these modifications

is the secure tethering of the Ras protein to the cell

membrane, which is essential for its association with

both upstream and downstream signaling partners

(Downward 2003).

RAS (H-, K-, N-RAS) 1599 R

Operating upstream, GEFs such as son of sevenless

(SOS) that interact with and activate Ras proteins are

themselves activated by various signals including

those arising from receptor tyrosine kinases (RTK)

such as the EGF Receptor. However, Ras may also be

activated more indirectly by signals from Integrins or

G protein-coupled receptors (GPCR) (Fig. 2). The

function of Ras as a major signaling node is attested

to by the multiple GEFs that activate it, as well as by

the even larger numbers of Ras GEF binding partners

that regulate their function.

R

Ras Signaling Pathways

MAP Kinase Signaling

Activated Ras interacts directly with the RAF1 protein,

a Ser-Thr kinase, and stimulates its kinase activity.

Most notable of the substrates of Raf are the mitogen-

activated protein kinase kinases (MAPK, also known

as MEKs) which phosphorylate the extracellular sig-

nal-regulated kinases (ERKs) to regulate

the transcription of target genes through the

E26-transcription factor proteins (ETS) (Fig. 2). The

Ras-MAP kinase effector pathway regulates cell pro-

liferation and is required for Ras-induced cell transfor-

mation (Khosravi-Far et al. 1995). Signaling through

the MAP kinase pathway is frequently enhanced in

cancers by the somatic acquisition of activating muta-

tions in the Ras proteins or their effector Raf proteins.

However, activating mutations in both Ras and Raf are

only rarely seen together, illustrating the importance of

abnormal signaling through the MAP kinase pathway

in cancer (Karnoub and Weinberg 2008; Downward

2003).

PI3 Kinase Signaling

Upon activation, Ras proteins are capable of

interacting with the catalytic subunit (p110) of the

class I phosphoinositide 3-kinases (▶PI3Ks), which

results in the production of phosphatidylinositol-3,4,5-

trisphosphate (PtdIns(3,4,5)P3). This second messen-

ger molecule binds a large number of proteins includ-

ing PDK1 and the Ras homology family protein Rac,

and regulates their activity. One of these pathways

downstream of PtdIns(3,4,5)P3 regulates cell survival

through the serine/threonine kinase AKT/Protein

Kinase B (AKT/PKB) (Fig. 2) (Downward 2003).

Like the MAP kinase pathway, the PI3 kinase

signaling pathway is also indispensible for Ras-

mediated cell transformation (Rodriguez-Viciana

et al. 1997).

Other Ras Effector Pathways

While Ras is capable of activating RalGDS, the GEF

for the Ras-like (RalA/B) small GTPases, conflicting

evidence exists for the involvement of the RalA/B

effector pathway in transformation. This pathway

was originally considered of minor importance to

transformation, but new evidence suggests that signal-

ing through RalA/B is sufficient for transformation of

some human cell types (Hamad et al. 2002). Rac1

(Khosravi-Far et al. 1995; Samuel et al. 2011), tumor

invasion and metastasis inducing protein (TIAM1,

a Rac GEF protein), the epsilon form of phosphoi-

nositide-specific phospholipase C (PLCe), and

RASSF have all been shown to be Ras effectors, but

much remains unknown of their functions downstream

of Ras.

Ras in Disease

Cancers are the disease manifestations most com-

monly associated with aberrant signaling through

Ras. Mutations in one or more of the Ras isoforms

are frequently observed in cancers, with codon 12/13

mutations making up the majority of these (Loriot et al.

2009). These forms of Ras are usually able to associate

with and activate effector proteins in the absence of

GTP binding and indeed mutant Ras has attenuated

affinity for GTP. Both the MAP kinase and PI3-kinase

effector pathways are required for Ras-mediated trans-

formation and increased signaling through both these

pathways been implicated in providing cancer cells

with a survival advantage.

Different isoforms of Ras are preferentially

expressed in different organs, leading to isoform-

specific disease manifestations resulting from aberra-

tions in Ras activity. For instance, most pancreatic

cancers exhibit K-Ras mutations, but H-Ras muta-

tions are almost never observed in this disease.

Furthermore, N-Ras mutations are common in skin

cancers, but K-Ras mutations are not. That this is

likely the result of differences in the expression levels

of Ras isoforms, which may nevertheless function

similarly in different cell types, is supported by

the observation that the H-Ras coding sequence

R 1600 RAS (H-, K-, N-RAS)

placed under the transcriptional control of the endog-

enous K-Ras promoter can rescue the embryonic

lethality resulting from K-Ras deficiency (Potenza

et al. 2005).

Ras in Development

Aberrant signaling downstream of Ras has been

identified as being the cause of several developmental

disorders. Collectively termed cardio-facio-

cutaneous diseases, neurofibromatosis type-1 and the

Noonan and Costello syndromes are characterized by

inherited lesions in effectors, GAPs or GEFs of Ras

(Loriot et al. 2009) or infrequently by acquired

somatic mutations in Ras genes. People suffering

from these syndromes generally exhibit abnormalities

in the bones of the face, insufficiency in cardiac func-

tion, stunted growth, and an increased cancer risk

(Schubbert et al. 2007). The fact that activating

germ-line K-Ras mutations are rarely observed in

genetic syndromes is strong evidence that unregulated

Ras activation is highly disruptive during develop-

ment and is consistent with the embryonic lethality

associated with inherited activating K-Ras mutations

in mice.

Therapeutic Inhibition of Ras Signaling

Antagonizing the activity of mutant Ras has always

been an attractive therapeutic modality for cancer.

Many of these therapies have focused on disrupting

the posttranslational modifications essential for the

localization of Ras to the cell membrane, but have

suffered from lack of selectivity. Agents such as

farnesyl transferase inhibitors (FTI) also interfere

with the processing of other proteins such as RhoB.

Approaches to therapeutic targeting of Ras expression

using antisense oligonucleotides are currently under

trial. These approaches have run into problems associ-

ated with delivery and the lack of efficient take-up into

cancer cells (Downward 2003). Small molecule inhib-

itors of the MAP kinase pathway protein Raf, such as

Sorafenib (Sebolt-Leopold et al. 1999; Iyer et al.

2010), have been more successful, but only target

a single Ras effector pathway. Sorafenib has been

approved for the treatment of renal cell and hepatocel-

lular carcinomas. Inhibitors of MEKs such as U0126

and PD98059 are effective in inhibiting activation

of the MAP kinase pathway and are also currently

under trial.

Summary

The Ras genes were first discovered in viruses that

possess cell transforming ability before it was realized

that very similar genes exist in untransformed cells. The

Ras proteins have important physiological functions in

transducing signals that regulate cell proliferation and

survival. They constitute signaling hubs that integrate

and link growth factor signals from upstream to the

appropriate effector pathways downstream. Activating

mutations in Ras genes or in components of Ras effector

pathways are commonly observed in cancer, leading to

increased cell proliferation and survival. Germ-line

mutations of Ras effector pathways underlie a group of

developmental disorders including theNoonan andCos-

tello syndromes, collectively termed cardio-facial-

cutaneous syndromes. Ras antagonizing therapies are

beginning to be used effectively to treat cancers.

References

Brunger AT, Milburn MV, Tong L, de Vos AM, Jancarik J,

Yamaizumi Z, et al. Crystal structure of an active form of

RAS protein, a complex of a GTP analog and the HRAS p21

catalytic domain. Proc Natl Acad Sci U S A. 1990;87:

4849–53.

Der CJ, Krontiris TG, Cooper GM. Transforming genes of

human bladder and lung carcinoma cell lines are homologous

to the ras genes of Harvey and Kirsten sarcoma viruses. Proc

Natl Acad Sci U S A. 1982;79:3637–40.

Downward J, Targeting RAS. Signalling pathways in cancer

therapy. Nat Rev Cancer. 2003;3:11–22.

Green M, Rokutanda H, Rokutanda M. Virus specific RNA in

cells transformed by RNA tumour viruses. Nat New Biol.

1971;230:229–32.

Hall A, Marshall CJ, Spurr NK, Weiss RA. Identification of

transforming gene in two human sarcoma cell lines as a new

member of the ras gene family located on chromosome 1.

Nature. 1983;303:396–400.

Hamad NM, Elconin JH, Karnoub AE, Bai W, Rich JN,

Abraham RT, et al. Distinct requirements for Ras oncogen-

esis in human versus mouse cells. Genes Dev. 2002;16:

2045–57.

Iyer R, Fetterly G, Lugade A, Thanavala Y. Sorafenib: a clinical

and pharmacologic review. Expert Opin Pharmacother.

2010;11:1943–55.

Karnoub AE, Weinberg RA. Ras oncogenes: split personalities.

Nat Rev Mol Cell Biol. 2008;9:517–31.

RASA1 1601 R

Khosravi-Far R, Solski PA, Clark GJ, Kinch MS, Der CJ. Acti-

vation of Rac1, RhoA, and mitogen-activated protein kinases

is required for Ras transformation. Mol Cell Biol.

1995;15:6443–53.

Loriot Y, Mordant P, Deutsch E, Olaussen KA, Soria JC. Are

RAS mutations predictive markers of resistance to standard

chemotherapy? Nat Rev Clin Oncol. 2009;6:528–34.

Potenza N, Vecchione C, Notte A, De Rienzo A, Rosica A,

Bauer L, et al. Replacement of K-Ras with H-Ras supports

normal embryonic development despite inducing cardiovas-

cular pathology in adult mice. EMBO Rep. 2005;6:432–7.

Rodriguez-Viciana P,Warne PH,Khwaja A,Marte BM, Pappin D,

Das P, et al. Role of phosphoinositide 3-OH kinase in cell

transformation and control of the actin cytoskeleton by Ras.

Cell. 1997;89:457–67.

Samuel MS, Lourenco FC, Olson MF. K-Ras mediated murine

epidermal tumorigenesis is dependent upon and associated

with elevated Rac1 activity. PLoS One. 2011;6:e17143.

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opmental disorders and cancer. Nat Rev Cancer.

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binding activity as an assay for src protein of rat-derivedmurine

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Wiland A, Gowan RC, et al. Blockade of the MAP kinase

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transforming proteins of Rous sarcoma virus, Harvey

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Shih TY, Weeks MO, Young HA, Scolnick EM. p21 of Kirsten

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R

Ras Associated with Diabetes

▶Ras-Related Associated with Diabetes

RAS D1/DEXRAS1 (AGS1)

▶Activators of G-Protein Signaling (AGS)

Ras Guanyl Nucleotide-ReleasingProtein 1

▶RasGRP1

Ras Guanyl-Releasing Protein

▶RasGRP1

Ras Guanyl-Releasing Protein 1

▶RasGRP1

Ras Homolog Gene Family, Member C

▶RhoC (RHOC)

Ras Oncogenes

▶RAS (H-, K-, N-RAS)

RASA1

Philip E. Lapinski and Philip D. King

Department of Microbiology and Immunology,

University of Michigan, Medical School, Ann Arbor,

MI, USA

Synonyms

p120 GAP; p120 RasGAP; RasGAP

Historical Background

The small, membrane-tethered G-protein Ras plays an

important role in many cellular processes, including

growth, differentiation, and survival (Wennerberg

et al. 2005). Ras acts as a molecular switch which is

bound to GDP in its inactive state, and GTP in its

active state. When Ras is active, it can directly associ-

ate with the serine/threonine kinase Raf, which can be

activated by phosphorylation upon recruitment to the

membrane. Raf can then activate the dual-specificity

protein kinase MEK1/2, which in turn activates the

R 1602 RASA1

Mitogen-Activated Protein Kinase (MAPK) ERK1/2.

Since Ras controls multiple cellular outcomes, its

activity is tightly regulated. Inactive, GDP-bound Ras

can be activated by interaction with Guanine-

nucleotide Exchange Factors (GEFs), which eject

GDP from the nucleotide binding site of Ras and

allow GTP to bind, which is present at a much higher

molar concentration than GDP in the cytoplasm.

Examples of Ras GEFs include Son of Sevenless

(SOS) and Ras Guanyl-Releasing Protein 1

(RasGRP1). Active, GTP-bound Ras is inactivated by

association with GTPase-Activating Proteins (GAPs),

which enhance the low intrinsic GTPase activity of Ras

by several orders of magnitude, resulting in hydrolysis

of GTP to GDP (Iwashita and Song 2008). Ras p21

Protein Activator 1 (RASA1) was the first GAP to be

characterized at the molecular level. The discovery of

RASA1 was initially based on the observation that

Ras-GTP levels in vivo were much lower than

expected based on the intrinsic GTPase activity of

Ras (Trahey and McCormick 1987). Subsequently,

the ubiquitously expressed RASA1 protein was puri-

fied and its cDNA cloned from human and bovine

tissue (Trahey et al. 1988; Vogel et al. 1988). At least

14 Ras GAPs have since been discovered in mammals,

including neurofibromin (NF1), Ca2+-promoted Ras

inactivator (CAPRI), and Synaptic Ras GTPase-

activating protein 1 (SYNGAP1) (Bernards 2003).

Structure

GAPs are modular proteins, with numerous distinct

domains in addition to the conserved, catalytic GAP

domain. The RASA1 molecule is composed of six

such modular domains. These include two Src-homol-

ogy-2 (SH2) domains and a Src-homology-3 (SH3)

domain (which recognize phospho-tyrosine residues

and proline-rich sequences, respectively), a pleckstrin

homology (PH), and PKC2 homology (C2) domain

(both implicated in membrane phospholipid binding,

the latter in a calcium-dependent manner), and a GAP

domain, which confers GTPase-enhancing activity

(Takai et al. 2001). The SH2-SH3-SH2 domains

of RASA1 are responsible for binding to cytoplasmic

proteins, which include p190 RhoGAP and Dok-1

(Iwashita and Song 2008). Dok-1 is an adapter protein

that plays a role downstream of tyrosine kinase

signaling, and p190 RhoGAP acts as a GAP for the

Rho family of G proteins. The number of protein-

binding and membrane-binding domains in RASA1

suggests that it is involved in a complex signaling

network.

The GAP domain of RASA1 contains three

conserved motifs that are shared with all GAP

proteins. These include an arginine-finger loop,

a phenylalanine-leucine-arginine region, and an

a7/variable loop, with the arginine residue in the argi-

nine-finger loop being critical for the transition state of

GTP hydrolysis (Iwashita and Song 2008).

RASA1 is known to be phosphorylated by a number

of protein tyrosine kinases in multiple cell types. How-

ever, the stoichiometry of phosphorylation is generally

low, and no effect of phosphorylation on GAP activity

or subcellular localization has been reported (Takai

et al. 2001).

Function

The only known enzymatic function of RASA1 is to

accelerate the hydrolysis of GTP to GDP by Ras.

While the isolated GAP domain of RASA1 is sufficient

to promote GTP hydrolysis of purified Ras in vitro, full

activity in vivo requires the SH2-SH3-SH2 domains,

suggesting that protein-protein interactions are critical

for RASA1 function (Marshall et al. 1989; Gideon

et al. 1992). Indeed, RASA1 interacts with active,

phosphorylated PDGF Receptor and EGF Receptor

via its SH2 domains, negatively regulating their activ-

ity by suppressing Ras signaling (Margolis et al. 1990;

Ekman et al. 1999). PDGFReceptor and EGFReceptor

are not closely related by sequence, suggesting that

RASA1 associates with a broad range of growth factor

receptors.

Despite being the prototypical RasGAP, RASA1 is

not simply a negative regulator of Ras. In addition to

controlling Ras activation, RASA1 controls certain

cellular functions in a GAP-domain-independent and

a Ras-independent manner. For example, RASA1 has

been implicated in the control of cell motility through

its interaction with p190 RhoGAP. In cell monolayer

wounding assays, RASA1-deficient embryonic fibro-

blasts were impaired in establishing cell polarity and

migration into the wound. These functions appear to

require the interaction of RASA1 with p190 RhoGAP,

and are independent of Ras regulation (Kulkarni et al.

2000). The Rho proteins, for which p190 RhoGAP

RASA1 1603 R

is a negative regulator, are known to control the for-

mation of focal adhesions and actin fibers necessary for

directed cell movement. This association between

RASA1 and p190 RhoGAP implies that RASA1 can

act as a positive mediator of signaling, in addition to its

negative-regulator role as a RasGAP.

Another GAP-domain-independent function of

RASA1 is the regulation of apoptotic cell death. In

fibroblasts subjected to mild apoptotic stress, RASA1

is cleaved by activated Caspase 3. The free N-terminal

fragment of RASA1 is able to directly activate AKT,

a major kinase in the apoptotic pathway, via its SH2

and SH3 domains (Yang et al. 2005). Under these

conditions, RASA1 is thought to provide anti-

apoptotic signals that permit survival of the stressed

cell. However, under more severe apoptotic stress, the

N-terminal fragment of RASA1 is further cleaved by

Caspase 3, resulting in two shorter N-terminal frag-

ments. The result of this cleavage is a reduction in

AKT activity, which leads to efficient apoptotic death

of the cell. Therefore, the second cleavage event of

RASA1 functions to abrogate the anti-apoptotic func-

tion of the longer N-terminal fragment.

R

RASA1-Deficiency

A null allele of mouse rasa1 has been generated, and

mice homozygous for the null allele die at embryonic

day 10.5 (E10.5) (Henkemeyer et al. 1995). These

embryos appear to develop normally until E9.25, at

which time they display a defect in posterior elonga-

tion. No abnormal cell proliferation is observed in

RASA1 deficient embryos, and in fact by E9.5 are

significantly smaller than littermate controls. This is

most likely due to a severe vascular developmental

defect, in which blood vessel endothelial cells fail to

organize into a vascular network in the yolk sac. The

blood vasculature in the embryo proper is also

affected, and ultimately develops local ruptures lead-

ing to leakage of blood into the body cavity. Eventually

the pericardial sac becomes distended, leading to

a labored heartbeat and reduced blood flow. RASA1-

deficient embryos also display extensive apoptotic cell

death in the brain, with large numbers of dead and

dying cells as early as E9.0 in the hindbrain, optic

stalk, and telencephalon.

A recently developed conditional RASA1 mouse

model has been used to define a role for RASA1 in

the development and survival of T cells (Lapinski et al.

2011). In this model, exon 18 of rasa1, which encodes

the catalytic arginine-finger loop of the GAP domain,

was flanked by LoxP sites (floxed). The floxed allele

permits normal RASA1 expression, and thus circum-

vents embryonic lethality. However, excision of the

floxed exon in mice by transgenic Cre recombinase

results in nonsense-mediated RNA decay, and

a complete loss of RASA1 expression. This system

permits the study of RASA1 deficiency in adult mice

with the use of transgenic tissue-specific or inducible

Cre recombinase. Specific deletion of RASA1 early in

thymocyte development by Cre expressed under the

control of the proximal LCK promoter resulted in

increased death of CD4+ CD8+ double positive thy-

mocytes. Surprisingly, RASA1-deficient thymocytes

showed increased positive selection on a Major Histo-

compatibility Complex (MHC) Class II background,

which was associated with increased Ras/MAPK

signaling. RASA1 was found to be dispensable during

T cell receptor stimulation by agonist peptide/MHC

complex in peripheral T cells, as measured by cytokine

secretion, proliferative capacity, and activation-

induced cell death. However, absence of RASA1 led

to substantially reduced numbers of naive T cells in the

peripheral lymphoid organs. This phenomenon was

due, at least in part, to a reduced sensitivity to the

pro-survival cytokine IL-7.

RASA1 in Disease

Mutations in Ras are closely linked to development of

human cancer, with up to 90% of certain tumors har-

boring an oncogenic Ras allele. Commonly, an onco-

genic Ras mutation renders it refractory to GAP

activity, which leaves Ras trapped in its active,

GTP-bound state (Scheffzek et al. 1997). In addition,

RASA1 nonsense mutations have been associated

with basal cell carcinomas in humans (Friedman

et al. 1993).

A recently described human clinical disorder

known as capillary malformation-arteriovenous mal-

formation (CM-AVM) has been shown to be caused

by mutations of the RASA1 gene (Boon et al. 2005;

Revencu et al. 2008). This condition is characterized

by multiple randomly distributed pink lesions that

result from the malformation of skin capillaries.

Approximately one third of patients develop fast-flow

R 1604 RASA1

vascular lesions, including Parkes Weber syndrome,

arteriovenous fistulas, and intracranial arteriovenous

malformations. Arteriovenous fistulas are abnormal

connections between arteries and veins, where the

two are directly connected without branching into

capillaries, and ParkesWeber Syndrome is character-

ized by cutaneous flush, and multiple underlying

arteriovenous fistulas. It is also associated with soft

tissue and skeletal hypertrophy, usually of an affected

limb. Thus far, 140 individuals with RASA1 muta-

tions have been identified, and all but 6 of these have

CM-AVM. Forty-two different mutations in the

RASA1 gene have been described, including inser-

tions and deletions resulting in frame-shifts, disrup-

tion of splice sites, and nonsense, missense, or

splice-site substitutions. The mutations are randomly

distributed throughout the RASA1 gene, and only one

germline RASA1 gene is affected in CM-AVM

patients (mutation of both alleles of RASA1 would

presumably result in embryonic lethality). CM-AVM

is hypothesized to arise from loss of function of

the intact RASA1 allele by somatic mutation, which

is consistent with the focal nature of the lesions.

At least two CM-AVM patients have developed

chylothorax or chylous ascites, rare conditions in

which lipid-laden lymph fluid, or chyle, leaks from

lymphatic vessels into the thoracic or abdominal

cavities, respectively. Thus, RASA1 has been found

to play a role in the formation and maintenance of

the blood and/or lymph vasculature in both mice

and humans.

Consistent with the blood vascular phenotype

of RASA1-null embryos and in RASA1-mutant

humans, RASA1 has been found to play a role in the

angiogenic switch (Anand et al. 2010). In this process,

new blood vessels are stimulated to grow from existing

ones in response to growth factor stimulation. RASA1

expression has been found to be suppressed by

microRNA (mIR)-132, a genome encoded, noncoding

RNA regulator of gene expression. In a human embry-

onic stem cell model of vasculogenesis, mIR-132 was

found to be highly expressed. mIR-132 was also

upregulated in the epithelium of human tumors and

hemangiomas, but not in normal epithelium. RASA1,

which was expressed in normal epithelium, but not

tumor epithelium, was found to be a major target of

mIR-132. Suppression of RASA1 expression by

mIR-132 led to increased Ras activation in endothelial

cells and the induction of neovascularization. This

result suggests that mIR-132 acts as an angiogenic

switch by downregulating RASA1 expression, and

consequently increasing Ras signaling in endothelial

cells. Anti-mIR-132, a specific antagonist of mIR-132,

was found to restore RASA1 expression in tumor

endothelium. Taken together, these results suggest

that mIR-132, or RASA1 itself, might be viable

targets for therapeutic anti-vasculogenesis treatments

in patients.

Summary

RASA1, the prototypical Ras GAP, plays an important

role in the negative regulation of Ras in growth factor

receptor signaling. In addition, a number of positive

regulatory roles for RASA1 have been described.

Despite being the first GAP discovered, there remain

many unanswered questions about its function. How

RASA1 controls angiogenesis and lymphangiogenesis

is unknown, but disregulated Ras signaling through

one or more growth factor receptors is the most likely

mechanism. The receptors VEGFR1, VEGFR2, and/or

VEGFR3 are strong candidates. In addition, the precise

mechanism by which RASA1 regulates naıve T cell

survival remains to be elucidated. With the use of

recently developed tools described above, these ques-

tions will likely be the subject of intensive study for

years to come.

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p120RasGAP activates the endothelium to facilitate patho-

logical angiogenesis. Nat Med. 2010;16(8):909–14.

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GTPase activating proteins in man and Drosophila. Biochim

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Boon LM, Mulliken JB, et al. RASA1: variable phenotype with

capillary and arteriovenous malformations. Curr Opin Genet

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Ekman S, Thuresson ER, et al. Increased mitogenicity of an

alphabeta heterodimeric PDGF receptor complex correlates

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Friedman E, Gejman PV, et al. Nonsense mutations in the

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RasGrf (RAS Protein-Specific Guanine Nucleotide-Releasing Factor) 1605 R

R

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cyte positive selection and survival of naive T cells.

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Ras-Associated Protein RAB8

▶Rab8

RasGAP

▶RASA1

RasGrf (RAS Protein-Specific GuanineNucleotide-Releasing Factor)

Eugenio Santos and Alberto Fernandez-Medarde

Centro de Investigacion del Cancer, IBMCC

(CSIC/USAL), University of Salamanca,

Salamanca, Spain

Synonyms

CDC25; CDC25L; CDC25Mm; GNRP; GRF

Historical Background

The Ras guanine nucleotide releasing factor (RasGrf)

proteins were isolated in an effort to find mammalian

homolog(s) of the yeast CDC25 Ras activator protein.

The search for mammalian Ras GEFs during the early

1990s led to the discovery and isolation of the Son of

sevenless (Sos) and the RasGrf proteins. Whereas the

Sos proteins had ubiquitous expression, RasGrf

expression was restricted mainly to the central ner-

vous system. Soon after the discovery of the first

member of the RasGrf family (RasGrf1) in neural

tissues, a second highly homologous member of this

family was isolated from embryonic stem cells

(RasGrf2). Later on, a number of distinct, alterna-

tively spliced isoforms have been described for both

genes in a variety of tissues or developmental stages.

RasGrf1 and RasGrf2 are large, modular proteins

composed by multiple functional domains accounting

for protein–protein or protein–lipid interactions

which are responsible for coupling to upstream and

downstream signaling as well as for fine regulation of

their intrinsic exchange activity (Santos and

Fernandez-Medarde 2008, 2009).

Early biochemical characterization of the RasGrfs

yielded identification of their specific targets in

different Ras GTPase families, as well as initial char-

acterization of their contribution to specific cellular

signal transduction pathways. Whereas both RasGrf1

and RasGrf2 are able to activate canonical

Ras proteins (H-Ras, N-Ras or K-Ras) and Rac1,

only RasGrf1 is able to activate members of

the R-Ras subfamily (R-Ras, TC21, M-Ras)

R 1606 RasGrf (RAS Protein-Specific Guanine Nucleotide-Releasing Factor)

(Santos and Fernandez-Medarde 2008, 2009). Fur-

thermore, both RasGrfs are known to be activatable

in response to different signals including increases of

intracellular levels of calcium or cAMP, activation of

heterotrimeric G-protein-coupled receptors, or NGF

receptor stimulation. More recent work using geneti-

cally modified animal models has provided significant

clues to physiological roles played by the RasGrfs.

Analysis of RasGrf1 and RasGrf2 knockout mice

show the implication of RasGrf1 in memory and

learning processes, light perception, glucose homeo-

stasis, and substance addiction (Brambilla et al. 1997;

Fernandez-Medarde et al. 2009; Font de Mora et al.

2003; Tonini et al. 2006), and the participation of

RasGrf2 in memory formation and immunological

responses (Li et al. 2006; Ruiz et al. 2007).

RasGrfs Protein Structure and DomainDistribution

The RasGrfs are large, highly homologous proteins

(sharing ca. 80% homology and 63% identity in their

sequences). RasGrf1 is slightly larger than RasGrf2

(respective molecular weights of the full-length pro-

teins: 140 kDa and 135 kDa in mice; 145 kDa and

140 kDa in humans). These two proteins share multiple

functional domains which are essential for fine regula-

tion and control of their intrinsic catalytic activity and

protein stability as well as subcellular localization and

functional link to upstream/downstream signals (Fig. 1).

From N- to C-terminus, these domains include:

PH1 domain- Although many PH domains are

reported to mediate protein targeting to membranes,

a RasGrf1 construct lacking this domain retains par-

tial plasma membrane localization. The PH1 domain

is reported to interact with G-protein b/g subunits, andis required for normal G-protein induced ERK1/2

activation (Innocenti et al. 1999). In addition,

together with the adjacent IQ and coiled coil domains,

the PH1 domain is involved in interaction with

scaffold proteins such as JIP2 or Spinophilin or with

ribosomal proteins (Santos and Fernandez-Medarde

2009). Separate studies have also shown that

this domain is necessary for complete calcium-

and LPA-mediated activation of RasGrf1, and it

is phosphorylated upon interaction with the TrkA

NGF receptor (Innocenti et al. 1999; Robinson et al.

2005).

Coiled coil (cc) domain- All functional roles for thisdomain have been described in association with the

flanking PH1 and IQ domains. As described above, its

contribution is important for complete ERK1/2 activa-

tion upon intracellular calcium increase and for inter-

action with the JIP2 scaffold protein (Santos and

Fernandez-Medarde 2009).

IQ domain- The main role of this domain is the

interaction with Calmodulin, which is essential for

activation of both RasGrfs by increases of intracellular

calcium concentration. Mutations in this domain abol-

ish ionomycin-mediated activation of RasGrf1 and

stimulation of the Ras-ERK pathway. It is also

involved, jointly with the PH1 and CC domains, in

the interaction with the JIP2 scaffold protein (Santos

and Fernandez-Medarde 2009).

DH domain- The DH-PH2 domain tandem consti-

tutes the typical catalytically active domain facilitating

GDP/GTP exchange on GTPases of the Rho

subfamily. In the RasGrf proteins, this region is

responsible for activation of Rac1 (Santos and

Fernandez-Medarde 2008, 2009). The DH domain

also exerts a regulatory role on Ras activation

by RasGrf1, and may also be important for

Ras-independent, ionomycin-induced ERK activation

(Freshney et al. 1997). This domain also mediates

protein–protein interactions of RasGrf1 with

b�tubulin and SCLIP and it is involved in homo- and

hetero-oligomerization of the RasGrf1 and RasGrf2

proteins.

PH2 domain- A defined functional role for this

domain remains unclear. In RasGrf1, some reports

indicate that it is required for proper Ras and ERK

activation, whereas studies in different cellular sys-

tems suggest that this domain is dispensable for Ras

or ERK activation. Further work is needed to clarify

these discrepancies analyzing RasGrf function in more

physiological environments.

REM motif- This region is common to all Ras Gua-

nine Nucleotide Exchange Factors and it is responsible

for GEF–Ras interaction. It is likely that the REM

motif has a similar role in all the Sos, ▶RasGRP, and

RasGrf1 families of GEF proteins.

CBD motif- This short domain usually targets pro-

teins for degradation by the proteasome. It is located

between the REM and CDC25H domains. Its involve-

ment with ubiquitination and subsequent proteolytic

degradation has been shown experimentally only for

RasGrf2.

PH1 PH2DH ND CDC25HREM

CC IQ ITIM ITIM CDB

PEST rich region

PH1 PH2DH CDC25HREM

CC IQ ITIM CDB

PEST rich region

mRasGrf1

mRasGrf2

588 635 74923 130 244 430 1027 1259

588 635 74923 135 243 429 934 1189

RasGrf (RAS Protein-Specific Guanine Nucleotide-Releasing Factor), Fig. 1 RasGrf1 and RasGrf2 domain dis-

tribution: PH pleckstrin homology domain, CC coiled coil

domain, IQ isoleucine (I)/glutamine (Q) motif, DH Dbl homol-

ogy domain, ITIM immuno tyrosine–based inhibition motifs,

REMRas exchanger motif,DB destruction box,ND neurological

domain, PEST Rich Region (P)-proline, (E)-glutamic acid,

(S)-serine, and (T)-threonine rich region, CDC25H CDC25

homology domain

RasGrf (RAS Protein-Specific Guanine Nucleotide-Releasing Factor) 1607 R

R

PEST motif rich region- PEST [Proline (P),

Glutamic acid (E), Serine (S), Threonine (T)] regions

are known targets for ▶ calpain-type protease degra-

dation, a role that has been experimentally demon-

strated in RasGrf1 (Santos and Fernandez-Medarde

2009). This area is also targeted in RasGrf1 for phos-

phorylation by PKA or upon muscarinic receptor–

mediated activation (Mattingly 1999). It is also phos-

phorylated by CDK5 in both RasGrfs, inhibiting the

GEF activity toward Rac1 (RasGrf2) or Ras (RasGrf1)

(Kesavapany et al. 2006).

Neuronal domain- Only found in RasGrf1, it is

responsible for RasGrf1 binding to the NR2B subunit

of the NMDA receptors and subsequent activation of

downstream signaling pathways (Krapivinsky et al.

2003).

CDC25H domain- This C-terminal domain contains

the catalytic region responsible for GDP/GTP

exchange (GEF activity) on Ras family members. It

is shared by all GEFs acting on canonical Ras proteins

and shows a markedly high degree of conservation

through evolution. This domain is necessary and suffi-

cient for “in vitro” Ras activation by RasGrf1, and it is

also responsible for Ras activation by RasGrf2. “In

vivo” modulation of the GEF activity of the CDC25H

domain in the context of full-length RasGrf proteins

can be exerted through various intramolecular interac-

tions or biochemical modifications (Santos and

Fernandez-Medarde 2009, 2008).

Signaling Through the RasGrfs

Activation of RasGrfs in Response to Increase in

Intracellular Calcium Concentration

Cellular calcium influx can modulate the activation of

the RasGrfs through a mechanism involving Calmod-

ulin binding to their IQ domains (Fig. 2). Mutations in

this domain abolish ionomycin-mediated activation of

RasGrf1 and stimulation of the Ras-ERK pathway. In

addition, the N-terminal region of RasGrf1 cooperates

synergistically with the IQ domain to potentiate

RasGrf1-mediated activation of ERK1/2 upon stimu-

lation by LPA or calcium (Santos and Fernandez-

Medarde 2009).

RasGrf2 can also be activated by increased intra-

cellular calcium concentration. In 293T cells, calcium

influx causes translocation of RasGrf2 to the cell

periphery, localizing it in close proximity to mem-

brane GTPases, through a mechanism not yet fully

understood. Although RasGrf2 interacts with calmod-

ulin through its IQ domain, a full, functionally pro-

ductive interaction appears to need the additional

cooperation of other N-terminal domains (de Hoog

et al. 2000). Interestingly, a RasGrf2 construct

lacking the IQ domain is still able to activate Ras,

suggesting that interaction with calmodulin is not

necessary for RasGrf2 GEF activity, although both

the IQ domain and Calmodulin interaction with

RasGrf2 are indispensable for ERK activation.

RasGrf 1

ERK

a b

c

p38Ras

Rac1

Neuriteextension

Neuronalsoma

morphology

TrkA

P

NGF

NR 1 NR2A NR 1

Ca2+ Ca2+

Glut

CamK KCaMK I

CaM

RasGrf 1

NR2B

p38

LTD

?*

RasGrf 2

**

p38

Ras

Rac1

CalciumIonophores

Rac1 Ras

ERK

LTP

ERK

LTP

Glut

Agonist

cAMP

α

αββ γγ

PKA

Ca2+CaM

RasGrf 1

Ras

Rac1P

? SrcP

?

PACK

RasGrf (RAS Protein-Specific Guanine Nucleotide-Releasing Factor), Fig. 2 The main upstream factors activat-

ing RasGrf proteins include the NGF receptor (a), NMDA

receptors, intracellular increases of calcium (b), non-receptor

protein kinases, PKA or G-protein coupled receptors (c).*CaMK1 activation of RasGrf1 has been proposed, but not

demonstrated. **No physical interaction has been demonstrated

between NR2A and RasGrf2

R 1608 RasGrf (RAS Protein-Specific Guanine Nucleotide-Releasing Factor)

These observations suggest the existence of Ras-

independent mechanisms of ERK1/2 activation, or

the need of other interacting partners for full coupling

of Ras and ERK1/2 activation (Santos and Fernandez-

Medarde 2008).

Activation of RasGrfs in Response to G-Protein

Coupled Receptors (GPCR)

The intrinsic activity of intracellular RasGrf1 can be

enhanced by stimulation with LPA or serum, but not

with PDGF. This activation is inhibited by

pretreatment with pertussis toxin, but not with genis-

tein, suggesting that GPCRs, but not receptor tyrosine

kinases (RTK) play a role in serum activation of

RasGrf1 (Zippel et al. 1996). LPA treatment of

NIH3T3 cells overexpressing RasGrf1 induces phos-

phorylation of RasGrf1 in serine residues, and calcium

is also needed for full GEF activation (Fig. 2). Besides

LPA, other GPCRs can also activate RasGrf1.

Overexpression of subtype 1 human muscarinic recep-

tor induces RasGrf phosphorylation and its GEF activ-

ity upon carbachol stimulation. This activation is

prevented by phosphatases and G-protein a-subunitoverexpression, and is constitutively established

when G-protein bg subunits are overexpressed. The

5-HT4 serotonin receptor is another GPCR able to

activate RasGrf1. Its overexpression induces RasGrf1

phosphorylation by PKA (at Serine 916) and

RasGrf (RAS Protein-Specific Guanine Nucleotide-Releasing Factor) 1609 R

IQ-dependent activation upon serotonin stimulation,

suggesting that the complete activation of RasGrf1 by

these receptors involves both cAMP and calcium/

calmodulin-dependent signaling (Fig. 2) (Norum

et al. 2007).

Overexpression of G-protein bg subunits and LPA

treatment are also known to induce RasGrf1 GEF

activity toward Rac1, leading to JNK and c-fos pro-

moter activation, but to fully activate Rac1, RasGrf1

needs to be phosphorylated in tyrosine by ▶ Src

(Kiyono et al. 2000) (Fig. 2).

R

Activation of RasGrfs in Response to Receptor and

Non-receptor Tyrosine Kinases

In PC12 cells, the TrkA nerve growth factor (NGF)

receptor has been reported to interact with, and induce

phosphorylation of, RasGrf1 in its PH1 domain

(Robinson et al. 2005) (Fig. 2). In addition, RasGrf1

potentiates NGF-induced differentiation of PC12 cells

in a process dependent on H-Ras and ERK1/2, but

independent of Rac1 or ▶ PI3K pathways. On the

other hand, RasGrf1 coordinates H-Ras and Rac1 path-

ways in a PI3K/AKT-dependent manner, to induce

soma expanded morphology in PC12 cells (Santos

and Fernandez-Medarde 2009).

The Src tyrosine kinase can also mediate transduc-

tion of G-protein-dependent signals from RasGrf1 to

Rac1 proteins, but not to Ras canonical proteins

(Kiyono et al. 2000). Other tyrosine kinases, such as

▶ACK1 and Lck, are also able to phosphorylate

RasGrf1, resulting in enhanced Ras GEF activity

(Santos and Fernandez-Medarde 2009). In particular,

ACK1 is known to be activated by Cdc42, which in its

inactive GDP-Cdc42 conformation is also able to

inhibit RasGrf1 activation of Ras (Arozarena et al.

2001). A similar mechanism may be applicable to

RasGrf2, as the expression of dominant negative

Cdc42 (Cdc42N17) abolishes RasGrf2 recruitment

to the plasma membrane, Ras activation and ERK

phosphorylation (Santos and Fernandez-Medarde

2008). RasGrf2 and RasGrf1 are activated upon

T-cell receptor stimulation, a process requiring the

contribution of tyrosine kinase(s) of the Src family.

Activation of RasGrf2 induces Ras-dependent and

PLC-g1-mediated signaling pathways, producing the

activation of ▶NF-AT, a transcriptional factor cru-

cial for T-cell activation and differentiation (Ruiz

et al. 2007).

Control of RasGrf Expression and CellularProtein Levels

Transcriptional Control

RasGrf1 is an imprinted gene expressed only after

birth. In mice, the paternal allele of the RasGrf1 locus

is methylated on a differentially methylated domain

(DMD) located 30 kbp 50 of the promoter. In mouse

neonatal brain, RasGrf1 expression occurs exclusively

from the paternal allele and accounts for ca. 90% of the

total RasGrf1 expressed. A repeat sequence located

immediately downstream of the DMD controls its

methylation and is therefore required to establish

RasGrf1 methylation in the male germ line. CTCF (a

CCCTC-binding factor) binds to the DMD in

amethylation-sensitivemanner, acting as an “enhancer

blocker.” In the unmethylated maternal allele, CTCF is

bound to DMD thus silencing expression, whereas

CTCF cannot bind to the methylated paternal allele,

thus allowing expression. The repeats and the DMD

thus constitute a dual switch regulating RasGrf1

imprinting and timing of expression (Yoon et al. 2005).

Although RasGrf2 appears not to be an imprinted

gene, genomic methylation may still play a significant

role in control of expression of this locus. The shortage

of specific studies on RasGrf2 expression determines

that the mechanisms controlling expression in the post-

natal brain remain largely unknown. Interestingly, in

colon, pancreatic and lung (NSCLC) tumors and cell

lines, hypermethylation of RasGrf2 locus and reduced

protein expression are frequently associated (Santos

and Fernandez-Medarde 2008).

Some external factors are also known to modulate

postnatal RasGrf1 expression. Cocaine induces

overexpression in dorsal and ventral striatum, whereas

the Alzheimer-related amyloid precursor protein with

the Swedish mutation (APPSw) or oncogenic ErbB2/

Neu and luteinizing hormone repress its expression in

hippocampus and mammary gland, respectively (San-

tos and Fernandez-Medarde 2009).

Control of Proteolytic Degradation

The intracellular concentration of the RasGrf proteins

is also posttranslationally regulated by cellular prote-

ases. Both RasGrf1 and RasGrf2 contain a type

A cyclin destruction box (CDB), located between the

REM and CDC25 domains (Fig. 1). These domains

have been reported to trigger ubiquitination and deg-

radation of RasGrf2 by the proteasome upon Ras

R 1610 RasGrf (RAS Protein-Specific Guanine Nucleotide-Releasing Factor)

binding. There is no direct experimental evidence

showing a similar role of this domain in RasGrf1,

although the interaction between RasGrf1 and the

deubiquitinating enzyme mUBPy results in increased

RasGrf1 half-life, and in M2 melanoma cells, the

actin-binding protein Filamin A induces destabiliza-

tion and ubiquitination of RasGrf1 with a subsequent

reduction of MMP9 expression (Santos and

Fernandez-Medarde 2009).

Calpain can also cleave RasGrf1, but the functional

significance of such cleavage is unclear. Some reports

described that this cleavage increases RasGrf1 GEF

activity toward Ras proteins by releasing the

C-terminus from inhibition by the N-terminal portion,

whereas other studies show that phosphorylation by

p35/CDK5 targets RasGrf1 for proteolysis by ▶m-

calpain, resulting in reduced Ras activation and AKT

phosphorylation (Santos and Fernandez-Medarde

2009; Kesavapany et al. 2006). Phosphorylation of

RasGrf2 by p35/CDK5 is also reported to result in

accumulation and increased local concentration of

RasGrf2 protein in the body of neurons (Santos and

Fernandez-Medarde 2008).

Genetically Modified Animal Models of theRasGrfs

RasGrf1

Analysis of the phenotypes of a number of independent

RasGrf1 KO strains generated in different laboratories

has contributed to a better understanding of the func-

tional “in vivo” roles of RasGrf1. Two main types of

phenotypes have been reported: those related to defects

in memory consolidation and learning, and those

related to growth retardation and glucose homeostasis

responses.

An initial report on RasGrf1-KO mice (Brambilla

et al. 1997) described LTP defects and associated

impairment of amygdala-dependent learning, whereas

later studies on a separate KO strain reflected LTD

defects and impairment of hippocampus-dependent

learning (Li et al. 2006). The discrepancy may be due

to the use of different gene targeting strategies or

different mouse genetic backgrounds. In any event,

a role of RasGrf1 in memory and learning is supported

by separate studies of the hippocampus of RasGrf1 KO

mice that showed specific transcriptional alterations

involving genes related to those neural processes.

RasGrf1 also shows a role in cannabinoid tolerance,

as the KOmice show lower tolerance toD9-tetrahydro-cannabinoid, probably through alterations in cannabi-

noid receptor- and cAMP-mediated signaling (Tonini

et al. 2006). Finally, RasGrf1 is also important to

maintain normal photoreception, as the RasGrf1 KO

mice develops light perception problems worsening

with age progression (Fernandez-Medarde et al. 2009).

Analysis of the knockout mice has also revealed the

role of RasGrf1 in the control of postnatal growth.

Adult RasGrf1 null mice are 15–25% smaller than

wild-type controls. The reduced size is probably

directly associated to the lower levels of growth hor-

mone and circulating plasma insulin observed in

RasGrf1-deficient mice pituitary. The association

between hypoinsulinemia and reduced pancreatic

beta-cell mass observed in the KO mice is indicative

of a role of RasGrf1 in control of beta cell proliferation

and neogenesis (Font de Mora et al. 2003).

RasGrf2

RasGrf2 is dispensable for mouse development, post-

natal growth, fertility, and normal aging, as the

RasGrf2 KO mice are morphologically indistinguish-

able from their wild-type controls. Amore subtle, brain

phenotype is suggested by studies of a different

RasGrf2 KO strain, pointing to lower levels of ERK

activation upon NMDA-induction, and linking the

absence of RasGrf2 to defective LTP in the CA1 region

of the hippocampus (Li et al. 2006). Additional

insights into the role of RasGrf2 “in vivo” have been

obtained through the analysis of mice harboring com-

binations of a disrupted RasGrf2 locus with null muta-

tions for other GEFs. Thus, elimination of both

RasGrf2 and RasGrf1 results in higher sensitivity to

the neurotoxic effects of ischemia in the mouse brain

(Santos and Fernandez-Medarde 2009). Furthermore,

analysis of RasGrf2/▶Vav3 and RasGrf2/▶Vav1 null

mice suggest a role for RasGrf2 in T-receptor signaling

responses in lymphocytes (Ruiz et al. 2007).

Summary

TheRasGrfs are themain GEF activators ofmammalian

Ras GTPases in the adult central nervous system. The

experimental in vivo evidence indicates that both

RasGrfs are able to activate the canonical Ras proteins

(H-, N- and K-Ras) and Rac1. RasGrf1 and RasGrf2 are

RasGrf (RAS Protein-Specific Guanine Nucleotide-Releasing Factor) 1611 R

R

large, highly homologous proteins sharing a modular

structure composed of multiple distinct, functional

domains which are instrumental for regulation of their

intrinsic GEF activity and for modulation of their par-

ticipation in signal transduction connecting a variety of

upstream signals to their respective, specific down-

stream targets and elicited cellular responses. The GEF

activity of the RasGrfs becomes activated in response to

a variety of cellular signals including LPA, increased

cytosolic concentration of calcium or cAMP, and acti-

vation of cell surface receptors for various signaling

molecules such as ▶NMDA, AMPA, serotonin, mus-

carinic agonists (G-protein coupled receptors), or NGF

(trkA) (Fig. 2). Known downstream effects of the par-

ticipation of the RasGrfs in cellular signaling pathways

include control of cellular shape and nuclear organiza-

tion, neurite extension, neuronal synaptic plasticity,

induction of LTP or LTD, and neuroprotection against

ischemia. Analysis of genetically modified animal

models has uncovered the specific functional roles of

RasGrf1 in memory and learning, postnatal growth,

pancreatic beta cell proliferation, retinal photoreception,

and neuroprotection against ischemia. Likewise,

RasGrf2 has also been implicated inmemory formation,

neuroprotection, and immunological responses in lym-

phocytes. The participation of the RasGrfs in processes

leading to human disease is also suspected. Increased

RasGrf2 gene methylation is frequently observed in

various human tumors and cancer cell lines. Other

observations suggest the implication of RasGrf1 in

visual defects, drug addiction, and Alzheimer-like neu-

rodegenerative diseases.

In spite of the vast amount of published information

on the RasGrf proteins, a number of key questions still

remain unanswered. Because of their prevalent expres-

sion in the CNS, most functional studies on the RasGrfs

have been restricted to neural tissues and cell lineages.

However, as bothRasGrfs are also expressed outside the

CNS, their functional roles at those external locations

remain less defined and require further, extensive stud-

ies. Another poorly understood area is the functional

significance of the great variety of small RasGrf

transcripts and peptides detected for both RasGrf1 and

RasGrf2 in many tissues and/or states of development.

An interesting hypothesis would be that such an assort-

ment of RasGrf isoforms may contribute to the fine-

tuned regulation of the activation of their cellular

Ras/Rho targets at the spatial and temporal level.

A better understanding of the mechanisms linking

functional observations made for the RasGrfs at the

cellular level with those made at the organism level

would also be desirable. This pertains questions such

as: Is the participation of RasGrf1 in cytoskeleton

remodeling necessary for neuritogenesis? Is there a con-

nection between the role of RasGrf1 in neuritogenesis

and its contribution to memory formation processes?

Finally, future work efforts should be strongly aimed

at getting definitive answers to the current hints linking

the RasGrf proteins to the development of different

human illnesses and pathological processes.

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RasGRP

▶RasGRP1

RasGRP1

John J. Priatel1, Kevin Tsai1 and Kenneth W. Harder2

1Department of Pathology and Laboratory Medicine,

Child and Family Research Institute, University of

British Columbia, Vancouver, BC, Canada2Department of Microbiology and Immunology,

Life Sciences Institute, University of British

Columbia, Vancouver, BC, Canada

Synonyms

Calcium and DAG-regulated guanine nucleotide

exchange factor II; CalDAG-GEFII; Guanine

nucleotide exchange factor, calcium- and DAG-regu-

lated; MGC129998; MGC129999; Ras guanyl nucleo-

tide-releasing protein 1; Ras guanyl-releasing protein;

Ras guanyl-releasing protein 1; RasGRP

Historical Background

Antigen receptor stimulation of T cells and B cells

results in the rapid conversion of the small GTPase

Ras from its “inactive” GDP-bound form to its “active”

GTP-bound form (Alberola-Ila and Hernandez-Hoyos

2003). However, the mechanisms of Ras regulation in

lymphocytes, particularly by phorbol esters, had long

remained a mystery as known signaling pathways

could not account for its activation. The discovery of

the Ras guanyl-nucleotide exchange factor RasGRP1,

cloned by two groups searching for novel Ras activa-

tors in rat brain and murine T cells, was a major

advance leading to a better understanding of Ras reg-

ulation in lymphocytes (Stone 2011). Ectopic expres-

sion of RasGRP1 in rodent fibroblasts was found to be

capable of activating Ras and inducing cellular trans-

formation. Subsequently, investigations of human

T cells and RasGRP1-deficient mice have corroborated

key roles for RasGRP1 in both T-cell receptor- and

phorbol ester-induced Ras activation (Stone 2011).

Originally coined RasGRP (Ras guanyl nucleotide-

releasing protein), RasGRP1 is the prototypic member

of the RasGRP family of proteins that share a number

of conserved elements that have been implicated in its

function and regulation (Stone 2011), including REM

(Ras exchange motif) domain,▶CDC25 (cell division

cycle 25)-related domain, calcium-binding “EF-hand”

motif, and diacylglycerol-binding “C1” domain

(Fig. 1). Besides RasGRP1, the RasGRP family also

comprises of RasGRP2, RasGRP3, and RasGRP4.

RasGRP1 Functions in Developing T Cells

The small GTPase Ras acts as a molecular switch,

cycling between GDP-bound “off” and a GTP-bound

“on” conformations, and serves to link signals from

cell surface receptors to intracellular effector path-

ways. Ras activation can be modulated by its own

intrinsic GTPase activity, converting GTP to GDP,

and guanine nucleotide exchange. However, the Ras’

rates of GTP hydrolysis and nucleotide exchange are

N- REM CDC25 E F C1 -C

RasGRP1, Fig. 1 Conserved domains of RasGRP1. Domain

structure of human RASGRP1 protein (NCBI accession:

AAH67298; 765 aa), annotated by NCBI Blast Search, is sche-

matically shown. REM (Ras Exchange Motif; E-value:

1.44e-15) and ▶CDC25 (also known as RasGEF; E-value:

1.44e-15) domains participate in the catalytic activity of

the exchange factor. The Protein kinase C conserved region 1

(C1; E-value: 1.54e-12) domain binds diacylglycerol and

phorbol esters. EF-hand domains (E-value: 8.42e-04) are

suspected to act as calcium sensors and calcium signal

modulators

RasGRP1 1613 R

R

very low unless paired with catalytic proteins. Guanine

nucleotide exchange factors (Ras GEFs) control the

activation of Ras by catalyzing GDP release from

Ras and facilitating its association with more prevalent

cellular GTP. Conversely, Ras GTPase-activating pro-

teins (Ras GAPs) accelerate GTP hydrolysis reaction

converting Ras-GTP to its inactive GDP-bound form.

Tight control of Ras activity is essential for regulating

cell activation, proliferation, differentiation, and apo-

ptotic programs in multiple cell types. By associating

with various effector proteins, activated Ras initiates

signaling through multiple downstream pathways such

as the mitogen-activated protein kinase (MAPK)

cascade.

T-cell receptor (TCR) stimulation results in the

rapid activation of the small GTPase Ras whose signals

are essential for the development of T cells in the

thymus (Alberola-Ila and Hernandez-Hoyos 2003).

Taking cues from Ras studies on non-lymphocytes,

the Ras GEF SOS (Son of Sevenless) has been

postulated to regulate Ras upon TCR activation. SOS

proteins are ubiquitously expressed and their functions

are modulated through their association with the adap-

tor GRB2 (growth factor receptor-bound protein 2).

Moreover, the SH2 domain of GRB2 targets SOS to

phosphorylated tyrosine residues of surface receptors

and adaptor proteins.

According to the SOS-based model, the following

sequence of events leads to Ras activation in T cells:

TCR ligation activating the ▶ Src protein tyrosine

kinases (PTKs) LCK (lymphoid cell kinase) and Fyn

leading to the phosphorylation of CD3s ITAMs

(immunoreceptor tyrosine-based activation motifs),

these phosphorylated ITAMs mobilizing the Syk

PTK ▶ZAP-70 (zeta-associated protein – 70 kDa) by

way of ▶ZAP-70s SH2 domain and becoming acti-

vated through the action of ▶ Src PTKs, activated

▶ZAP-70 phosphorylating multiple tyrosine residues

on the docking adaptor transmembrane protein▶LAT

(Linker for Activated T cells) and phosphorylated

▶LAT recruiting the GRB2/SOS complex in close

proximity of plasma membrane–bound Ras and facil-

itating its displacement of GDP. However, a SOS

based model of Ras activation cannot explain at least

two T-cell phenomena: (1) Why phorbol esters or their

analogs activate Ras? and (2) Why PKC inhibitors

dampen Ras activation?

The cloning of RasGRP1 identified the first of

a novel class of Ras guanyl nucleotide-releasing pro-

teins that possessed calcium- and diacylglycerol

(DAG)-responsive elements and elucidated a critical

mechanism by which TCR signal transduction and

phorbol ester stimulation is linked to the activation of

the Ras-MAPK cascade in T cells (Stone 2011). TCR

signaling incorporates RasGRP1 function through the

mobilization and enzymatic activities of PLCg1. Sim-

ilarly to GRB2/SOS, PLCg1 is recruited to phosphor-

ylated ▶LAT through its SH2 domain and becomes

phosphorylated by Tec family protein kinases. Subse-

quently, the action of activated PLCg1 converts PIP2

(phosphatidylinositol 4, 5 bisphosphate) into IP3 (ino-

sitol 3, 4, 5 triphosphate) and DAG, a second messen-

ger previously thought to solely activate PKC through

its DAG-binding C1 domain. However, DAG also

causes RasGRP1 to become membrane localized

through its own DAG-binding C1 domain. Further-

more, DAG indirectly impacts RasGRP1 as activated

PKC regulates RasGRP1 activity through its phosphor-

ylation (Roose et al. 2005). As a consequence, the

integration of RasGRP1 into T-cell signaling provides

an explanation for the well-documented activation of

Ras by DAG analogs and phorbol esters such as PMA

(phorbol myristate acetate). In addition, the integration

of RasGRP1 also provides a mechanism for why PKC

inhibitors block Ras activation.

Ras-MAPK signaling downstream of the pre-TCR

and TCR is critical for two developmental checkpoints

as thymocytes undergo an ordered series of maturation

steps within the thymic microarchitecture (Alberola-

Ila and Hernandez-Hoyos 2003). Thymocyte devel-

opment is most often tracked through the variable

expression of the cell surface markers CD4 and CD8.

After productive rearrangement of the TCRb chain andpairing with the pre-TCRa, pre-TCR expression by

the most immature CD4- CD8- double-negative (DN)

thymocytes drives ligand-independent Ras-MAPK

R 1614 RasGRP1

signaling at the first developmental checkpoint and

differentiation into CD4+ CD8+ double-positive (DP)

thymocytes. Accompanying rearrangement and

expression of the TCRa chain, TCR-dependent Ras-

MAPK signaling at the second developmental

checkpoint (called “thymocyte selection”) becomes

contingent upon the recognition of self-antigens

(i.e., self-peptides presented in the context of self-

MHC molecules). The intensity of TCR interaction

with self-antigens on thymic cortical epithelial cells

and bone marrow–derived cells is presumed to deter-

mine the strength of signal and the fate of the devel-

oping DP thymocyte. According to the strength of

signal hypothesis, cells that do not recognize self-

antigens fail to receive TCR signaling resulting in

death by neglect; cells that recognize self-antigens

robustly receive strong TCR signaling dying via active

apoptosis (negative selection); and cells that recognize

self-antigens weakly receive moderate TCR signaling

differentiating into mature CD4+ CD8- or CD4- CD8+

single-positive (SP) T cells (positive selection).

DP thymocytes discriminate graded TCR-

dependent Ras-MAPK signals and translate them into

a cell fate decision (Alberola-Ila and Hernandez-

Hoyos 2003). TCR-induced Ras signaling results in

the activation of three distinct families of MAPKs:

ERK (extracellular signal-regulated kinases), JNK

(c-Jun N-terminal kinases), and p38. The activation

of the MAPKs plays qualitatively and quantitatively

distinct roles in thymocyte selection: ERK has been

most often paired with positive selection whereas JNK

and p38 are associated with negative selection. How

might different MAPKs selectively pair and become

activated upon TCR-induced Ras signaling was not

clear. To elucidate the role of RasGRP1 in T-cell

development, analyses of RasGRP1�/� mice revealed

a near-normal number of DN and DP thymocytes but

a severe deficiency in mature thymocytes, suggesting

a block in thymocyte selection (Stone 2011). Further-

more, RasGRP1�/� thymocytes failed to activate both

Ras and ERK upon phorbol ester stimulation. In addi-

tion, one-month old RasGRP1�/� mice had very few

splenic T cells. As a consequence of the T-cell pheno-

type present in RasGRP1�/� mice along with the

governing role that TCR signaling plays in T-cell

development, RasGRP1 was hypothesized to link Ras

activation with TCR signaling.

To examine the role of RasGRP1 in thymocyte

selection, two lines of RasGRP1�/� TCR transgenic

mice were generated to determine the effect of

RasGRP1 under conditions of defined TCR signaling

strength (Priatel et al. 2002). Results from these exper-

iments indicated that positive selection, particularly

a weakly selecting TCR, and TCR-induced ERK acti-

vation are critically dependent on RasGRP1. By con-

trast, RasGRP1-deficiency had no effect on negative

selection or JNK and p38 MAPK activation. These

conclusions were consistent with complementary

findings from another study investigating Grb2+/�

mice (Gong et al. 2001). Halving of the amount of

GRB2/SOS led to decreased Ras, JNK, and p38 acti-

vation and impaired negative selection. However,

ERK activation and positive selection were unaffected

by Grb2 haploinsufficiency, suggesting that the

RasGRP1-ERK pathway may have a lower threshold

of activation than GRB2/SOS. The rationale for TCR

signaling to employ two different Ras GEFs may be to

subject Ras to differential regulation or to pair it with

a unique subset of effectors. Based on the findings

from the above studies, a hypothesis was formulated

that the Ras GEFs RasGRP1 and GRB2/SOS may

serve to selectively pair Ras activation with differen-

tial MAPKs pathways. According to this model, DP

thymocytes expressing a positively selecting TCR will

activate Ras and solely the MAPK ERK via RasGRP1

whereas DP thymocytes expressing a negatively

selecting TCR will activate Ras and the full range of

MAPKs (ERK, JNK, and p38) via the use of both

GRB2/SOS and RasGRP1 pathways.

RasGRP1 Functions in Other Blood Cells

RasGRP1 was originally envisioned to have very

restricted expression, transcripts being detected

solely in T cells and some neuronal cell lineages

(Stone 2011). More recent studies have found that

RasGRP1 has a much broader tissue distribution

than previously thought. RasGRP1 is also expressed

in B cells and is presumed to couple the B-cell antigen

receptor (BCR) to Ras-ERK signaling in an analogous

fashion to the way it functions in TCR signal trans-

duction (Coughlin et al. 2005). However, RasGRP1

function in BCR signaling appears to be partially

masked through the coexpression of RasGRP3 and

the sharing of some redundant functions with

this RasGRP family member. Studies using the

immature B cell line WEHI-231 have linked

RasGRP1 1615 R

a RasGRP1-pathway that is ERK-independent to BCR-

induced apoptosis (Guilbault and Kay 2004). Analyses

of human NK (natural killer) cells using RNA interfer-

ence have demonstrated that RasGRP1 regulates

ITAM-dependent cytokine production and NK cell

cytotoxicity (Lee et al. 2009). In addition, RasGRP1

knockdown in NK cells was found to result in damp-

ened Ras, ERK, and JNK activation.

RasGRP1 is also expressed by mast cells and sig-

nals downstream of the high affinity IgE receptor

FceR1 (Liu et al. 2007). Moreover, FceR1 degranula-

tion and cytokine production were greatly reduced in

RasGRP1�/� mast cells relative to wild type and

RasGRP1�/�mice failed to elicit anaphylactic allergic

reactions. Interestingly, RasGRP1 in mast cells

was found to link FceR1-mediated Ras signaling to

▶ PI3K (phosphatidyl inositol 3-kinase) pathway

rather than to ERK activation. By contrast,

a concentrated effort to establish a connection between

RasGRP1 signaling and ▶PI3K activation in lympho-

cytes has been unsuccessful (Stone 2011). In addition,

RasGRP1 expression and function has been recently

described outside of the hematopoietic system,

although those studies are described elsewhere (Stone

2011).

R

RasGRP1 Activity and SubcellularLocalization

The most controversial issue regarding RasGRP1 is its

subcellular locale when active. Long-held dogma

asserts that Ras operates at the plasma membrane,

however, a number of reports have challenged this

belief by arguing that TCR-induced Ras activation

occurs at the Golgi in a PLCg1- and RasGRP1-

dependent fashion (Mor and Philips 2006). As Ras

proteins transit through endomembranes on their way

to the plasma membrane, there is the potential for

subcellular compartmentalization of Ras activation to

add another dimension of complexity to Ras signaling

and possible cellular responses (Fig. 2). Nevertheless,

other studies have found evidence solely for plasma

membrane-associated-Ras and -RasGRP1 upon TCR

and BCR ligation (Stone 2011), raising the question of

whether observations of active RasGRP1 and activated

Ras at internal membranes are an artifact of protein

overexpression or the tracking of fluorescently tagged

rather than native endogenous molecules.

An exquisite investigation monitoring endogenous

signaling molecules has suggested that subcellular

compartmentalization of the Ras/RasGRP1/ERK path-

way plays a key role in developing thymocytes under-

going selection (Daniels et al. 2006). Using OT-1 TCR

transgenic preselection CD4+CD8+ thymocytes and

specific peptides that span the boundary of positive

and negative selection, it was revealed that negatively

selecting peptides targeted Ras, ▶Raf-1, and

RasGRP1 to the plasma membrane, whereas these

molecules colocalized to endomembranes when

thymocytes were stimulated with peptides mediating

positive selection. Notwithstanding, as this study

determined the localization of total Ras rather than

active Ras (i.e., Ras-GTP), it is conceivable

that sufficient signaling necessary to mediate positive

selection is initiated via trace amounts of RasGRP1

and Ras that is situated at the plasma membrane.

A recent study utilizing novel high affinity probes

for Ras-GTP imaged in live Jurkat T cells was capable

of discerning the accumulation of endogenous Ras-

GTP solely at the plasma membrane (Rubio et al.

2010). In addition, the failure of a palmitoylation-

defective mutant of N-Ras that is restricted to

endomembranes to become activated upon TCR

stimulation further asserts that plasma membrane

localization is required for Ras activation. Future stud-

ies will require sophisticated tools like the one

discussed above and high-resolution microscopic

imaging to settle the debate over the localization of

active RasGRP1 and Ras-GTP.

RasGRP1 and Autoimmunity

T cells are a vital component of the body’s defense

system and their capacity to differentiate self- from

foreign-antigens is crucial to protect against both path-

ogenic challenge and autoimmune-mediated self-

destruction. The dependence of T-cell development,

T-cell function, and T-cell tolerance on TCR signaling

suggests that mutations affecting TCR signal transduc-

tion may cause a multitude of deleterious health-

related effects. Immunodeficiency may arise from

alterations to the TCR repertoire and T-cell function.

In addition, aberrant TCR signaling may promote auto-

immunity by influencing central- (deletion of

autoreactive T cells in the thymus) and peripheral-

T-cell tolerance (T-cell anergy, activation-induced

PKC

LCK PLCγ1

DAGCa2+

RasGRP1

RasGRP1 RasRas

RasGolgiapparatus

Nucleus

?

SOSGRB2

LAT

ZAP70

TCRCD3

RasGRP1

GTP GTP

GTP

GDP GDP

GDP

RasGRP1, Fig. 2 The activation of Ras by T-cell receptor

signal transduction. T-cell receptor stimulation induces the acti-

vation of the protein tyrosine kinases LCK (lymphoid cell

kinase) and ▶ZAP-70 (zeta-associated protein – 70 kDa) and

subsequently, phosphorylation of ▶LAT (Linker for Activated

T cells). Phosphorylated ▶LAT results in the GRB2 (Growth

factor Receptor-Bound protein 2)/SOS (Son of sevenless)

complex being targeted to the plasma membrane through

Grb2’s SH2 (Src homology 2) domain. Simultaneously, phos-

phorylated LAT also recruits PLCg1 (phospholipase Cg1),resulting in its activation. Activated PLC-g1 leads to the

production of DAG (diacylglycerol) and increases in cytosolic

calcium. DAG and perhaps rises in cytosolic calcium cause the

cytoplasmic protein RasGRP1 to become translocated, localiz-

ing at the plasma membrane or endomembranes such as the

Golgi apparatus. The concentrations of these two second mes-

sengers along with phosphorylation of RasGRP1 by PKC (pro-

tein kinase C) are speculated to determine the cellular site of

RasGRP1’s mobilization. The subcellular compartmentalization

of Ras signaling may serve to subject Ras to differential regula-

tion or to pair it with a unique subset of effectors

R 1616 RasGRP1

cell death [AICD] and suppression by regulatory

T cells). Importantly, abnormal Ras-ERK signaling in

T cells has been described in a number of autoimmune

diseases in humans and animal models.

One reported consequence of decreased activation

of Ras-ERK pathway in T cells is reduced DNA

methyltransferase I (DNMT1) expression causing the

derepression of autoimmune genes (Gorelik et al.

2007). Recently, two microRNAs, miRNA-21 and

miRNA-148a, overexpressed in T cells from both

patients with systemic lupus erythematosus (SLE)

and lupus-prone MRL/lpr mice have been found to

downmodulate DNMT1 directly and indirectly by

turning down Ras-ERK signaling and targeting

RasGRP1 transcripts (Pan et al. 2010). By contrast,

defective RasGRP1 expression in a subset of SLE

patients has been proposed to result from aberrant

RNA splicing (Stone 2011). Additionally,

dysregulated RasGRP1 expression has been implicated

in another autoimmune disease through genome-wide

association studies linking RasGRP1 variants to type 1

diabetes (Stone 2011).

The severely impaired T-cell maturation in the

thymus of young RasGRP1-deficient mice is corre-

lated with a small but activated population of

peripheral T cells, particularly of the CD4 lineage

(Layer et al. 2003; Priatel et al. 2007). However,

with age, RasGRP1-deficient mice (on a mixed

RasGRP1 1617 R

R

C57BL/6:129SvJ genetic background), derived

through a classical gene targeting approach

(RasGRP1�/�) and a spontaneous mouse mutant of

RasGRP1 (RasGRP1lag; lag is an acronym

representing lymphoproliferation-autoimmunity-

glomerulonephritis), were found to exhibit massive

lymphoproliferation and autoimmunity with similarity

to SLE (Layer et al. 2003). At 5 months of age,

RasGRP1lag and RasGRP1�/� mice displayed spleno-

megaly, lymphadenopathy, glomerulonephritis, lym-

phocytic infiltrates within many organs, elevated

antinuclear antibodies (ANAs), anorexia, and lethargy.

However, the penetrance of a severe autoimmune phe-

notype within one animal colony of RasGRP1�/�mice

disappeared after successive backcrossing of the

targeted mutation onto the C57BL/6 background.

C57BL/6 RasGRP1�/� mice remained lymphopenic

and free of severe autoimmune disease up to 1 year

of age despite high-serum ANA levels (Priatel et al.

2007). It is possible that genetic modifiers from the

129/SvJ genetic background or environmental factors,

such as distinct microfloral, may synergize with

RasGRP1-deficiency to promote fulminant disease.

The lack of RasGRP1 in developing thymocytes

may push the balance toward autoimmunity. It has

been proposed that DP thymocytes capable of matur-

ing into mature SP thymocytes need to express more

strongly self-reactive TCRs if they lack RasGRP1 to

overcome their signaling deficits (Priatel et al. 2002;

Layer et al. 2003). As TCR transgenic studies have

argued that RasGRP1 is not necessary for central tol-

erance (Priatel et al. 2002), the affinity/avidity of TCRs

expressed by RasGRP1�/� mature SP thymocytes

perhaps straggle the boundary between positive and

negative selection. In addition, RasGRP1 has been

shown to play a critical role in the formation of natural

Foxp3-expressing regulatory T cells, suggesting

that impaired development or function of this lineage

may contribute to disease in RasGRP1 mutant mice

(Stone 2011).

There are also a number of peripheral mechanisms

by which RasGRP1-deficiency may collude with

defective thymocyte development to cause disease.

Firstly, the lymphopenic compartment within

RasGRP1�/� mice that results from decreased thymic

output may favor oligoclonal T-cell outgrowth and

generation of T-cell effectors through abundance of

cytokines like IL-7 and increased availability of self-

peptides/self-MHC molecules. Notably, RasGRP1�/�

T cells have a distinct TCR repertoire relative to wild

type animals resulting from altered T-cell development

or peripheral T-cell homeostasis (Priatel et al. 2007).

Secondly, aberrant TCR signaling or TCR repertoire in

mature T cells may lead to weakened immune

responses, chronic infections, and proinflammatory

conditions. Viral challenge experiments demonstrated

that RasGRP1�/�mice generate drastically fewer anti-

gen-specific T cells and delayed pathogen clearance as

compared to wild type mice (Priatel et al. 2007).

Thirdly, the resistance to AICD exhibited by

RasGRP1-deficient T cells in vitro has been postulated

to enhance their pathogenicity in vivo by escaping

apoptosis (Layer et al. 2003). Fourthly, the function

or maintenance of regulatory T cells may be impacted

by diminished IL-2 production observed for

RasGRP1�/� T-cell effectors (Layer et al. 2003;

Priatel et al. 2010). Collectively, these findings suggest

multiple means by which aberrant RasGRP1 signaling

may enhance susceptibility to immunologic disease.

RasGRP1 and Cancer

Ras signaling regulates proliferation, differentiation

and survival and activating Ras mutations are present

in approximately 30% of all human cancers. As the

original descriptions of RasGRP1 documented

its capacity to transform rodent fibroblasts in vitro

(Stone 2011), it raises the question as to whether

altered RasGRP1 expression or activity can lead to

tumorigenesis. To date, findings from several studies

have supported this hypothesis.

The observation that the RasGRP1 is a frequent site

of proviral insertion in retrovirus-induced murine T-

cell lymphomas suggests that RasGRP1 can act as an

oncogene (Stone 2011). Corroborating these findings,

overexpression of RasGRP1 in the thymus was able to

initiate thymic lymphomas in a pre-TCR/TCR-

independent manner (Klinger et al. 2005). An investi-

gation into genes able to induce acute myeloid

leukemia (AML) found that RasGRP1 can act alone

as a leukemic initiator/driver or act in concert with

other leukemic causing genes to cause disease

(Vassiliou et al. 2011). In addition, therapeutically

targeting the Ras-ERK pathway using MEK inhibitors

in a mouse model of AML revealed that increased

RasGRP1 expression correlated with tumor resistance

to the drug, although the precise mechanism of action

R 1618 RasGRP1

remained unclear (Lauchle et al. 2009). Besides

its association with blood cancers, RasGRP1

overexpression in murine epidermal keratinocytes

led to spontaneous development of squamous cell

papillomas in the absence of chemical tumor initiators

(Diez et al. 2009). Consequently, RasGRP1 is

relevant to tumorigenesis in hematopoietic and

non-hematopoietic cells.

Summary

RasGRP1 is the prototypical member of the RasGRP

family of guanyl nucleotide exchange factors whose

function is to couple surface receptor signaling to the

activation of the small GTPase Ras and downstream

MAPK pathways. The activity of RasGRP1 is regu-

lated through its mobilization to membranes and

pairing with Ras by the second messenger DAG

(diacylglycerol). RasGRP family members are com-

posed of a REM (Ras exchange motif) and ▶CDC25

(cell division cycle 25)-related domains, functioning in

Ras recognition and catalysis of GDP exchange, and

“EF-hand” and DAG-binding “C1” motifs, serving to

modulate its recruitment to membranes. Initial inves-

tigations revealed that RasGRP1 links TCR signaling

to the activation of Ras-ERK pathway, playing crucial

roles in both T-cell development and mature T-cell

function. More recent studies have found that

RasGRP1mediates critical regulation of surface recep-

tor signaling and effector functions in B cells, NK

cells, and mast cells. Perturbations in RasGRP1 func-

tion are suspected to underlie immunodeficiency, auto-

immunity, and blood cell malignancies.

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2010;185:3536–43.

Ras-Related Associated with Diabetes 1619 R

Stone J. Regulation and function of the RasGRP family of Ras

activators in blood cells. Genes Cancer. 2011;2(3):320–34.

Vassiliou GS, Cooper JL, Rad R, Li J, Rice S, Uren A, Rad L,

Ellis P, Andrews R, Banerjee R, et al. Mutant nucleophosmin

and cooperating pathways drive leukemia initiation and

progression in mice. Nat Genet. 2011;43:470–5.

Rasl2-8

▶TLR4, Toll-Like Receptor 4

Ras-Related Associated with Diabetes

Jose-Luis Gonzalez de Aguilar

Laboratory of Molecular Signaling and

Neurodegeneration, INSERM, Faculty of Life

Sciences, University of Strasbourg, Strasbourg, France

Synonyms

Rad; Ras associated with diabetes; RRAD

R

Historical Background

Ras associated with diabetes (Rad) was identified in

the beginning of the 1990s as a clone differentially

expressed in two subtraction cDNA libraries prepared

from skeletal muscle of normal individuals and

patients with Type II (non-insulin-dependent) diabetes

mellitus (Reynet and Kahn 1993). Analysis of

the newly identified clone revealed about 50% identity

at the nucleotide level with members of the Ras super-

family, which consists of more than a hundred low-

molecular-weight guanine nucleotide–binding pro-

teins, also referred to as small GTPases. The major

feature of this class of molecules is their ability to

cycle between a GDP-bound inactive and a

GTP-bound active conformation. Small GTPases are

divided into six subfamilies: Ras, Rho, Arf, Rab,

▶Ran, and RGK, in which Rad is included. They

participate in important cellular processes such as

growth and differentiation, cytoskeletal dynamics,

membrane trafficking, vesicle transport, and signal

transduction. In their seminal report, Reynet and

Kahn observed that the expression of Rad is typically

highest in skeletal muscle, cardiac muscle, and lung of

normal individuals. They also found that Rad mRNA

levels appeared increased in Type II diabetes muscle as

compared to Type I diabetes or nondiabetic muscle

(Reynet and Kahn 1993). These findings provided the

framework for follow-up studies on the comprehension

of the biology of Rad in relation to muscle (patho)

physiology and cancer.

Structure and Regulation of the Activityof Rad

The human Rad gene is localized in chromosome

16q22, spans 3.75 kb, and is composed of five exons

and four introns. Translation presumably starts from an

in-frame ATG codon in the second exon, which gives

rise to a protein of 269 amino acids with a predicted

molecular weight of 29,266 kDa (Caldwell et al. 1996).

Rad is the prototype member of the small GTPases in

the RGK family, which also includes Rem, Rem2, and

Gem/Kir. From a structural point of view, Rad

possesses the five highly conserved GTPase domains

G1 to G5, characteristic of the Ras-related proteins, but

it also displays particular features distinct from that

commonly observed in other small GTPases. First, Rad

exhibits several nonconserved amino acids in G1, G2,

and G3 domains that may affect its GTPase function.

Second, Rad shows longer NH2 and COOH termini

of 88 and 31 amino acids, respectively. Third, The

COOH terminus of Rad does not present the typical

CAAX isoprenylation motif that usually facilitates the

attachment to cell membranes (Reynet and Kahn 1993).

Rad has been shown to interact with a variety of

proteins that can thereby determine its function and/or

subcellular distribution. For example, overexpressing

Rad in a neuroblastoma cell line triggers cellular flat-

tening and neurite extension, and it has been proposed

that these effects are the consequence of binding of

Rad to the Rho-associated protein kinase ROCK,

which is known to regulate the shape and movement

of cells by acting on the cytoskeleton. Such an inter-

action impedes ROCK activity which, in turn, results

in the inhibition of contraction and retraction (Ward

et al. 2002). The subcellular distribution of Rad greatly

depends on the interactions with the Ca2+-binding

protein calmodulin (CaM) and the multifunctional

regulatory protein 14-3-3. It has been reported that

R 1620 Ras-Related Associated with Diabetes

the lack of binding to CaM induces the accumulation

of Rad in the nucleus, whereas the association with

14-3-3 maintains Rad within the cytoplasm

(Mahalakshmi et al. 2007). Interestingly, Rad presents

several consensus phosphorylation sites in the

extended COOH terminus, which are close to the

CaM-binding domain. Rad can be phosphorylated on

serine residues by Ca2+-/CaM-dependent protein

kinase II (CaMKII), PKA, ▶ casein kinase II and

PKC. These post-translational modifications do not

seem to affect the ability of Rad to hydrolyze GTP

but alter its affinity to bind CaM and 14-3-3, thus

interfering with the subcellular localization of the pro-

tein (Moyers et al. 1998; Mahalakshmi et al. 2007).

Little is known about the mechanisms controlling

the expression of Rad at the transcriptional level. Early

studies indicated the localization of Rad to thin

filaments in skeletal muscle, as well as its increased

expression during myoblast fusion (Paulik et al. 1997).

In agreement with these observations, subsequent stud-

ies showed that several transcription factors involved

in myogenesis, including MEF2, MyoD, and Myf5,

stimulate the transcriptional activity of the Rad pro-

moter (Hawke et al. 2006). Increased Rad expression

has also been observed in experimental conditions

characterized by acute accumulation of toxic reactive

oxygen species in skeletal muscle, such as hind limb

ischemia–reperfusion and sciatic nerve axotomy–

induced muscle denervation (Halter et al. 2010).

2+

Role of Rad in Tissue Remodeling

In vascular proliferative diseases, such as atherosclero-

sis, the formation of vascular lesions ismainly provoked

by aberrant migration, attachment, and proliferation of

vascular smooth muscle cells. Multiple factors may

contribute to produce this pathological remodeling.

Using adenovirus-mediated gene delivery, it has been

reported that the expression of Rad is able to inhibit the

migration and attachment of vascular smooth muscle

cells, and that this effect is dependent on GTP loading.

Experimental evidence supports that Rad reduces the

formation of stress fibers and focal contacts necessary to

the remodeling process, by interfering with the Rho/

ROCK signaling pathway (Fu et al. 2005).

Pathological remodeling is also observed in heart in

response to injury and stress, and it leads to myocardial

hypertrophy and fibrosis, and subsequent heart failure.

The expression of Rad is normally high in the myocar-

dium but decreases in diseased heart. Transgenic mice

lacking the Rad gene are more prone to develop car-

diac hypertrophy, and this is associated with an

increase in the phosphorylation of CaMKII. Through

its interaction with CaMKII, it has been proposed that

Rad decreases the phosphorylation and activity of this

kinase, and hence reduces cardiac hypertrophy (Chang

et al. 2007). Rad-deficient mice also show severe myo-

cardial fibrosis. By binding the transcription factor

C/EBP-d, Rad impedes the expression of connective

tissue growth factor, which is a key stimulator of the

production of extracellular matrix leading to fibrosis.

Rad therefore acts as a negative regulator of fibrosis

in the heart (Zhang et al. 2011).

Role of Rad in Cancer

First reports indicated that Rad may be an oncogenic

protein, since it increases the rate of growth of breast

cancer cells in vitro. In addition, the ability of these

cells to trigger the formation of tumors in nude mice

is also exacerbated in the presence of Rad. The

oncogenic potential of Rad resides within the NH2

and COOH termini and seems not to be dependent on

its GTPase activity. However, the metastasis suppres-

sor gene, nm23, which stimulates GTP hydrolysis by

Rad, is able to diminish its tumor-promoting effect

(Tseng et al. 2001).

Other studies postulated that Rad is a tumor

suppressor factor, because its expression appears

frequently inhibited in lung and breast cancers

(Suzuki et al. 2007). In support of this notion, it has

been shown that Rad is a direct transcriptional target of

the tumor suppressor p53 and, thus, can reduce migra-

tion and invasiveness of cancer cells by acting

on cytoskeleton reorganization (Hsiao et al. 2011).

Moreover, Rad triggers the apoptotic death of

cardiomyocytes in vitro by stimulating the phosphor-

ylation of p38 MAPK and by decreasing the levels of

the pro-survival molecule Bcl-xL (Sun et al. 2011).

Role of Rad in Ca Channel Activity

Rad is a potent inhibitor of Ca2+ currents through

L-type voltage-dependent channels. This occurs via

the interaction of Rad with auxiliary CaVb subunits,

Ras-Related Associated with Diabetes 1621 R

which are involved in the trafficking toward the cell

surface of the principal subunits forming the channels.

The interaction between Rad and the CaVb subunit

prevents channel expression on the cell surface, by

sequestering CaVb subunits to the nucleus. The inter-

action depends on critical amino acid residues in the

COOH terminus, and is facilitated by the absence of

14-3-3 and CaM binding, both of which allow

relocalization of Rad within the cytoplasm (Finlin

et al. 2003; Beguin et al. 2006). On the other hand,

heart-specific overexpression of a dominant negative

form of Rad, that binds GDP but not GTP, leads to an

increase in the number of L-type voltage-dependent

Ca2+ channels on the cell surface and, subsequently,

cardiac arrhythmogenesis (Yada et al. 2007).

R

Role of Rad in Skeletal Muscle Metabolism,Development, and Disease

Rad was initially identified as being upregulated in

diabetic patients. Follow-up studies did not confirm

this finding, since Rad expression appeared normal in

particular populations of diabetic patients and in the

Zucker rat model of diabetes and obesity (Paulik et al.

1997). Of note, however, additional in vitro and in vivo

experimental evidence strongly suggests the implica-

tion of Rad in glucose metabolism. First,

overexpressing Rad in C2C12 and L6 myocyte cell

lines reduces insulin-stimulated glucose uptake

(Moyers et al. 1996). Second, using mice that

overexpress Rad in muscle, it was shown that the

increase in the expression of Rad acts synergistically

with a high-fat diet to induce insulin resistance as

observed in Type II diabetes (Ilany et al. 2006).

tumorige

migration ofsmooth muscle

and cancer cells

Ras-Related Associatedwith Diabetes,Fig. 1 Biological Functions

of Rad. Rad interacts with

many different proteins and

participates in a variety of

cellular processes. Interacting

proteins are shown in boxes.

Arrows indicate inhibitory (�)

or stimulatory (+) actions. See

text for further details

Rad is expressed during normal rat muscle devel-

opment and in regenerating muscle in response to

injury. Such an expression is located in the myogenic

progenitor cell population, as well as in the newly

regenerated myofibers as occurs in the mdx mouse

model of Duchenne muscular dystrophy (Hawke

et al. 2006). Extending these observations, it was also

found that Rad is upregulated in skeletal muscles

affected by the chronic neuromuscular degenerative

condition, amyotrophic lateral sclerosis. In this

disease, however, the upregulation of Rad is intimately

associated with muscle atrophy, since it takes place

within the myofibers that suffer from the degenerative

process (Halter et al. 2010).

Summary

Rad is a multifunctional GTPase involved in many

different molecular and cellular processes, mainly in

muscle tissue, during development, and under normal

adult and disease conditions. A major feature of Rad is

the ability to interact with a variety of proteins, which

determine not only the activity but also the specific role

of this GTPase in a given physiological or pathological

context (see Fig. 1). Rad can exert different inhibitory

and stimulatory actions and, intriguingly, it can exhibit

opposite roles for a particular situation. Thus, studies

on the contribution of Rad to cancer showed both pro-

and antitumoral activity. Not all the functions of Rad

have been completely deciphered, such as its implica-

tion in muscle metabolism. In addition, the molecular

mechanisms underlying Rad functions remain unclear

and sometimes controversial. Finally, further efforts

are needed to elucidate the regulatory factors, either

apoptosis

nicity

Rock

CaMKII

insulin resistance

glucose uptake

cardiacfibrosis

cardiachypertrophy

musclecontractionRad

GTPase

C/EBP−δ

Cavβ

- -

-

-

-

+

+ +

R 1622 Ras-Related Protein Rab-7a

extra- or intracellular, that control Rad actions. From

a pathological point of view, a better knowledge of

these aspects of the biology of Rad would provide the

basis for future therapeutic interventions.

References

Beguin P, Mahalakshmi RN, Nagashima K, Cher DH, Ikeda H,

Yamada Y, et al. Nuclear sequestration of beta-subunits by

Rad and Rem is controlled by 14-3-3 and calmodulin and

reveals a novel mechanism for Ca2+ channel regulation.

J Mol Biol. 2006;355:34–46.

Caldwell JS, Moyers JS, Doria A, Reynet C, Kahn RC. Molec-

ular cloning of the human rad gene: gene structure and

complete nucleotide sequence. Biochim Biophys Acta.

1996;1316:145–8.

Chang L, Zhang J, Tseng YH, Xie CQ, Ilany J, Br€uning JC, et al.Rad GTPase deficiency leads to cardiac hypertrophy.

Circulation. 2007;116:2976–83.

Finlin BS, Crump SM, Satin J, Andres DA. Regulation

of voltage-gated calcium channel activity by the Rem

and Rad GTPases. Proc Natl Acad Sci USA. 2003;100:

14469–74.

Fu M, Zhang J, Tseng YH, Cui T, Zhu X, Xiao Y, et al. Rad

GTPase attenuates vascular lesion formation by inhibition of

vascular smooth muscle cell migration. Circulation.

2005;111:1071–7.

Halter B, Gonzalez de Aguilar JL, Rene F, Petri S, Fricker B,

Echaniz-Laguna A, et al. Oxidative stress in skeletal muscle

stimulates early expression of Rad in a mouse model of

amyotrophic lateral sclerosis. Free Radic Biol Med.

2010;48:915–23.

Hawke TJ, Kanatous SB, Martin CM, Goetsch SC, Garry DJ.

Rad is temporally regulated within myogenic progenitor cells

during skeletal muscle regeneration. Am J Physiol Cell

Physiol. 2006;290:C379–87.

Hsiao BY, Chen CC, Hsieh PC, Chang TK, Yeh YC, Wu YC,

et al. Rad is a p53 direct transcriptional target that inhibits

cell migration and is frequently silenced in lung carcinoma

cells. J Mol Med (Berl).. 2011;89:481–92.

Ilany J, Bilan PJ, Kapur S, Caldwell JS, Patti ME, Marette A,

et al. Overexpression of Rad in muscle worsens diet-induced

insulin resistance and glucose intolerance and lowers plasma

triglyceride level. Proc Natl Acad Sci USA. 2006;103:

4481–6.

Mahalakshmi RN, NgMY, Guo K, Qi Z, Hunziker W, Beguin P.

Nuclear localization of endogenous RGK proteins and mod-

ulation of cell shape remodeling by regulated nuclear trans-

port. Traffic. 2007;8:1164–78.

Moyers JS, Bilan PJ, Reynet C, Kahn CR. Overexpression of

Rad inhibits glucose uptake in cultured muscle and fat cells.

J Biol Chem. 1996;271:23111–16.

Moyers JS, Zhu J, Kahn CR. Effects of phosphorylation on

function of the Rad GTPase. Biochem J. 1998;333:609–14.

Paulik MA, Hamacher LL, Yarnall DP, Simmons CJ, Maianu L,

Pratley RE, et al. Identification of Rad’s effector-binding

domain, intracellular localization, and analysis of expression

in Pima Indians. J Cell Biochem. 1997;65:527–41.

Reynet C, Kahn CR. Rad: a member of the Ras family

overexpressed in muscle of type II diabetic humans. Science.

1993;262:1441–4.

Sun Z, Zhang J, Zhang J, Chen C, Du Q, Chang L, et al. Rad

GTPase induces cardiomyocyte apoptosis through the

activation of p38 mitogen-activated protein kinase. Biochem

Biophys Res Commun. 2011;409:52–7.

Suzuki M, Shigematsu H, Shames DS, Sunaga N, Takahashi T,

Shivapurkar N, et al. Methylation and gene silencing of the

Ras-related GTPase gene in lung and breast cancers. Ann

Surg Oncol. 2007;14:1397–404.

Tseng YH, Vicent D, Zhu J, Niu Y, Adeyinka A, Moyers JS,

et al. Regulation of growth and tumorigenicity of breast

cancer cells by the low molecular weight GTPase Rad and

nm23. Cancer Res. 2001;61:2071–9.

WardY,YapSF,RavichandranV,Matsumura F, ItoM, Spinelli B,

et al. The GTP binding proteins Gem and Rad are negative

regulators of the Rho-Rho kinase pathway. J Cell Biol.

2002;157:291–302.

Yada H, Murata M, Shimoda K, Yuasa S, Kawaguchi H, Ieda M,

et al. Dominant negative suppression of Rad leads to QT

prolongation and causes ventricular arrhythmias via

modulation of L-type Ca2+ channels in the heart. Circ Res.

2007;101:69–77.

Zhang J, Chang L, Chen C, Zhang M, Luo Y, Hamblin M, et al.

Rad GTPase inhibits cardiac fibrosis through connective

tissue growth factor. Cardiovasc Res. 2011;91:90–8.

Ras-Related Protein Rab-7a

▶Rab7a in Endocytosis and Signaling

Ras-Related Protein Rab-8A

▶Rab8

Ras-Related Rab8

▶Rab8

RCN-1

▶Regulator of Calcineurin 1 (RCAN1)

Receptor Related to FPR (RFP)

▶ FPR2/ALX

2+

Recoverin 1623 R

Recoverin

Pavel P. Philippov and Evgeni Yu Zernii

Department of Cell Signalling, A.N. Belozersky

Institute of Physico-Chemical Biology,

M.V. Lomonosov Moscow State University,

Moscow, Russia

Synonyms

A-protein; Cancer-associated retinopathy antigen;

CAR-antigen; 23 kDa photoreceptor cell-specific

protein; p26; p26; S-modulin

R

Historical Background

In 1989, P. Philipopov’s group from M.V. Lomonosov

Moscow State University invented a method for puri-

fication of the visual G-protein transducin (Gt) and

some other G-proteins. The idea of the method

was based on the ability of visual rhodopsin to bind

and to release transducin in the absence and in the

presence of GTP, respectively. For this aim, a column

with delipidated visual rhodopsin immobilized on

Concanavalin A Sepharose was used. Chromatography

of a crude extract of bovine rod outer segments on

the column allowed one to obtain a set of transducin

subunits with a slight contamination of cGMP-

phosphodiesterase. Also, an admixture of an unknown

protein with an apparent molecular weight of 26 K

could be seen on the electrophoregram. The unknown

protein attracted the attention of the group since

the capability of binding to rhodopsin had been

a characteristic feature of several key photoreceptor

proteins, such as transducin, ▶ rhodopsin kinase, and

arrestin. That is why the group decided to study this

protein in more detail. The protein named “p26” was

purified to a homogeneous state and used to arise

specific antibodies. Screening of the retina and

a number of other tissues for the presence of p26 with

the use of the antibodies detected this protein only in

the retina, in particular in the photoreceptor layer. It

was also demonstrated that the amino acid sequence of

p26 exhibited several calcium binding sites of the

EF-hand type and the ability of p26 to bind Ca2+ was

confirmed by experiments with calcium-45. In

addition, p26 was suggested to be a Ca -specific regu-

lator of photoreceptor ▶ guanylate cyclase, a key

enzyme of photoreceptors recovery, and due to this

ability it was rechristened as “▶ recoverin.” Afterward,

a 26 K protein named “S-modulin” was purified from

frog rod outer segments and shown to have a primary

structure similar to bovine recoverin. Later it became

clear that the binding of recoverin to rhodopsin is not

quite specific as recoverin, due to its Ca2+-myristoyl

switch, is capable of binding to hydrophobic substances,

e.g., to Phenyl-Sepharose, in a Ca2+-dependent manner.

That recoverin is capable of activating guanylate

cyclase was, however, disproved in subsequent works.

The mistake in the initial assignment of the recoverin

function might apparently be explained by the presence

of endogenous guanylate cyclase activator(s), GCAP1

and/or GCAP2, in the recoverin preparations used in the

preceding works. Nevertheless, recoverin continues to

be considered as a participant of the photoreceptor

recovery but now as a Ca2+-sensor of rhodopsin kinase,

the enzyme catalyzing phosphorylation and thus desen-

sitization of the visual receptor rhodopsin (for reviews,

see Senin et al. 2002; Philippov et al. 2006). After the

discovery of recoverin, a large number of other EF-

hand-containing Ca2+-binding proteins were described,

which form a family of the neuronal calcium sensor

(NCS) proteins. The expression of the NCS proteins is

restricted within neurons and neuroendocrine cells, in

which these proteins provide a Ca2+-sensitivity to a

number of protein targets (for a review, see Burgoyne

and Weiss 2001; Philippov et al. 2006).

Another line of the recoverin research was started in

1987, when an antigen with an apparent molecular

weight of 23 K was found in sera of patients with

cancer-associated retinopathy (CAR). This antigen

named “CAR-antigen” was then purified from bovine

rod outer segments and shown to be identical to

recoverin. These works have sprung an intensive

recoverin study as a paraneoplastic antigen in cancer

(for reviews, see Senin et al. 2002; Adamus 2006;

Philippov et al. 2006). More recently, recoverin has

become the first member of a new group of cancer-

specific antigens designated as “cancer-retina anti-

gens.” In addition to recoverin, this group includes

several key retinal proteins, such as rhodopsin,

transducin, rhodopsin kinase, and some others. In

health, these proteins are highly specific for the retina,

but in cancer they can be expressed inmalignant tumors

localized outside the retina (Bazhin et al. 2007).

R 1624 Recoverin

Tissue and Cellular Distribution of Recoverin

Immunochemical analysis demonstrated the presence of

recoverin in the adult retina of all investigated species.

Among these are: man, bull, monkey, mouse, rat, rabbit,

frog, chameleon, and newt. In the case of the chicken

retina, contradictory data were obtained: recoverin-

positive immunoreaction was described in one case,

but it was not found in another work. In addition to the

retina, recoverin immunoreactivity was observed in the

ocular ciliary epithelium, pinealocytes of the pineal

organ, and rat olfactory epithelium. Within the retina,

recoverin-positive reaction was found in photoreceptor

cells as well as in higher order neurons (bipolar and

ganglion cells) of a number of species and in amacrine

cells of lamprey Lampetra fluviatilis. As already noted,recoverin is suggested to function in photoreceptors as a

Ca2+-sensor of rhodopsin kinase, but its role in neurons

different from photoreceptors and in tissues different

from the retina remains unknown (for reviews, see

Senin et al. 2002; Philippov et al. 2006).

Within photoreceptors, recoverin is detected in the

outer and inner segments, cell bodies, and synaptic

pedicles. Most of the protein is localized in rod inner

segments, with approximately 12% present in the

outer segments in the dark and less than 2% remaining

in that compartment in the light (Strissel et al. 2005).

Thus, light causes a reduction of recoverin in rod outer

segments, accompanied by its redistribution toward

rod synaptic terminals.

Recoverin Structure

Recoverin is a compact (23.4 K, 201 amino acids)

protein consisting of two globular N- and C-terminal

domains, separated by a short linker. N-terminal gly-

cine of recoverin is acylated predominantly with the

myristic acid residue (C14:0) or, to a lesser extent, with

one of the following fatty acid residues: C14:1 (5-cis),C14:2 (5-cis, 8-cis), or C12:0. Each of recoverin

domains contains a pair of potential Ca2+-binding

sites of the EF-hand type: in total, the recoverin

molecule contains four potential Ca2+-binding sites

that are disposed evenly along the amino acid chain

of the protein. Of these, only two – EF-hand 2 and

EF-hand 3 – are capable of binding calcium ions.

Whereas EF-hand 1 and EF-hand 4 are inactive in

this respect due to the following structural “defects”:

(1) in the sequences of EF-hand 1 and EF-hand 4,

residues of negatively charged amino acids critical

for the coordination of Ca2+ are missing in the 1st

and 3rd positions of the 12-mer Ca2+-binding loops,

(2) EF-hand 1 cannot accept the conformation needed

for the binding of calcium as P40 is present in the

fourth position of the EF-hand 1 loop, (3) EF-hand 4

contains a salt bridge between the side chains of the

K161 and G171 in the 2nd and 12th positions of

the Ca2+-binding loop, and (4) the highly conserved

glycine at position 6 of the loop is replaced by the

aspartic acid residue (D165) (for reviews, see Senin

et al. 2002; Philippov et al. 2006).

The comparison of the structural data for apo- and

Ca2+-containing forms of myristoylated recoverin

obtained by X-ray diffraction and NMR-spectroscopy

revealed structural changes in the protein molecule,

accompanying the binding of calcium (Fig. 1). In the

apo-form, the myristoyl moiety of recoverin is buried

into a deep hydrophobic cavity or hydrophobic

“pocket,” consisting of a cluster of aromatic and

other nonpolar amino acid residues (L28, W31, Y32,

F35, I44, F49, I52, Y53, F56, F57, Y86, L90, W104,

and L108) of the protein molecule. Binding of calcium

by recoverin leads to a 45� rotation of the N- and

C-terminal domains around G96 and to significant

conformational changes in the N-terminal domain. As

a result (1) initially antiparallel a-helices of EF-hand 2become perpendicular one to another and (2) a-helicesof EF-hand 1 turn around G42, allowing myristoyl

group to move outward from the hydrophobic environ-

ment. The consequence of these changes is the

exposure of the hydrophobic amino acid cluster of

the pocket and the myristoyl group of recoverin in

solution – the so-called “Ca2+-myristoyl switch”

mechanism. The exposed myristoyl group allows

recoverin to associate to membranes, while the

amino acid cluster of the pocket participates in the

interaction with the target enzyme, rhodopsin kinase

(G-protein-coupled receptor kinase 1, GRK-1) (for

reviews, see Senin et al. 2002; Philippov et al. 2006).

It should be added that the C-terminus of recoverin,

in addition to ten a-helices “A-J” normally present in

other NCS proteins, contains a variable C-terminal seg-

ment with an extra a-helix “K.” Recent studies have

demonstrated that C-terminal segment in recoverin is

involved in regulating its Ca2+-binding properties, as

well as in recognizing and regulating of rhodopsin

kinase (Weiergr€aber et al. 2006; Zernii et al. 2011).

2+

Recoverin, Fig. 1 Three-dimensional structures of recoverin.Ribbon diagrams represent different recoverin forms: (a) Ca2+-free recoverin. Image of 1iku.pdb (Tanaka et al. 1995) created

with PyMol v.0.99 (DeLano Scientific LLC); (b) Ca2+-boundrecoverin. Image of 1jsa.pdb (Ames et al. 1997) created with

PyMol v.0.99 (DeLano Scientific LLC); (c) recoverin in

a complex with peptide 1–25 of rhodopsin kinase. Image of

2i94.pdb (Ames et al. 2006) created with PyMol v.0.99 (DeLano

Scientific LLC). Structural elements are drawn in different

colors: EF-hand 1 and EF-hand 4 (blue), EF-hand 2 and

EF-hand 3 (red), N-terminal myristoyl group (green), calciumions (yellow), nonpolar amino acid residues of the hydrophobic

pocket (orange), and peptide 1–25 of rhodopsin kinase

(magenta)

Recoverin 1625 R

R

Molecular Properties of Recoverin

Recoverin molecule is characterized by a set of key

properties required for the signaling activity of the

protein. Among them the most important are calcium

binding, N-terminal myristoylation and the ability to

bind to phospholipid membranes. In recombinant

non-myristoylated recoverin, the binding of calcium

to EF-hands 2 and 3 occurs independently with

different affinities: Kd ¼ 6.9 and 0.11 mM, respec-

tively. In contrast, the binding of calcium to

recombinant myristoylated recoverin is

a cooperative sequential process (Hill coefficient

¼ 1.75), wherein EF-hand 3 is occupied first, facili-

tating the subsequent filling of EF-hand 2 (an apparent

Kd of the complex formed is equal to 17 mM). Thus,

N-terminal myristoylation confers onto recoverin the

cooperativity in calcium binding to EF-hands 2 and 3.

Also, the myristoyl residue significantly stabilizes

the conformation of the Ca2+-free protein during the

stepwise transition toward the fully Ca -occupied

state (for reviews, see Senin et al. 2002; Philippov

et al. 2006).

Myristoylated recoverin is capable of binding to

hydrophobic surfaces, such as the photoreceptor and

artificial lipid membranes. Depending on calcium

concentration, compartmentalization of recoverin

reversibly changes from a soluble Ca2+-free form to

a membrane-bound Ca2+-containing form. This pro-

cess is due to the mechanism of the Ca2+-myristoyl

switch that operates in recoverin: after EF-hand 3 is

filled by calcium, EF-hand 2 is subsequently filled,

which triggers the exposition of the myristoyl group

that attaches recoverin to the membrane. Solid-state

nuclear magnetic resonance studies revealed that the

Ca2+-bound protein is positioned on the membrane

surface so that its long molecular axis is oriented 45�

with respect to the normal membrane. The myristoyl

group is buried inside the membrane, whereas the

N-terminal region of recoverin points toward the

R 1626 Recoverin

membrane surface, with close contacts formed by

basic residues K5, K11, K22, K37, R43, and K84.

This orientation of the membrane-bound protein

allows an exposed hydrophobic crevice, near the mem-

brane surface, to serve as a binding site for the target

protein, rhodopsin kinase (Valentine et al. 2003). The

half-maximal binding of recoverin to photoreceptor

membranes in vitro occurs at 2.5 mM of a free calcium

concentration ([Ca2+]f), which is slightly out of the

physiological range of cytoplasmic [Ca2+]f. However,

extrapolation to in vivo conditions in rod outer

segments, which bear stacks of densely packed mem-

branes, reveals that the apparent affinity of recoverin

to calcium is in the submicromolar (i.e., physiological)

range of [Ca2+]f (for reviews, see Senin et al. 2002;

Philippov et al. 2006). The binding of recoverin to

membranes depends on their lipid composition: it is

enhanced with the elevation of the content of

phosphatidylserine (Senin et al. 2007), polyunsatu-

rated phospholipids (Calvez et al. 2011), and most

notably cholesterol. High cholesterol content in pho-

toreceptor disk membranes found at the base of rod

outer segments might favor the affinity of recoverin

to the membranes and shift its binding to the physio-

logical range of [Ca2+]f (for a review, see Philippov

et al. 2006). The Ca2+-dependence of the recoverin

binding to photoreceptor or artificial lipid mem-

branes is also regulated by the C-terminal segment of

recoverin, which serves as an internal modulator of its

Ca2+-sensitivity and functional activity (Weiergr€aberet al. 2006; Senin et al. 2007; Zernii et al. 2011).

Along with Ca2+-binding, which is a key molecular

property of recoverin, the protein can bind Zn2+ with

stoichiometry of 1:1 and apparent Kd of 30 and 7.1 mMfor apo- and Ca2+-loaded protein forms, respectively

(Permyakov et al. 2003). Also, recoverin molecules are

able to form a disulfide dimer and thiol oxidized mono-

mer under mild oxidizing conditions, using unique

C39 highly conserved within NCS family (Permyakov

et al. 2007). It is unclear yet, whether the above prop-

erties have the physiological significance.

Targets and Functions of Recoverin

A major intracellular target of recoverin in rod outer

segments is suggested to be rhodopsin kinase

(GRK-1). The filling of EF-hand 2 with calcium

(in myristoylated recoverin the filling of EF-hand 2

occurs only after EF-hand 3 is already filled) results

in the exposition of a cluster of the hydrophobic amino

acids that provides recoverin with an ability to interact

with rhodopsin kinase and thus inhibits the activity of

the enzyme. According to the surface plasmon reso-

nance studies, the half-maximal binding of rhodopsin

kinase to immobilized recoverin occurs at approxi-

mately 0.51 mM of rhodopsin kinase. Myristoylation

has a little effect on the binding of recoverin to the

kinase, but it shifts the half-maximal effect of calcium

on the binding from 150 nM for non-acylated recoverin

to 400 nM for myristoylated recoverin (for reviews,

see Senin et al. 2002; Philippov et al. 2006). Recoverin

binds to a region of residues 1–15 at the N-terminus

of rhodopsin kinase (Higgins et al. 2006). Nuclear

magnetic resonance studies of the complex between

Ca2+-bound recoverin and a N-terminal fragment of

rhodopsin kinase, residues 1–25 (RK1-25) revealed that

the hydrophobic face of the RK1–25 helix (L6, V9, V10,

A11, A14, and F15) interacts with an exposed hydro-

phobic groove on the surface of recoverin, lined

by side chains of the residues W31, F35, F49, I52,

Y53, F56, F57, Y86, and L90. In that structure,

the first eight residues of recoverin at the N-terminus

are solvent-exposed, enabling the N-terminal

myristoyl group to interact with target membranes

(Ames et al. 2006). The half-maximal inhibition

of rhodopsin kinase by recoverin is observed at

2–3 mM and 1.5–1.7 mM of calcium in the case of

non-acylated and myristoylated recoverin, respec-

tively. At saturating calcium concentrations, the

half-maximal inhibition of rhodopsin kinase occurs at

6.5–8 mM of non-myristoylated recoverin and at

0.8–3 mM of myristoylated recoverin, suggesting that

photoreceptor membranes enhance inhibitory effect of

recoverin upon rhodopsin kinase. The inhibition of

rhodopsin kinase by recoverin is facilitated when the

cholesterol content of membranes is increased. As the

cholesterol content in photoreceptor disk membranes

changes along the axis of rod outer segment from 5%

at the tip to 30% at the base, the above-mentioned

effect of cholesterol might be of physiological

importance (for reviews, see Senin et al. 2002;

Philippov et al. 2006). The activity of recoverin as

a Ca2+-sensor of rhodopsin kinase is also regulated

by the C-terminal segment of recoverin (Weiergr€aber

et al. 2006; Zernii et al. 2011).

Therefore, a number of the in vitro data suggest that

recoverin functions as a Ca2+-sensor of rhodopsin

Recoverin 1627 R

R

kinase in photoreceptor cells. According to these data,

at high calcium, corresponding to a dark state of pho-

toreceptor cells, rhodopsin kinase forms a complex

with recoverin and becomes inactive; at low calcium,

corresponding to the bleached state of photoreceptor

cells, the complex dissociates allowing activation of

the enzyme (for reviews, see Senin et al. 2002;

Philippov et al. 2006). However, the in vivo data on

recoverin function are contradictive. On the one hand,

recoverin is suggested to be implicated in the light

adaptation of photoreceptor cells by Ca2+-dependent

prolongation of the photoresponse due to (1) regulating

the lifetime of photoactivated rhodopsin through

feedback on rhodopsin kinase and (2) regulating the

speed of the light-induced change of [Ca2+]f through

a Ca2+-buffering mechanism (for a review, see

Philippov et al. 2006). On the other hand, there are

data suggesting that the effect of recoverin on the

photoresponse could not be explained by its effect

on phototransduction as such. Instead, the prolonged

signal transmission that enhances visual sensitivity is

the effect of recoverin downstream of photo-

transduction in rods (Sampath et al. 2005). Recent

data have advanced a new argument in favor of

recoverin as a Ca2+-sensor of rhodopsin kinase

in vivo (Chen et al. 2010). According to these data,

(1) background light accelerates inactivation of photo-

excited rhodopsin and (2) recoverin is required for the

light-dependent modulation of the photoexcited rho-

dopsin lifetime, probably due to its capability to regu-

late rhodopsin kinase in a Ca2+-dependent manner.

The function(s) of recoverin in the structures differ-

ent from photoreceptor outer segments still remains

unknown, but it is possible that the following data

could help to provide a clue to this issue. In the ribbon

synapse of photoreceptors, recoverin is co-localized

with membrane palmitoylated protein-4 (MPP4),

a retina-specific scaffolding protein, which has been

implicated in organizing presynaptic protein com-

plexes. Western blot analysis of bovine retinal

anti-recoverin precipitates detects co-precipitating

MPP4, supporting an association between the MPP4-

containing protein complex and recoverin in vivo.

However, immunoprecipitation experiments do not

show a direct interaction between recoverin and

MPP4 in 293-BNA cells co-transfected with both pro-

teins (F€orster et al. 2009). More recently, pull-down

assay and surface plasmon resonance study have

revealed a neuron-specific Ca2+-binding protein

caldendrin as a potential target for recoverin in retinal

bipolar cells and pineal gland. In particular, both

proteins are co-localized in these structures and an

increase of intracellular calcium facilitates the translo-

cation of caldendrin to intracellular membranes,

which is under control of the complex formation with

recoverin (Fries et al. 2010).

Recoverin in Cancer

In health, the expression of recoverin is mainly

restricted within the retina. In cancer, recoverin can

also be a paraneoplastic (or onconeural) antigen which

expressed in tumors localized outside the nervous

system. The aberrant expression of recoverin in malig-

nant cells causes an autoimmune response in some

cancer patients what is followed by the development

of paraneoplastic retina degeneration or cancer-

associated retinopathy, CAR. Autoantibodies against

recoverin (AAR) are detected in patients with different

kinds of cancer (for reviews, see Adamus 2006; Bazhin

et al. 2007).

A model of antibody-induced apoptosis of photore-

ceptor cells, underlying the CAR syndrome, has been

proposed (for a review, see Adamus 2006). Serum

AAR should be in sufficiently high titers to enter the

eye and cause retinopathy. Circulating AAR cross the

blood-retinal barrier and penetrate into retinal layers,

where AAR attack photoreceptors which express

recoverin. AAR then penetrate into retinal cells by an

active process of endocytosis. Once in the cell, AAR

block the recoverin function in phototransduction,

which results in the enhancement of rhodopsin phos-

phorylation and an increase in the concentration

of intracellular calcium ions. The high intracellular

calcium activates the mitochondria-dependent and

caspase-9-dependent activation of caspase 3, leading

to DNA fragmentation and cell death.Massive death of

photoreceptor cells leads to retinal dysfunction and

degeneration.

The CAR syndrome, similar to other paraneoplastic

neurological syndromes, is a very rare event: its occur-

rence is of the order of 1%. However, underlying AAR

might occur much more frequently (Bazhin et al.

2004). An important feature of the CAR syndrome, as

well as other paraneoplastic syndromes, is that it can

be manifested long before the clinical diagnosis of the

underlying tumor (for a review, see Adamus 2006).

R 1628 Recoverin

Such a feature of the CAR syndrome and underlying

AAR could be useful to clinicians to predict the future

development of a particular cancer.

Summary

Recoverin, initially named “p26,” is a Ca2+-binding

protein with a predominantly retinal localization,

which belongs to the neuronal calcium sensor (NCS)

protein family. The recoverin molecule consists of

201 amino acid residues and contains four poten-

tial EF-hand Ca2+-binding sites, of which only two –

EF-hands 2 and 3 – are capable of binding calcium.

The N-terminus of recoverin is acylated, mainly

myristoylated. Due to the mechanism of the Ca2+-

myristoyl switch, compartmentalization of recoverin

is changed from a soluble Ca2+-free form to

a membrane-bound Ca2+-containing form, and vice

versa, depending on an external calcium concentration.

In the Ca2+-free form, the N-terminal myristoyl moiety

of recoverin is buried into the hydrophobic pocket of

the protein; on calcium binding, the myristoylated

N-terminus is exposed, providing membrane associa-

tion of recoverin. Recoverin is suggested to operate as

a Ca2+-sensor of rhodopsin kinase (G-protein-coupled

receptor kinase 1, GRK-1), which catalyzes phosphor-

ylation and thus desensitization of the visual receptor

rhodopsin. In cancer, recoverin can also be

a paraneoplastic (or onconeural) antigen, the aberrant

expression of which in malignant tumors of some

patients causes an autoimmune response and the devel-

opment of paraneoplastic retina degeneration or

cancer-associated retinopathy. An important feature

of the CAR syndrome and underlying autoantibodies

against recoverin is that they can be detected long

before the clinical diagnosis of the corresponding

tumor. Such a feature of the autoantibodies could be

useful to clinicians to predict the future development

of a particular cancer.

References

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Philippov PP, Koch KW, editors. Neuronal calcium sensor

proteins. New York: Nova; 2006. p. 181–99.

Ames JB, Ishima R, Tanaka T, Gordon JI, Stryer L, Ikura M.

Molecular mechanics of calcium-myristoyl switches. Nature.

1997;389:198–202.

Ames JB, Levay K, Wingard JN, Lusin JD, Slepak VZ. Struc-

tural basis for calcium-induced inhibition of rhodopsin

kinase by recoverin. J Biol Chem. 2006;281:37237–45.

Bazhin AV, Savchenko MS, Shifrina ON, Demoura SA, Chikina

SY, Jaques G, Kogan EA, Chuchalin AG, Philippov PP.

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occurrence of anti-recoverin autoantibodies in sera and

recoverin in tumors. Lung Cancer. 2004;44:193–8.

Bazhin AV, Schadendorf D, Willner N, De Smet C,

Heinzelmann A, Tikhomirova NK, Umansky V,

Philippov PP, Eichm€uller SB. Photoreceptor proteins as

cancer-retina antigens. Int J Cancer. 2007;120:1268–76.

Burgoyne RD, Weiss JL. The neuronal calcium sensor family

of Ca2+-binding proteins. Biochem J. 2001;353:1–12.

Calvez P, Demers E, Boisselier E, Salesse C. Analysis of the

contribution of saturated and polyunsaturated phospholipid

monolayers to the binding of proteins. Langmuir. 2011;27:

1373–9.

Chen CK, Woodruff ML, Chen FS, Chen D, Fain GL. Back-

ground light produces a recoverin-dependent modulation of

activated-rhodopsin lifetime in mouse rods. J Neurosci.

2010;30:1213–20.

F€orster JR, Lochnit G, St€ohr H. Proteomic analysis of the mem-

brane palmitoylated protein-4 (MPP4)-associated protein

complex in the retina. Exp Eye Res. 2009;88:39–48.

Fries R, Reddy PP, Mikhaylova M, Haverkamp S, Wei T,

M€uller M, Kreutz MR, Koch K-W. Dynamic cellular trans-

location of caldendrin is facilitated by the Ca2+-myristoyl

switch of recoverin. J Neurochem. 2010;113:1150–62.

Higgins MK, Oprian DD, Schertler GF. Recoverin binds exclu-

sively to an amphipathic peptide at the N terminus of

rhodopsin kinase, inhibiting rhodopsin phosphorylation

without affecting catalytic activity of the kinase. J Biol

Chem. 2006;281:19426–32.

Permyakov SE, Cherskaya AM, Wasserman LA, Khokhlova TI,

Senin II, ZargarovAA, ZinchenkoDV, Zernii EY, LipkinVM,

Philippov PP, Uversky VN, Permyakov EA. Recoverin is

a zinc-binding protein. J Proteome Res. 2003;2:51–7.

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Zinchenko DV, Lipkin VM, Uversky VN, Permyakov EA.

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Philippov PP, Senin II, Koch K-W. Recoverin: a calcium-

dependent regulator of the visual transduction. In: Philippov

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Sampath AP, Strissel KJ, Elias R, Arshavsky VY, McGinnis JF,

Chen J, Kawamura S, Rieke F, Hurley JB. Recoverin

improves rod-mediated vision by enhancing signal transmis-

sion in the mouse retina. Neuron. 2005;46:413–20.

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control of rhodopsin phosphorylation: recoverin and rhodop-

sin kinase. Adv Exp Med Biol. 2002;514:69–99.

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Biochem. 2007;8:24.

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Regulator of Calcineurin 1 (RCAN1) 1629 R

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435:441–50.

Reggie-1 (Reg-1)

▶ Flotillin-2 (FLOT2)

Reggie-2 (Reg-2)

▶ Flotillin-1 (flot1)

R

Regulator of Calcineurin 1 (RCAN1)

Masakazu Fujiwara and Mohammad Ghazizadeh

Department of Molecular Pathology, Institute of

Development and Aging Sciences, Graduate School of

Medicine, Nippon Medical School, Kawasaki,

Kanagawa, Japan

Synonyms

ADAPT78; Calcipressin1; CBP1; Down syndrome

candidate region 1 (DSCR1); Down syndrome critical

region 1 (DSCR1); MCIP1; Nebula; RCN-1; Sarah

Historical Background

Regulator of calcineurin 1 (RCAN1) was first isolated

by Fuentes et al. in 1995 during a search for genes

associated with clinical features of Down syndrome

(e.g., mental retardation and congenital heart disease)

(Fuentes et al. 1995). Coding sequences of RCAN1

were identified from the 21q22.1–q22.2 region of

human chromosome 21 by an Alu-splice PCR method.

It was initially thought that RCAN1 was significantly

associated with the Down syndrome phenotype

(Fuentes et al. 1997b), thus RCAN1 was first desig-

nated Down Syndrome Critical Region 1 (DSCR1).

Future studies showed that the DSCR1 gene product

is a calcineurin regulator, and the new name “regulator

of calcineurin (RCAN)” was adopted to describe the

gene function. Although RCAN1 is still referred to as

DSCR1, ADAPT78, MCIP1, Calcipressin1, RCN-1,

Nebular, Sarah, or CBP1, both the HUGO Gene

Nomenclature Committee (HGNC) and the Mouse

Genomic Nomenclature Committee (MGNC) (Davies

et al. 2007) have adopted the RCAN1 nomenclature.

RCAN1 is an endogenous inhibitor of the serine

phosphatase calcineurin (Fuentes et al. 2000; Gorlach

et al. 2000; Kingsbury and Cunningham 2000).

Calcineurin is a heterodimer that is composed of the

catalytic subunit calcineurin A (CnA) and regulatory

subunit calcineurin B (CnB). RCAN1 directly binds

to CnA and inhibits the catalytic activity of

calcineurin. Calcineurin controls the phosphorylation/

dephosphorylation of several transcription factors,

such as the nuclear factor of activated T cells

(NFAT) (Crabtree and Olson 2002), CREB (Liu and

Graybiel 1996), and the MEF2 transcription factor

families (Mao and Wiedmann 1999). Dephosphoryla-

tion of NFAT by activated calcineurin promotes

translocation of NFAT to the nucleus, where the tran-

scription factor binds to DNA and activates gene

transcription (Crabtree and Olson 2002). A wide vari-

ety of physiological processes, such as lymphocyte

activation (Fruman et al. 1995), neurite outgrowth

(Chang et al. 1995), aging (Mair et al. 2011), heart

development (Yang et al. 2000), skeletal muscle fiber

type differentiation (Olson and Williams 2000), and

cardiac function (Ryeom et al. 2003) are regulated by

calcineurin-activated transcription factors. Thus,

RCAN1, which controls calcineurin activity, regulates

various physiological functions.

RCAN1 is highly conserved fromunicellular eukary-

otes to multicellular animals, and orthologs have

been identified in Saccharomyces cerevisiae (Kingsbury

and Cunningham 2000), Cryptococcus neoformans(Gorlach et al. 2000), Caenorhabditis elegans

1 5432 76

4

1

RCAN1-1L

RCAN1-4

RCAN1-1S

252 amino acids

197 amino acids

197 amino acids

Regulator of Calcineurin 1 (RCAN1), Fig. 1 The human

RCAN1 mRNA isoforms. The 252-amino acid RCAN1-1 L,

197-amino acid RCAN1-1 S, and 197-amino acid RCAN1-4

each have different ATG start codons

R 1630 Regulator of Calcineurin 1 (RCAN1)

(Lee et al. 2003), and Drosophila melanogaster (Chang

et al. 2003). The human RCAN1 gene contains seven

exons, where exons 1–4 can be alternatively transcribed

or spliced to produce differentmRNA isoforms (Fuentes

et al. 1997a). Among the four potential transcripts, the

major transcripts are RCAN1-1 and RCAN1-4, which

have exon 1 and exon 4 for the first exon, respectively

(Fig. 1). Additionally, there are two start codons in exon

1; the long formofRCAN1-1,which encodes 252 amino

acids, is referred to as RCAN1-1 L (Genesca et al.

2003), and the short form of RCAN1-1, which encodes

197 amino acids, is referred to as RCAN1-1 S (Fuentes

et al. 1997a).

RCAN1 Expression

RCAN1 is highly expressed in the human fetal brain

and adult heart. Lower expression levels have been

detected in the adult brain, lung, liver, skeletal muscle,

kidney, and pancreas. The fetal lung, liver, kidney, and

placenta also have low RCAN1 expression (Fuentes

et al. 1995). RCAN1 expression in rat and mice is

similar to the associated human tissues, and there is

high expression in the rodent brain and heart (Fuentes

et al. 1995, 1997a). In the rat brain, the in situ hybrid-

ization signal for RCAN1 expression was high in the

olfactory bulb, the piriform cortex, the dentate granule

cell layer, the pyramidal cell layer of the hippocampus,

the striatum and the cerebellar cortex. No signal for

RCAN1 expression was detected in the white matter

(Fuentes et al. 1995). The brains of 2–7-day-old

neonatal rats had higher RCAN1 expression in the

neocortex and the hypothalamus compared to adults

rat older than 16 days (Fuentes et al. 1995).

RCAN1 expression is diverse throughout many

different cell and tissue types and is regulated by

various stimuli. Vascular endothelial growth factor

(VEGF) (Minami et al. 2004; Yao and Duh 2004),

thrombin (Minami et al. 2004), oxidative stress

(Crawford et al. 1997), Ca2+-mediated stress (Cano

et al. 2005), ß-amyloid fragments (Ermak et al.

2001), TNFa (Minami et al. 2004; Yao and

Duh 2004), and endoplasmic reticulum stress (Zhao

et al. 2008) have been reported to induce RCAN1

expression. Oxidative stress in hamster HA-1 cells

induced RCAN1 expression as early as 90 min after

peroxide exposure, indicating that the transcriptional

response of RCAN1 is rapid and robust. A previous

study indicated that the maximal increase in RCAN1

expression was 7.8-fold after 5 h of initial exposure

(Crawford et al. 1997). Similarly, rapid and robust

RCAN1 expression occurs with other types of stimuli,

such as VEGF and thrombin. After 1 h of treatment,

VEGF and thrombin stimulation increased RCAN1

expression in human endothelial cells by 22.3-fold

and 17.7-fold, respectively (Minami et al. 2004).

RCAN1 transcription is regulated via a negative

feedback loop with RCAN1-4, which is an isoform

that is abundant in the fetal kidney, adult heart,

placenta, and skeletal muscle (Ermak et al. 2002;

Fuentes et al. 1997a, 2000). RCAN1 expression is

induced by the transcription factor NFAT, and NFAT

activation and nuclear translocation are regulated by

calcineurin phosphatase activity. Therefore, an exces-

sive amount of RCAN1 inhibits calcineurin and atten-

uates the calcineurin–NFAT signaling pathway,

thereby establishing a negative feedback loop that

suppresses its own expression. Calcineurin/NFAT

signal-dependent RCAN1-4 transcription is controlled

by a promoter region that is located upstream of exon 4

(between nucleotides �350 and �166); this promoter

region also contains putative NFAT and AP-1 binding

Regulator of Calcineurin 1 (RCAN1) 1631 R

sites (Cano et al. 2005; Yang et al. 2000; Zhao et al.

2008). In contrast, RCAN1-1 L, which is the isoform

that is predominantly expressed in the fetal and adult

brains (Ermak et al. 2002; Fuentes et al. 1997a, 2000)

is controlled by a conserved muscle-specific CAT

(M-CAT) site located 1,426 bp upstream of exon 1

(Liu et al. 2008). Transcription enhancer factor 3

directly interacts with the M-CAT site in the

promoter and is required for RCAN1-1 L expression

(Liu et al. 2008).

R

Inhibitory and Stimulatory Effects of RCAN1

RCAN1 is an endogenous inhibitor of calcineurin;

RCAN1 overexpression inhibits NFAT-mediated

calcineurin signaling by directly binding to the cata-

lytic subunit of calcineurin (Fuentes et al. 2000).

However, RCAN1 gene disruption also inhibits

calcineurin–▶NFAT signaling instead of stimulating

the signaling. In yeast, disruption of the RCAN1orthologus gene (RCN1) results in significantly

decreased calcineurin–NFAT signaling (Kingsbury

and Cunningham 2000). In RCAN1�/� mice,

calcineurin activity is decreased in response to cardiac

hypertrophy induced by pressure overload (Vega et al.

2003). These conflicting results suggest that reciprocal

effect of RCAN1 to the calcineurin activities, as

high RCAN1 expression is associated with calcineurin

inhibition, while physiological levels of RCAN1

expression are associated with calcineurin stimulation.

A number of reports have indicated that phosphor-

ylation of RCAN1 is important for RCAN1-mediated

biphasic effects on calcineurin. Using both human

and yeast RCAN1, Hilioti et al. showed that RCAN1-

mediated calcineurin stimulation requires phosphory-

lation at the conserved FLISPPxSPP motif when

RCAN1 is expressed at low concentrations (Hilioti

et al. 2004). Moreover, Liu et al. also showed that

phosphorylation of low concentrations of RCAN1 acti-

vates calcineurin–NFAT signaling (Liu et al. 2009).

Because the phosphorylated FLISPPxSPP RCAN1

motif is rapidly degraded by the SCFCdc4 ubiquitin

ligase complex (Genesca et al. 2003; Kishi et al.

2007), it has been hypothesized that phosphorylated

RCAN1 rapidly degrades and releases calcineurin

from the inactive complex (Kishi et al. 2007).

RCAN1-mediated stimulation of calcineurin activity

is observed only at physiological RCAN1 expression

levels because the net balance of phosphorylated

RCAN1 is much higher. On the other hand,

when RCAN1 is overexpressed, a majority of the

protein is not phosphorylated; this stable RCAN1

binds to calcineurin, which covers the catalytic domain

and inhibits phosphatase activity. Overexpressed

RCAN1 also increases calcineurin proteolysis

(Lee et al. 2009), which also contributes to RCAN1-

mediated inhibition. Collectively, RCAN1 inhibits

calcineurin signaling by binding to calcineurin and

interfering with substrate binding; phosphorylated

RCAN1 has a shorter half-life and appears to be stim-

ulatory when expressed at low levels.

Domains and Motifs of RCAN1

Structural and functional analysis of RCAN1 indicates

that specific domains or motifs are required to modu-

late calcineurin activity (Fig. 2). Both the N- and

C-terminals of RCAN1 contain motifs that can bind

to calcineurin and inhibit calcineurin catalytic activity

(Fuentes et al. 2000; Vega et al. 2002). In the

C-terminal, the PxIIxT motif at exon 7 is required for

calcineurin binding and inhibition (Chan et al. 2005).

The CIC motif (consensus sequence in mammals is

LGPGEKYELHA(G/A)T(D/E)(S/T)TPSVVVHVC

(E/D)S) at the C-terminal is also necessary for inhibi-

tion of calcineurin–NFAT signaling (Mulero et al.

2009). The PxIxIT-like motif within the CIC motif

efficiently blocks calcineurin activity by binding to

the calcineurin surface with the help of the LxxP

motif when RCAN1 is overexpressed. At the N-termi-

nal, a domain that resembles three-dimensional struc-

tures of RNA recognition motifs binds and inhibits

calcineurin activity (Mehta et al. 2009).

The PxIxIT-like and LxxP motifs appear to be

important for the stimulation of calcineurin signals

at low RCAN1 expression level in addition to their

roles in inhibition at high expression level. The TxxP

motif, which is adjacent to the PxIIxT motif at exon 7,

is only required for the stimulatory effects (Mehta

et al. 2009). Together with the FLISPPxSPP motif

phosphorylation site (see section “Inhibitory and

Stimulatory Effects of RCAN1”), these motifs are

important for RCAN1-mediated stimulation of

calcineurin signaling (Fig. 2).

Regulator of Calcineurin 1 (RCAN1), Fig. 2 The RCAN1

motifs and aligned amino acid sequences of the LxxP,

FLISPPxSPP, PxIxIT-like, PxIIxT, and TxxP motifs in mouse

RCAN1-4 (mRCAN1-4), rat RCAN1 (rRCAN1), human

RCAN1-4 (hRCAN1-4), mouse RCAN1-1 (mRCAN1-1), and

human RCAN1-1 (hRCAN1-1)

R 1632 Regulator of Calcineurin 1 (RCAN1)

Role of RCAN1 in Angiogenesis

Patients with Down syndrome have an extremely low

incidence of solid tumors, which indicates that RCAN1

plays an important role in both angiogenesis and tumor

development (Hasle 2001). RCAN1 gain-of-function

studies show that constitutive expression of RCAN1 in

endothelial cells impairs NFAT nuclear localization,

proliferation, and tube formation. RCAN1 also reduces

vascular density in Matrigel plugs and melanoma tumor

growth inmice (Minami et al. 2004).Another studywith

transgenic mice with three copies of RCAN1 showed

that these mice have significantly suppressed growth of

Lewis lung carcinoma and B16F10 melanoma cells

in vivo. Moreover, a study by Baek et al. showed that

a modest increase in RCAN1 expression (2.4-fold

increase in mRNA relative to littermate controls) is

sufficient for tumor growth suppression (Baek et al.

2009). In Xenopus laevis, overexpression of RCAN1

decreased the number of branching points that sprouted

from intersomitic vessels and decreased the vascular

density of the microvessels (Fujiwara et al. 2011).

Consistent with the previous loss-of-function

studies, studies in RCAN1�/� mice have shown that

RCAN1 inhibits angiogenesis. RCAN1 deletion

suppressed subcutaneous and metastatic tumor growth,

and endothelial cells isolated from these knockout

mice showed decreased VEGF-induced proliferation

(Ryeom et al. 2008). Specific knock down of RCAN1

expression by antisense oligonucleotides also inhibited

VEGF-stimulated migration of endothelial cells

(Iizuka et al. 2004). Researchers have hypothesized

that the appropriate RCAN1 expression level, as well

as the phosphorylation state influences RCAN1

regulation of calcineurin activity. High RCAN1

expression may reduce calcineurin activity and block

proliferation of endothelial cells, whereas low RCAN1

expression may hyperactivate calcineurin activity and

trigger apoptosis (Ryeom et al. 2008).

RCAN1 and Down Syndrome

Because RCAN1 is highly expressed in the brains of

Down syndrome patients, it is thought to be associated

with the Down syndrome phenotype (Fuentes et al.

1995). Overexpression of the Drosophila ortholog of

RCAN1 (nebula) causes neuronal defects that are

similar to Down syndrome, such as impaired synaptic

development, synaptic terminal structure, vesicle

recycling, and locomotor activity (Chang and Min

2009). Additionally, reduced or overexpression of neb-

ula impairs mitochondrial enzyme activity, the number

and size of mitochondria, and accumulation of toxic

reactive oxygen species (ROS) in the fly brains, which

are all characteristic of pathologies associated with

Down syndrome (Chang and Min 2005). These results

strongly suggest that altered expression of RCAN1

contributes to the neurological defects in Down

syndrome. Furthermore, cardiac defects, which are

another common feature associated with Down syn-

drome, have been reported in RCAN1/DYRK1A double

transgenic mice. A craniofacial defect was also

reported in Nfatc2�/�/Nfatc4�/� double-knockout

mice, which have impaired RCAN1-calcineurin–

NFAT signaling (Arron et al. 2006). These results

indicate that RCAN1 is an important gene that is asso-

ciated with the Down syndrome phenotype.

Regulator of Calcineurin 1 (RCAN1) 1633 R

RCAN1 and Alzheimer’s Disease

Overexpression of RCAN1 has been observed in

Alzheimer’s disease patient brains, where RCAN1

expression was twofold higher in the cerebral cortex

and threefold higher in the hippocampus (Ermak et al.

2001). Of the RCAN isoforms that are expressed in the

brain, RCAN1-1 L is upregulated in the neurons of

Alzheimer’s disease patients (Harris et al. 2007).

Overexpressed RCAN1 colocalizes with the neurode-

generative disease-associated proteins huntingtin

(Q148) and ataxia-3 (Q84) in cultured primary neurons

(Ma et al. 2004). Additionally, RCAN1 expression is

directly stimulated by the aggregated amyloid Aß

peptide, which is a peptide that plays a role in neuronal

degeneration in Alzheimer’s disease and human

neuroblastoma cell lines (Ermak et al. 2001).

Functional analyses of RCAN1 further indicate

that it is closely associated with Alzheimer’s

disease. Hypomorphic fly mutants with RCAN1

overexpression have decreased long-term memory

compared to control D. melanogaster, which display

40% memory retention 24 h after training. Corre-

spondingly, calcineurin activity is 40% higher in

these RCAN1 mutants (Chang et al. 2003). Inside the

cell, RCAN1 regulates the number of vesicles

undergoing exocytosis and the speed of vesicle fusion,

which opens and closes the pore (Keating et al. 2008).

These data indicate that RCAN1 is highly associated

with neuronal memory and learning and is thus a strong

candidate for Alzheimer’s disease neuropathology.

R

Other Unique Functions of RCAN1

Interestingly, female-specific RCAN1 functions have

been reported in non-vertebrates, such as C. elegansand D. melanogaster. In C. elegans, the RCAN1

ortholog (RCN-1) is expressed in the vulva epithelial

and muscle cells, and overexpression of RCN-1 results

in egg retention (Lee et al. 2003). Calcineurin null

mutants, which carry a large deletion in the calcineurin

B-regulatory subunit gene, have similar defects in fer-

tility and egg-laying. In D. melanogaster, the RCAN1

ortholog (sarah) is expressed in the oocytes and nurse

cells of normal flies and is critical for ovulation and

female courtship behavior. Inhibition of sarah expres-

sion decreases the number of eggs laid, and a

majority of the eggs arrest at metaphase I of meiosis

(Ejima et al. 2004). Moreover, misexpression affects

female courtship behavior, andD. melanogaster virgin

mutant females frequently display extrusion behavior

(Ejima et al. 2004). These studies indicate that RCAN1

may exert female-specific effects via regulation of

calcineurin signaling.

Summary

RCAN1 plays an important role in the cell by regulating

the multifunctional phosphatase calcineurin. RCAN1

function remains an area of ongoing investigation,

although a number of reports have identified several

RCAN1molecular mechanisms. However, there is little

information regarding the functional difference(s)

between the RCAN1 isoforms, which are differentially

expressed by various human tissues. Previous reports

have indicated that RCAN1-1 L overexpression acti-

vates the transcription factor NFAT and promotes path-

ologic angiogenesis in human endothelial cells, while

RCAN1-4 inhibits angiogenesis (Qin et al. 2006). The

mechanism for these opposing effects of the two

isoforms remains unknown because this data does not

agree with the current model of dual RCAN1 function.

Moreover, additional analyses of the nonconserved

N-terminal are needed. The N-terminus of RCAN1 con-

tains an aggregation-prone domain, and overexpression

of RCAN1 results in the formation of aggresome-like

aggregates in cultured primary neurons, and the number

of synapses is reduced in these neurons (Ma et al. 2004).

Therefore, studies of the RCAN1 N-terminus may be

informative for neurodegenerative diseases, such as

Down syndrome or Alzheimer’s disease. In conclusion,

RCAN1, which is an endogenous inhibitor of

calcineurin, has wide variety of physiological functions;

future studies are necessary to identify the molecular

mechanisms of RCAN1.

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Regulator of G-Protein Signaling 13

▶RGS13

Regulators of G-Protein Signaling

▶RGS Protein Family

Relaxin Family Peptide Receptors (RXFP)1 and 2

Roger J. Summers1, Michelle L. Halls2 and

Emma T. van der Westhuizen3

1Drug Discovery Biology, Monash Institute of

Pharmaceutical Sciences, Monash University,

Parkville, VIC, Australia2Department of Pharmacology, University of

Cambridge, Cambridge, UK3Institut de Recherche en Immunologie et

Cancerologie, Universite de Montreal, Montreal,

QC, Canada

Synonyms

GREAT; RXFP1: LGR7; RXFP2: LGR8

Historical Background: Relaxin FamilyPeptides and Their Receptors

Relaxinwas one of the first reproductive hormones to be

identified, following the observation that a factor in the

serum of pregnant guinea pigs induced relaxation of the

birth canal (Hisaw 1926). Until recently, relaxin was

considered purely a hormone of pregnancy and littlewas

known of its potential roles in males and nonpregnant

females; the purification of relaxin from animal sources

led to the determination of its peptide structure, biolog-

ical actions, and development of reliable bioassays

(Schwabe and McDonald 1977; James et al. 1977;

John et al. 1981), and this knowledge precipitated the

use of recombinant DNA techniques to clone the rat

(Hudson et al. 1981) and pig (Haley et al. 1982) relaxin

R 1636 Relaxin Family Peptide Receptors (RXFP) 1 and 2

genes, followed soon after by human gene-1 (RLN1)

(Hudson et al. 1983) and gene-2 relaxin (RLN2) (Hud-

son et al. 1984). The identification of additional relaxin

peptides (including the neuropeptide relaxin-3), in addi-

tion to the recent de-orphanization of multiple

G protein-coupled receptors (GPCRs) for relaxin, more

than 75 years after the identification of the peptide

itself, stimulated a resurgence of interest in this

pleiotropic hormone.

Relaxin is a two-chain peptide with a high level of

structural homology to insulin. This established the

structural determinants of the insulin/relaxin peptide

family, and precipitated a search for related peptides

containing similar structural motifs. Additional mam-

malian peptides were revealed: insulin-like growth

factor (IGF)-I and IGF-II, and the insulin/relaxin-like

peptides (INSL) INSL3, INSL4, INSL5 and INSL6.

Since the publication of the human genome, many

orphan GPCRs including the relaxin family peptide

receptors were de-orphanized following sequence-

based prediction of a seven transmembrane-spanning

domain structure. A search of the human genome

for paralogs of the orphan GPCR, leucine-rich repeat-

containing GPCR (LGR) 7 (RXFP1), identified an

additional orphan GPCR, LGR8 (RXFP2) (Hsu et al.

2002). Based upon the phenotypic similarities of mice

lacking either INSL3 or the mouse LGR8 (GREAT)

gene, the insulin/relaxin peptides were tested for activ-

ity at the two orphan receptors; both LGR7 and LGR8

responded to relaxin with a dose-dependent increase in

cAMP production, and the expression patterns of

LGR7 were consistent with known relaxin binding

sites (Hsu et al. 2000). The de-orphanization of

LGR7 led the way for ligand identification for the

highly similar orphan GPCR, LGR8; thus INSL3 was

identified as the specific peptide ligand that bound

LGR8 and stimulated cAMP production (Hsu et al.

2002; Kumagai et al. 2002). In addition, receptor

mRNA expression was found in tissues that express

INSL3, and humans with a deletion in the LGR8 gene

displayed cryptorchidism phenotypically identical to

that seen in INSL3-knockout mice. These receptors,

and two additional orphan GPCRs, were later renamed

▶ relaxin family peptide receptors (RXFP) 1–4, where

RXFP1 is the relaxin receptor, RXFP2 is the receptor

for INSL3, RXFP3 is the receptor for relaxin-3, and

RXFP4 is the INSL5 receptor (see RXFP3/4 page for

further information; reviewed in [Bathgate et al. 2006;

Halls et al. 2007; Ivell and Anand-Ivell 2009]).

Molecular Biology of RXFP1 and RXFP2

Human RXFP1 is located on chromosome 4q32.1, and

RXFP2 is located on 13q13.1. The receptors share 60%

amino acid sequence identity and 80% homology.

Both RXFP1 and RXFP2 have multiple alternatively

spliced isoforms (reviewed in [Halls et al. 2007]). Thus

far, 29 splice variants of RXFP1 and RXFP2 have been

identified; four of these variants have been studied in

greater detail, and show a wide range of tissue expres-

sion, but cannot bind either relaxin or INSL3 or

increase cAMP accumulation. Although one isoform

was highly expressed (RXFP2.1; deletion of exon 11,

corresponding to leucine-rich repeat [LRR] 7), others

were expressed either at very low levels (RXFP1.10;

deletion of exon 3, flanking the low-density lipoprotein

class a [LDLa] module), retained within the cell

(RXFP1.2; deletion of exon 12 and 13, corresponding

to LRRs 8 and 9), or in the case of one variant

(RXFP1.1; stop codon in exon 6, resulting in only the

LDLa module and two LRRs), secreted, raising inter-

esting questions regarding their role in endogenous

regulation of full-length RXFP1 (reviewed in [Halls

et al. 2007]). Another even smaller secreted variant,

RXFP1-truncate, has been identified in mouse, rat, and

pig (reviewed in [Halls et al. 2007]). RXFP1-truncate

consists of the receptor signal peptide, the LDLa mod-

ule, 33 residues of the LRR flanking sequence, and

a nonhomologous sequence of seven residues. When

a construct encoding the truncate peptide was tran-

siently expressed in HEK293T cells, RXFP1-truncate

was secreted and inhibited relaxin-stimulated cAMP

signaling mediated by the full-length receptor, acting

as a functional “antagonist” of relaxin. Expression of

RXFP1-truncate is increased during pregnancy in both

mouse and rat, suggesting a functional role of localized

antagonism of relaxin.

Structural Features and Functional Domainsof RXFP1 and RXFP2

Although all receptors of the RXFP family are classi-

fied as family A GPCRs, similar to the rhodopsin

receptor, they can be further subdivided into two dis-

tinct subgroups: the LGRs (Fig. 1a), and the small

peptide GPCRs (Fig. 1b). Generally, LGRs comprise

a LRR domain, a hinge region, seven transmembrane-

spanning domains, and a C-terminal tail. The type

C-terminal tail C-terminal tail

N-terminal tail

LDLamodule

LRR

Uniquehinge

region

7TMD7TMD

a b

Relaxin Family Peptide Receptors (RXFP) 1 and 2,Fig. 1 Schematic diagram of the structure of relaxin familypeptide receptors (RXFP). (a) The relaxin and INSL3 receptors,(RXFP1 and RXFP2, respectively) are G protein-coupled recep-

tors composed of the seven transmembrane spanning domains

(7TMD), with a large N-terminal ectodomain consisting of

a unique hinge-like region, 10 leucine-rich repeats (LRR),and a low-density lipoprotein class A (LDLa) module. (b) Therelaxin-3 and INSL5 receptors (RXFP3 and RXFP4,

respectively) are also G protein-coupled receptors that contain

the 7TMD but lack the LRR structure found in RXFP1 and

RXFP2

Relaxin Family Peptide Receptors (RXFP) 1 and 2 1637 R

R

C LGRs, RXFP1 and RXFP2, are differentiated from

the type A and type B LGRs by the presence of a low-

density lipoprotein class A (LDLa) module at the

extreme N-terminus, leading into 10 LRRs and a

unique hinge region.

While relaxin binds to both RXFP1 and RXFP2,

INSL3 is unable to bind RXFP1. Furthermore, rat

relaxin is unable to bind RXFP2 suggesting that the

relaxin interaction with RXFP2 is a species-specific

event (Halls et al. 2005). RXFP1 and RXFP2

form constitutive homodimers (and may form

heterodimers); dimerization is dependent upon the

transmembrane region, with stabilization provided by

the ectodomain (Svendsen et al. 2008a, b). Both recep-

tor homodimers exhibit negative cooperativity in

ligand binding (INSL3 at RXFP2; relaxin at RXFP1)

(Svendsen et al. 2008a, b). RXFP1 is also glycosylated,

and this plays a role in receptor cell surface expression

and activation of cAMP (reviewed in [Bathgate et al.

2006; Halls et al. 2007]).

Both RXFP1 and RXFP2 contain two ligand-

binding sites: a high-affinity site within the

ectodomain, and a lower-affinity site within the trans-

membrane region (Halls et al. 2005; Sudo et al. 2003).

Molecular modeling of the LRR region of RXFP1 has

revealed a likely binding cassette for relaxin, which

occurs at an angle of 45� across five of the parallel

LRRs (Bullesbach and Schwabe 2005). Deletion of

any of these residues within the receptor abolished

relaxin binding (Bullesbach and Schwabe 2005).

Despite the RXFP2 sequence containing all of the

residues required for the binding of relaxin to RXFP1,

a number of recent studies have suggested that these

amino acids are not essential for INSL3 binding

(Bullesbach and Schwabe 2006; Rosengren et al. 2006;

Scott et al. 2007). Thus, at RXFP2, relaxin and INSL3

use subtly different B-chain residues and thus bind to

different, but overlapping sites of the receptor. Although

five of the identified RXFP2 residues that interact with

INSL3 are conserved in RXFP1, the only two non-

conserved residues interact with the residues within

INSL3 (ArgB20 and TrpB27) that are critical for peptide

binding and activity (Scott et al. 2007), thus providing

an explanation for the lack of INSL3 binding at RXFP1.

Mutagenesis studies of the LDLa region highlighted

the essential role of this module in receptor signaling.

Mutation of conserved residues, those that compro-

mise correct folding, or entire deletion of the LDLa

module from either RXFP1 or RXFP2, results in recep-

tors that retain binding of their cognate peptide but

are unable to signal by increasing cAMP (Hopkins

et al. 2007; Kern et al. 2007). The LDLa module also

plays a role in receptor maturation and delivery to the

cell surface (Kern et al. 2007).

R 1638 Relaxin Family Peptide Receptors (RXFP) 1 and 2

Signal Transduction Pathways of RXFP1 andRXFP2

Constitutively active mutants of both RXFP1 and

RXFP2 (transmembrane helix 6: D637Y) increase

cAMP accumulation in a ligand-independent manner

(Hsu et al. 2000; Hsu et al. 2002). Consequently, much

research has focused on the cAMP accumulation mod-

ulated by these two receptors (reviewed by [Bathgate

et al. 2006; Halls et al. 2007; van der Westhuizen et al.

2008; Du et al. 2010]) (Figs. 2 and 3).

Both RXFP1 and RXFP2 couple to Gas to increase

cAMP, which is negatively modulated by coupling

to GaoB. Only RXFP1 can also couple to Gai3 to

activate further cAMP accumulation via a Gbg-phosphatidylinositol 3-kinase (PI3K)-protein kinase

C (PKC) z pathway to stimulate adenylyl cyclase 5.

Activation of the Gai3 pathway is dependent upon the

final 10 amino acids of the RXFP1 C-terminal tail

(requiring Arg752) and localization within lipid-rich

membrane domains.

For RXFP1, there is also evidence for activation of

other signaling pathways in response to relaxin

(reviewed in [Bathgate et al. 2006; Halls et al. 2007;

van der Westhuizen et al. 2008; Du et al. 2010]).

In THP-1 and human endometrial stromal cells, both

of which endogenously express RXFP1, there is evi-

dence for tyrosine kinase or mitogen-activated protein

kinase (MAPK)-dependent cAMP accumulation,

which may involve relaxin-mediated inhibition of

a phosphodiesterase. A number of cell types that

express RXFP1 rapidly activate extracellular regulated

kinase (ERK)1/2 (< 5 min) upon relaxin stimulation,

including human endometrial stromal cells, THP-1

cells, and primary cultures of human coronary artery

cells, pulmonary artery smooth muscle cells, and

renal myofibroblasts. There is also evidence for

relaxin-mediated increases in nitric oxide both acutely

and chronically, and in renal myofibroblasts, activation

of a nitric oxide pathway mediates the effects of

relaxin/RXFP1 on differentiation and collagen produc-

tion. Relaxin has also been reported to interact with the

glucocorticoid receptor.

In cell systems that endogenously express RXFP2,

the receptor appears to mediate a variety of responses

(reviewed in [Bathgate et al. 2006; Halls et al. 2007;

van der Westhuizen et al. 2008; Ivell and Anand-Ivell

2009]). In gubernacular cells, stimulation with INSL3

causes an increase in cAMP. However, and conversely,

in both male and female germ cells, stimulation of

RXFP2 causes inhibition of cAMP accumulation

mediated by pertussis toxin (PTX)-sensitive

G-proteins. Thus, it appears that the signaling out-

comes mediated by these receptors may vary greatly

depending upon the cell type, and perhaps reflecting

differences in the population of G proteins expressed.

Localization of RXFP1 and RXFP2 Receptors

RXFP1 is present in a variety of tissues (reviewed in

[Bathgate et al. 2006; Halls et al. 2007; van der

Westhuizen et al. 2008]). Northern blots of human tissue

identified relaxin receptor mRNA in ovary, uterus, pla-

centa, testis, prostate, brain, kidney, heart, lung, liver,

adrenal gland, thyroid gland, salivary glands, muscle,

peripheral blood cells, bonemarrow, and skin (Table 1).

An additional shorter length receptor mRNA was also

identified in oviduct, uterus, colon, and brain. Human

relaxin receptor protein expression has been identified

by immunohistochemical analysis in uterus, cervix,

vagina, nipple, and breast (Table 1). Studies using

Northern blots of rat tissue have additionally identified

receptor mRNA in small intestine and oviduct, and

LacZ reporter expression was also identified in oviduct

in a relaxin receptor knockout mouse model.

INSL3 receptor mRNA expression in human

(reviewed in [Bathgate et al. 2006; Halls et al. 2007;

van der Westhuizen et al. 2008; Ivell and Anand-Ivell

2009]) occurs in the uterus, testis, brain, pituitary,

kidney, thyroid, muscle, peripheral blood cells, and

bone marrow as determined by reverse transcriptase-

polymerase chain reaction (RT-PCR). Additional

expression was identified in the ovary and

gubernaculum in the mouse as determined by RT-PCR

and specifically in themesenchymal and cremaster mus-

cle of the gubernaculum using immunohistochemistry,

in the rat gubernaculum using RT-PCR and Northern

blot analysis, and in the rat ovary using RT-PCR,

Northern blot analysis, and in situ hybridization

(Table 1).

Physiological Roles of Relaxin/RXFP1 andINSL3/RXFP2

Initially, the principal role of relaxin was thought to

involve preparation of the birth canal for parturition, as

ATP

cAMP

AC

PKA

CREB

CRE

GRE

TK

GR NOS II

NO

eNOs

ERK 1/2

ATP

ACV

cAMPAkt

PI3k

Movement into lipid-richsignalling platforms

PKCζ

IκBIκB

NFκB

GαoB Gαi3 β γGαs

Relaxin Family Peptide Receptors (RXFP) 1 and 2,Fig. 2 Signaling pathways activated by RXFP1. Activation of

RXFP1 by relaxin promotes receptor coupling to Gas (to stim-

ulate) and GaoB (to inhibit) cAMP accumulation by modulating

adenylyl cyclase (AC) activity. The receptor may also increase

cAMP accumulation by a tyrosine kinase (TK) dependent mech-

anism. The cAMP downstream of Gas and GaoB activates pro-

tein kinase A (PKA) and cAMP response element binding

protein (CREB) to increase the transcriptional activity of the

cAMP response element (CRE) transcription factor. PKA may

also inhibit inhibitor kB (IkB), allowing increased activity of thenuclear factor kB (NFkB), and production of nitric oxide (NO)via nitric oxide synthase II (NOSII). RXFP1 also couples to Gai3(dependent upon lipid-rich microdomains) allowing activation

of a Gbg, phosphoinositide 3-kinase (PI3K), protein kinase

C (PKC)z pathway, to increase cAMP via activation of AC5.

As a result of Gai3 coupling, PI3K activation may also activate

an Akt, eNOS pathway to increase NO, and PKCz may activate

extracellular regulated kinase (ERK) 1/2, or activate IkB to

inhibit the activity of NkB

Relaxin Family Peptide Receptors (RXFP) 1 and 2 1639 R

R

first described in pregnant guinea pigs (Hisaw 1926).

Although this special endocrine function is found in

species such as rodents and some mammals, it is now

apparent that it is of reduced importance in higher spe-

cies. Currently it is established that in women, maxi-

mum circulating levels of relaxin occur during the first

trimester of pregnancy, and thus the role of relaxin in

pregnancy is likely to be associated with first trimester

physiological events such as embryo implantation. The

peptide also causes a number of additional physiological

changes associated with pregnancy, including uterine

growth and development, myometrial contractility, cen-

tral control of plasma osmolality, and cardiovascular

adaptations (reviewed in [Sherwood 2004]).

Away from the reproductive system, relaxin is pos-

tulated to exert a local influence upon the circulation

by increasing vasodilation and passive compliance

(reviewed in [Du et al. 2010]). Recent observations

suggest that relaxin exerts these local effects upon

vasodilation through the synthesis and production of

nitric oxide, particularly in situations of natural or

induced myocardial infarction. Furthermore, recent

phase II clinical trials have shown efficacy for relaxin

as a vasodilator in acute heart failure.

Another physiological effect of relaxin with thera-

peutic potential is its effect upon connective tissue

regulation and fibrosis. Importantly from

a therapeutic viewpoint, relaxin is able to decrease

excess collagen deposition in fibrotic lesions, with

a conservation of endogenous connective tissue struc-

ture. Relaxin also exerts anti-inflammatory effects

(reviewed in [Bathgate et al. 2006; Halls et al. 2007;

van der Westhuizen et al. 2008]), can inhibit the acti-

vation of human neutrophils by pro-inflammatory

agents and prevents histamine and granule release by

activated basophils and mast cells. These studies

GαsAc

ATP

cAMP

PKA

CREB

CRE

GαoB β γ

Relaxin Family PeptideReceptors (RXFP) 1 and 2,Fig. 3 Signaling pathwaysactivated by RXFP2.Activation of RXFP2 by

INSL3 or relaxin causes

increased cAMP accumulation

via Gas-mediated adenylyl

cyclase (AC) activation. Thereceptor also couples to GaoB,and both Ga and Gbg subunitsnegatively modulate AC

activity. The cAMP pool

(downstream of Gas andGaoB) activates protein kinase

A (PKA) and cAMP-response

element binding protein

(CREB) to modulate the

transcriptional activity of the

cAMP response element

(CRE)

R 1640 Relaxin Family Peptide Receptors (RXFP) 1 and 2

indicate a protective effect of relaxin during allergic

inflammatory responses. In rodent models of angio-

genesis and wound healing, relaxin treatment resulted

in increased vascularization and neoangiogenesis of

ischemic wound sites. There was no effect of relaxin

upon cytokine expression in cells taken from non-

wound sites, indicating promising specificity for ther-

apeutic applications.

In a manner that mirrors its effects in fibrosis, evi-

dence suggests that relaxin is recruited as an endoge-

nous factor for tissue remodeling in cancer cells

(reviewed in [Klonisch et al. 2007]). Increased expres-

sion of relaxin (but not RXFP1) has been detected in

both tissue from prostate carcinoma and prostate can-

cer cell lines. Interestingly, downregulation of either

relaxin or RXFP1 caused significant inhibition of

growth and invasiveness, in addition to increased apo-

ptosis. A human prostate cancer cell line engineered

to express relaxin exhibited increased tumor volume

and vascularization compared to controls. Relaxin is

associated with increased invasiveness of endometrial,

breast, and thyroid carcinomas. Serum relaxin

concentrations are also elevated in patients with bone

metastasis, and relaxin is a potent stimulator of

osteoclastogenesis (Ferlin et al. 2010).

The major physiological effects of INSL3 are

observed within the reproductive system (reviewed in

[Bathgate et al. 2006; Halls et al. 2007; van der

Westhuizen et al. 2008; Ivell and Anand-Ivell 2009]).

Lack of INSL3 (or alternatively, lack of RXFP2) inmice

results in cryptorchidism, or failure of the testes to

descend during development. Female INSL3-knockout

mice exhibit only a mild phenotype of disturbed cycle

length and increased ovarian apoptosis, whereas

overexpression of the peptide leads to ovarian descent

and bilateral inguinal hernia. The influence of the pep-

tide extends beyond early development, as INSL3 serum

levels increase with the onset of puberty in males, and

Relaxin Family Peptide Receptors (RXFP) 1 and 2, Table 1 Tissue localization of RXFP1 and RXFP2 receptors

RXFP1 RXFP2

Tissue/species Rat Mouse Human Rat Mouse Human

Ovary mRNAa Proteinf mRNAb mRNAa,e

Oviduct mRNAa mRNAd Proteinf

Uterus mRNAa mRNAd,b,a mRNAb mRNAb

Proteinc,f Proteinc

Uterine smooth muscle Proteinf mRNAd Proteinc

Proteinc

Endometrium mRNAb Proteinc mRNAa

Proteinf,c

Cervix,vagina Proteinc,f mRNAd,a,b Proteinc

Proteinb

Placenta mRNAa,b mRNAb

Nipple Proteinc mRNAd,a,b Proteinc

Breast Proteinc Proteinc Proteinc

Testis mRNAa,b mRNAd,b,a mRNAb mRNAa,e mRNAb mRNAb

Prostate mRNAb mRNAb

Gubernaculum mRNAb,a mRNAb

Brain mRNAa,b mRNAd,a,b mRNAb mRNAe mRNAb mRNAb

Proteinf

Brain regions mRNAe mRNAe mRNAe mRNAe

Proteinf Proteinf Proteinf Proteinf

Pituitary mRNAd

Kidney mRNAa mRNAb mRNAe mRNAb

Heart mRNAa,b mRNAd,a,b mRNAb

Proteinf

Lung mRNAa,b mRNAb

Liver mRNAb

Intestine mRNAa mRNAa,b

Colon mRNAa

Adrenal mRNAa mRNAb

Thyroid mRNAb mRNAb

Thymus mRNAa

Salivary glands mRNAb

Muscle mRNAb mRNAb

Blood cells mRNAb mRNAb

THP-1 monocytes mRNAb mRNAb

Proteing

Bone marrow mRNAb mRNAb

Skin mRNAa,b mRNAb

Italics ¼ low levels. For detailed references see Bathgate et al. (2006)aNorthern blotbRT-PCRcImmunohistochemistrydlacZ reporter expressioneIn situ hybridizationfReceptor autoradiographygReceptor binding

Relaxin Family Peptide Receptors (RXFP) 1 and 2 1641 R

R

are dependent upon the level of luteinizing hormone

stimulation of Leydig cells. Luteinizing hormone also

stimulates expression of INSL3 in ovarian theca and

testicular Leydig cells, which through activation of the

INSL3 receptor, causes meiotic progression of arrested

oocytes in preovulatory follicles, and suppresses male

germ cell apoptosis. Interestingly, in amanner similar to

relaxin, the INSL3/RXFP2 system has also been

R 1642 Relaxin Family Peptide Receptors (RXFP) 1 and 2

identified in human prostate carcinoma cell lines and

human thyroid carcinomas, and treatment of the tumor

cell lines with INSL3 resulted in increased tumor cell

motility. INSL3 and RXFP2 may also have a role in

osteoporosis.

Summary

RXFP1 is coupled to cAMP and many other signaling

pathways in different cell types. Linking these effec-

tors of RXFP1 activation to specific physiological end

points should allow the design and development of

targeted therapies. RXFP1 has therapeutic potential

for treatment of fibrosis, cancer metastasis, and is

currently in Phase III clinical trials for heart failure.

Although not as extensively studied, RXFP2 appears to

have a simpler physiological role, and fewer down-

stream effectors compared to RXFP1. RXFP2 has pos-

sible therapeutic potential for the treatment of some

types of cryptorchidism and could be used to control

fertility; however, more extensive research is required

to assess its true therapeutic potential.

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Relaxin Family Peptide Receptors (RXFP)3 and 4

Emma T. van der Westhuizen1, Michelle L. Halls2 and

Roger J. Summers3

1Institut de Recherche en Immunologie et

Cancerologie, Universite de Montreal, Montreal, QC,

Canada2Department of Pharmacology, University of

Cambridge, Cambridge, UK3Drug Discovery Biology, Monash Institute of

Pharmaceutical Sciences, Monash University,

Parkville, VIC, Australia

R

Synonyms

GPR100; RXFP3: GPCR135, SALPR; RXFP4:

GPCR142

Historical Background: Relaxin FamilyPeptides and Their Receptors

Relaxin family peptides including the relaxins 1–3,

insulin-like peptides (INSL) 3–6, and insulin-like

growth factors I and II have a similar architecture to

insulin. These peptides are generally involved in the

regulation of cell growth and metabolism. Relaxin was

originally identified as a hormone important during

pregnancy but is also now known to have roles in

collagen remodeling, wound healing, cardiovascular

responses, and as a brain neuropeptide. In the human,

three independent genes produce three relaxin peptides,

named relaxin-1, relaxin, and the recently discovered

relaxin-3 (Bathgate et al. 2002). Relaxin-3 is primarily

expressed in the brain as a neuropeptide that mediates

stress and feeding responses in rats (Tanaka et al.

2005; McGowan et al. 2005, 2007). Relaxin-3 peptide

sequences from different species are well conserved

(Bathgate et al. 2002; Wilkinson et al. 2005) and

the degree of sequence identity between the relaxin-3

peptides suggests that it is the ancestral relaxin peptide

from which the other relaxin and insulin-like peptides

evolved (Wilkinson et al. 2005).

The receptors for the relaxin family peptides are

G protein-coupled receptors (GPCRs), named relaxin

family peptide receptors (RXFP) 1–4, where RXFP1

is the relaxin receptor, RXFP2 is the INSL3 receptor,

RXFP3 is the relaxin-3 receptor, and RXFP4 is the

INSL5 receptor. RXFP3 was discovered by probing

a human cortical cDNA library (Matsumoto et al.

2000). Although it has high amino acid sequence

similarity to the somatostatin and the angiotensin II

receptors, it was not activated by these ligands, and

therefore it was initially named somatostatin and

angiotensin-like peptide receptor (SALPR). The

ligand for RXFP3 was later discovered by using the

receptor as bait to fish for peptides in extracts derived

from various rat tissues (Liu et al. 2003b). Only the

brain extracts increased GTPgS binding (Liu et al.

2003b), and purification led to the identification

of relaxin-3 (Liu et al. 2003b). RXFP4 was subse-

quently identified by searching the human genome

database (Genbank™) with the RXFP3 sequence

(Liu et al. 2003a). Since RXFP4 had 43% amino

acid sequence identity with RXFP3 it was hypothe-

sized that these receptors may share or have similar

ligands (Liu et al. 2003a). Relaxin-3 was also found to

activate RXFP4 (Liu et al. 2003a); however, RXFP4

was later identified as the receptor for INSL5 (Liu

et al. 2005).

Molecular Biology of RXFP3 and RXFP4

Human RXFP3 is located on chromosome

5p15.1-5p14 (Matsumoto et al. 2000) and human

RXFP4 is located on chromosome 1q22, and both

receptors are coded by a single exon sequence. In the

R 1644 Relaxin Family Peptide Receptors (RXFP) 3 and 4

rat RXFP3 gene, there are two potential start codons

(ATG) that are not seen in the human or the mouse

genes (Wilkinson et al. 2005). Transcription initiating

from the first potential start codon produces rat

RXFP3-long, with seven additional residues at the

amino terminus. Transcription initiation from the sec-

ond potential start codon produces rat RXFP3-short,

which is equivalent to the human and mouse RXFP3

sequences. The mouse RXFP4 sequence has 74%

homology with the human RXFP4 sequence, however,

the gene equivalent to RXFP4 in the rat is

a pseudogene that does not code a functional protein

(Chen et al. 2005), suggesting the INSL5-RXFP4

system is redundant in rats.

Structural Features and Functional Domainsof RXFP3 and RXFP4 Receptors

The relaxin family peptide receptors (RXFPs) belong

to two distinct subclasses of family A GPCRs, the

leucine-rich repeat-containing GPCRs (LGRs) and

the small peptide receptor-like GPCRs (RXFP3 and

RXFP4) (Fig. 1). RXFP3 and RXFP4 consist of

seven transmembrane spanning domains (7TMDs),

an extracellular amino-terminal tail, and an intracellu-

lar carboxy-terminal tail.

Receptor chimeras between RXFP3 and RXFP4

identified the amino-terminal tail and extracellular

loop 2 as important regions of the receptors for binding

to relaxin-3 or INSL5, respectively (Zhu et al. 2008).

These regions are also important for the activation of

RXFP3 by relaxin-3 (Zhu et al. 2008); however, trans-

membrane helicies 2, 3, 5 and extracellular loop 2 are

important for activation of RXFP4 by INSL5 (Zhu

et al. 2008).

Human relaxin-3 has a similar affinity for mouse,

rat, and human RXFP3 receptors, likewise, mouse/rat

relaxin-3 have similar affinities for human, mouse,

and rat RXFP3 (Chen et al. 2005). The potency of

human, mouse, and rat relaxin-3 at human, mouse,

and rat RXFP3 is similar in cAMP, GTPgS binding,

and calcium assays (Chen et al. 2005), suggesting the

minor variations in the ligand and receptor amino acid

sequences do not affect the function of the receptor.

The dog and rat INSL5 DNA sequences contain

a frameshift mutation, resulting in multiple stop

codons within the exonic sequence; therefore,

INSL5 is considered to be a nonfunctional

pseudogene in both the dog and the rat (Wilkinson

et al. 2005). The INSL5 gene is functional in mice;

however, mouse INSL5 is not expressed in the brain.

Since the expression pattern of RXFP4 and INSL5

overlap and are pseudogenes in the dog and rat this

suggests they are a ligand–receptor pair (Liu et al.

2005); however, the roles of both INSL5 and RXFP4

remain to be determined.

Signal Transduction Pathways of RXFP3 andRXFP4

Relaxin-3 interacts with both RXFP3 and RXFP4 to

increase GTPgS binding, suggesting that it initiates G

protein-mediated signaling events in cells that express

the receptors (Liu et al. 2003a,b). Unlike RXFP1 and

RXFP2 that increase cAMP in cells when treated with

relaxin or INSL3, respectively, none of the relaxin

family peptides tested increased cAMP levels in cells

expressing RXFP3 or RXFP4 (Liu et al. 2003a, b,

2005; van der Westhuizen et al. 2010). In fact,

relaxin-3 inhibited forskolin-stimulated cAMP accu-

mulation in RXFP3-expressing cell lines in

a concentration-dependent manner (Liu et al. 2003b;

van der Westhuizen et al. 2010) (Fig. 2). Weaker

inhibition of forskolin-stimulated cAMP accumulation

occurs in CHO-K1, HEK293, or SN56 cells expressing

RXFP3 receptors when treated with the B-chain of

relaxin-3 (Liu et al. 2003b), or with human relaxin or

porcine relaxin (van der Westhuizen et al. 2010).

INSL5 also inhibits forskolin-stimulated cAMP accu-

mulation in cells expressing RXFP4 but not in cells

expressing RXFP3 (Liu et al. 2005).

In addition to inhibition of the cAMP signaling

pathway, RXFP3 also activates ERK1/2 when stimu-

lated with relaxin-3, the B-chain of relaxin-3 or

relaxin. ERK1/2 activation involves Gai/o proteins

and either a PI 3-kinase or PKC-dependent pathway

(van der Westhuizen et al. 2007; 2010) (Fig. 2).

Increased transcription of AP-1 reporter genes is

also observed in RXFP3-expressing cells following

stimulation with relaxin-3, human relaxin, or porcine

relaxin in a cell-type and peptide-dependent manner,

potentially through activation of ERK1/2, ERK5, p38

MAPK, or JNK (van der Westhuizen et al. 2010)

(Fig. 2). Increased transcription of NF-kB reporter

genes was also observed in RXFP3-expressing cells

following stimulation with relaxin-3 but not with

LRR

Uniquehingeregion

LDLamodule

7TMD 7TMD

C-terminal tail

N-terminal tail

C-terminal tail

a b

Relaxin Family Peptide Receptors (RXFP) 3 and 4,Fig. 1 Schematic diagram of the structure of relaxin family

peptide receptors (RXFPs). (a) The relaxin and INSL3 receptors,(RXFP1 and RXFP2, respectively) are G protein-coupled recep-

tors composed of the seven transmembrane spanning domains

(7TMDs), with a large N-terminal ectodomain consisting of

a unique hinge-like region, 10 leucine-rich repeats (LRRs) and

a low-density lipoprotein class A (LDLa) module. (b) The

relaxin-3 and INSL5 receptors (RXFP3 and RXFP4, respec-

tively) are also G protein-coupled receptors that contain the

7TMDs but lack the LRR structure found in RXFP1 and RXFP2

AC

ERK1 / 2 PI3K PLC PKC

TK Src

NFκBAP-1

MAPK

IκB

β γ β γGαi / o Gαi / o

Relaxin Family PeptideReceptors (RXFP) 3 and 4,Fig. 2 Signaling pathways

activated downstream of

RXFP3. RXFP3 inhibits

cAMP production via

inhibitory G proteins (Gai/o)and activates extracellular

signal-regulated kinase (ERK)

1/2 via Gai/o proteins and

either a phosphatidylinositol

3-kinase (PI3K) or protein

kinase C (PKC)-dependent

pathway. RXFP3 is coupled to

increased gene transcription

from nuclear factor (NF)-kBand activator protein (AP)-1

transcription factors. MAPK

indicates either ERK, p38

MAPK, or JNK, and TK

indicates receptor tyrosine

kinase

Relaxin Family Peptide Receptors (RXFP) 3 and 4 1645 R

R

human relaxin, porcine relaxin, or human INSL3

(van der Westhuizen et al. 2010). Thus, RXFP3 cou-

ples to at least four different signaling pathways to

elicit responses in cells; ligand-biased signaling is

also observed at this receptor, with some pathways

activated exclusively by relaxin-3, while others are

activated by relaxin-3, human relaxin, porcine

relaxin, and human INSL3 (van der Westhuizen

et al. 2010).

RXFP4 is potentially coupled to the calcium

signaling pathway as INSL5 or relaxin-3 stimulation

of CHO-K1 cells co-transfected with RXFP4 and

AC

Ca2+

Gαi / o Gα16

Relaxin Family PeptideReceptors (RXFP) 3 and 4,Fig. 3 Signaling pathways

activated downstream of

RXFP4. RXFP4 inhibits

cAMP production via

inhibitory G proteins (Gai/o)and releases calcium via Ga16proteins

R 1646 Relaxin Family Peptide Receptors (RXFP) 3 and 4

Ga16 increased intracellular calcium, suggesting that

RXFP4 couples to Ga16 signaling pathways in cell

lines that express this G protein (Liu et al. 2003a,

2005) (Fig. 3). RXFP3 on the other hand, does not

activate calcium signaling (Liu et al. 2003b).

Localization of RXFP3 and RXFP4

RT-PCR and in situ hybridization studies showed that

RXFP3 is principally expressed in the rat brain

(Table 1), with strongest expression in the hypothala-

mus, paraventricular nucleus, cortex, septal nucleus,

preoptic area, supraoptic nucleus, periaqueductal gray,

nucleus insertus, and central gray regions in

the brainstem (Sutton et al. 2004; Ma et al. 2007).

The mouse ortholog of RXFP3 is also expressed in

the brain, as shown by Northern blots (Boels et al.

2004; Sutton et al. 2005; Lein et al. 2007). In the

periphery, however, RXFP3 mRNA was only detected

in the testis (Liu et al. 2003b). Direct radioligand

binding studies have also mapped RXFP3 binding

sites in the rat brain (Table 1) (Sutton et al. 2004; Ma

et al. 2007) (RXFP4 is a pseudogene in the rat, there-

fore not expressed). This study used an INSL5 A-

chain/relaxin-3 B-chain chimeric peptide (that does

not bind to RXFP1 or RXFP2) to identify RXFP3

binding sites in many regions involved in sensory

perception. The INSL5/relaxin-3 chimera binding

sites may have terminating projections from rat

relaxin-3-containing neurons, suggesting that relaxin-

3 produced locally by the brain, acts at regions where

RXFP3 is expressed (Sutton et al. 2004; Ma et al.

2007).

RT-PCR demonstrated RXFP4 expression in

human colon, thyroid, salivary gland, prostate, pla-

centa, thymus, testis, kidney, uterus, and brain

(Table 1) (Conklin et al. 1999; Liu et al. 2003a; Boels

and Schaller 2003). In situ hybridization did not show

INSL5 or RXFP4 mRNA expression in the mouse

brain and mouse INSL5 did not displace [125I]-

INSL5/relaxin-3 binding, suggesting that there were

very few if any RXFP4 sites in the mouse brain (Sutton

et al. 2005). RXFP4 mRNA is expressed in the human

colon, thyroid, salivary gland, prostate, placenta, thy-

mus, testis, kidney, and brain (Table 1) (Liu et al.

2003a; Boels and Schaller 2003).

Physiological Roles of Relaxin-3

The strongest expression of relaxin-3 mRNA occurs in

the nucleus incertus (Sutton et al. 2004). In neurons,

relaxin-3 is found in vesicles, suggesting that it is

a neurotransmitter released from activated neurons

(Tanaka et al. 2005). Based on the location of the

projections of relaxin-3-containing neurons, the pep-

tide is potentially important in motivational and emo-

tional behaviors, and may have a role in motor

function, sensory perception, processing sensory

Relaxin Family Peptide Receptors (RXFP) 3 and 4,Table 1 Tissue distribution of RXFP3 and RXFP4

RXFP3 RXFP4

Tissue/

species

Rat Mouse Human Human

Ovary mRNAc

Uterus mRNAc

Placenta mRNAb,c

Testis mRNAa mRNAb,c

Prostate mRNAb,c

Brain Proteine mRNAa,d mRNAb,c

Brain regions mRNAf,g

Proteinf,gmRNAh,i,j

ProteinimRNAa,d mRNAc

Pituitary mRNAd

Kidney mRNAb

Heart mRNAc

Intestine mRNAc

Colon mRNAb,c

Pancreas mRNAa,d

Adrenal mRNAa,d mRNAc

Thyroid mRNAb,c

Thymus mRNAa mRNAb,c

Salivary

glands

mRNAa,d mRNAb,c

Muscle mRNAc

Peripheral

blood cells

mRNAc

Bone marrow mRNAc

amRNA by RT-PCR (Liu et al. 2003b)bmRNA by RT-PCR (Liu et al. 2003a)cmRNA by multi-tissue expression array or Northern blot anal-

ysis (Boels and Schaller 2003)dmRNA by RT-PCR (Matsumoto et al. 2000)eReceptor autoradiography (Liu et al. 2005)fIn situ hybridization, receptor autoradiography (Sutton et al.

2004)gIn situ hybridization, receptor autoradiography (Ma et al. 2007)hIn situ hybridization (Boels et al. 2004)iIn situ hybridization, receptor autoradiography (Sutton et al.

2005)jIn situ hybridization (Lein et al. 2007)

Relaxin Family Peptide Receptors (RXFP) 3 and 4 1647 R

R

information and in learning and memory (Sutton et al.

2004; Tanaka et al. 2005). However, whether these

physiological roles are dependent upon the RXFP3

receptors around the terminal regions of the relaxin-

3-containing neurons remains to be determined. Injec-

tion of relaxin-3 into rat brain cerebral ventricles, the

paraventricular nucleus, supraoptic nucleus, arcuate

nucleus, or the anterior preoptic area increased food

intake in rats (McGowan et al. 2005, 2007), possibly

by acting at RXFP3 receptors in these areas.

Summary

RXFP3 activates many signaling pathways in various

cell lines and exhibits different but overlapping signal-

ing profiles dependent upon the activating ligand. On

stimulation with relaxin family peptides, RXFP3

inhibits cAMP production and activates MAP kinase

signaling pathways. RXFP3 also increases gene tran-

scription from AP-1 and NF-kB promoters, which may

be important in regulating feeding and stress responses

mediated by relaxin-3 in rats. Linking these signaling

pathways to pathophysiology should encourage the

design and development of antianxiety and antiobesity

therapies that specifically target particular RXFP3 sig-

naling pathways. Extensive study of RXFP4 is

required to determine its physiological role, its impor-

tance in pathophysiology, and to assess its potential as

a drug target.

References

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Reytomas IG, et al. Human relaxin gene 3 (H3) and

the equivalent mouse relaxin (M3) gene. Novel members

of the relaxin peptide family. J Biol Chem. 2002;277(2):

1148–57.

Boels K, Schaller HC. Identification and characterisation of

GPR100 as a novel human G-protein-coupled bradykinin

receptor. Br J Pharmacol. 2003;140:932–8.

Boels K, Hermans-Borgmeyer I, Schaller HC. Identification of

a mouse ortholog of the G-protein-coupled receptor SALPR

and its expression in adult mouse brain and during develop-

ment. Brain Res Dev Brain Res. 2004;152:265–8.

Chen J, Kuei C, Sutton SW, Bonaventure P, Nepomuceno D, Eriste

E, et al. Pharmacological characterization of relaxin-3/INSL7

receptors GPCR135 and GPCR142 from different mammalian

species. J Pharmacol Exp Ther. 2005;312(1):83–95.

Conklin D, Lofton-Day CE, Haldeman BA, Ching A, Whitmore

TE, Lok S, et al. Identification of INSL5, a new member of

the insulin superfamily. Genomics. 1999;60(1):50–6.

Lein ES, Hawrylycz MJ, Ao N, Ayres M, Bensinger A, Bernard

A, et al. Genome-wide atlas of gene expression in the adult

mouse brain. Nature. 2007;445:168–76.

Liu C, Chen J, Sutton S, Roland B, Kuei C, Farmer N, et al.

Identification of relaxin-3/INSL7 as a ligand for GPCR142.

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Liu C, Eriste E, Sutton S, Chen J, Roland B, Kuei C, et al.

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Liu C, Chen J, Kuei C, Sutton S, Nepomucendo D, Bonaventure

P, et al. Relaxin-3/insulin-like peptide 5 chimeric peptide,

a selective ligand for G protein-coupled receptor (GPCR)135

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and GPCR142 over leucine-rich repeat-containing G protein-

coupled receptor 7. Mol Pharmacol. 2005;67:231–40.

Ma S, Bonaventure P, Ferraro T, Shen PJ, Burazin TC, Bathgate

RA, et al. Relaxin-3 in GABA projection neurons of nucleus

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via G-protein-coupled receptor-135 in the rat. Neuroscience.

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Saito T, et al. The novel G-protein coupled receptor SALPR

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McGowan BM, Stanley SA, Smith KL, White NE, Connolly

MM, Thompson EL, et al. Central relaxin-3 administration

causes hyperphagia in male Wistar rats. Endocrinology.

2005;146(8):3295–300.

McGowan BM, Stanley SA,White NE, Spangeus A, PattersonM,

Thompson EL, et al. Hypothalamic mapping of orexigenic

action and Fos-like immunoreactivity following relaxin-3

administration in male Wistar rats. Am J Physiol.

2007;292(3):E913–9.

Sutton SW, Bonaventure P, Kuei C, Roland B, Chen J,

Nepomuceno D, et al. Distribution of G-protein-coupled

receptor (GPCR)135 binding sites and receptor mRNA in

the rat brain suggests a role for relaxin-3 in neuroendocrine

and sensory processing. Neuroendocrinology. 2004;80(5):

298–307.

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Zhu J, et al. G-protein-coupled receptor (GPCR)-142 does

not contribute to relaxin-3 binding in the mouse brain:

further support that relaxin-3 is the physiological

ligand for GPCR135. Neuroendocrinology. 2005;82(3–4):

139–50.

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Ozawa H, et al. Neurons expressing relaxin 3/INSL 7 in the

nucleus incertus respond to stress. Eur J Neurosci. 2005;21:

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Resistance to Inhibitors ofCholinesterase 8

▶Ric-8

ret-GC

▶Guanylyl Cyclase Receptors

Retinoic Acid Receptors (RARA, RARB,and RARC)

Scott A. Busby and Thomas P. Burris

Department of Molecular Therapeutics, The Scripps

Research Institute, Jupiter, FL, USA

Synonyms

NR1B1; NR1B2; NR1B3; RARa; RARb; RARg

Historical Background

The retinoic acid receptors (RARs) are ligand-

dependent transcription factors that belong to the

NR1B subtype of the nuclear receptor (NRs) super-

family and have broad roles in development, cell

growth and survival, vision, spermatogenesis, inflam-

mation, and neural patterning. These receptors act in

transmainly as heterodimers with retinoid X receptors

(RXRs). The actions of RARs are stimulated by the

binding of cognate natural ligands (all trans retinoic

acid and 9-cis retinoic acid) as well as a number of

synthetic ligands. In the presence or absence of ligand,

RAR/RXR heterodimers associate with retinoic acid

DNA response elements (RAREs) present in the

promoter or enhancer regions of target genes.

When no ligand is present, corepressor proteins and

histone deacetylases interact with the receptor DNA

complexes and prevent transcription from occurring.

When retinoid ligands are present, they bind

and activate the RARs by initiating a conformation

more favorable to the association of coactivator

proteins and subsequent recruitment of histone

acetyltransferases (HATs) and the components of

the basal transcriptional machinery that initiate

transcription of the RARE (Fig. 1). These consensus

site DNA sequences generally consist of two directly

repeated half sites of AGGTCA separated by two or

five base pair spacers (DR2 or DR5 elements). Due to

Retinoid ligands

HATs

RAR/RXR

Corepressors

HDACs

Transcription off

Coactivatorcomplex

BasalTranscription

Complex

Transcription On

Retinoid ligands

RARE

RAR/RXR

Retinoic Acid Receptors (RARA, RARB, and RARC),Fig. 1 Activation model of RARs. In the presence or absence

of ligand, RAR/RXR heterodimers associate with retinoic acid

response elements (RAREs) present in the promoter or enhancer

regions of target genes. When no ligand is present, corepressor

proteins and histone deacetylases interact with the receptor DNA

complexes and prevent transcription from occurring. When ret-

inoid ligands are present, they bind and activate the RARs by

initiating a conformation more favorable to the association of

coactivator proteins and subsequent recruitment of histone

acetyltransferases (HATs) and the components of the basal tran-

scriptional machinery to initiate transcription

Retinoic Acid Receptors (RARA, RARB, and RARC) 1649 R

their broad action in diverse cell and tissue

populations, RARs are essential signaling proteins for

basic and clinical research.

R

Role of RARs in Embryonic Development

Research from the 1950s implicated vitamin A defi-

ciencies as a cause for a number of congenital

malformations and defects observed in the develop-

ment of animals. These results were known long before

the discovery of retinoic acids as the most biologically

active forms of vitamin A. Later research then investi-

gated the role of retinoic acid in early embryonic

development and in the early 1990s led to the discov-

ery of the retinoic acid receptors (RARs). Since then

much work has been done to determine the role that

RARs play in embryonic development.

Genetic studies were conducted in the early 1990s

to investigate the role of all three RAR isoforms in

embryonic development by generating single and

double RAR mutant mice. While mice with

a mutation deleting only one RAR isoform contained

certain developmental abnormalities, they were still

viable, indicating a certain redundancy among

isoforms (Mark et al. 2009). However, RAR double

null mutants, containing genetic deletions of any two

of the RAR isoforms, died in utero or at birth due to

severe developmental deficits mainly in the vertebrae,

brain, and limbs indicating the importance of the

expression of the RARs on the formation of these

structures. The same deficits were observed by

a number of investigators where various components

of retinoic acid signaling were knocked out such as

retinaldehyde-synthesizing enzyme RDH10 (Sandell

et al. 2007), RA-synthesizing enzyme RALDH2

(Halilagic et al. 2007), or RALDH3 (Dupe et al.

2003). In addition, treatment of wild-type animals

with synthetic pan-specific RAR antagonists also pro-

duced the same defects (Kochhar et al. 1998;Wendling

et al. 2001). This demonstrated that not only expres-

sion of RARs was essential for normal development

NeurogenesisGene Expression

Zic2

X-shh

N-tubulinRARs + RAs

Otx2

Anterior genes(Spinal Cord)

Posterior genes(Hindbrain)

Anterior-PosteriorPatterning

Anterior Neural Tissue

Naive Ectoderm

Addition of NeuralInducers

Krox20Wnt1EnPax2XIF-3Xlim1Hox

XCG-1XAG-1XA-1EMX-1EMX-2Dtx1

XINK-2

X-ngnr-1

X-MyT-1

X-delta-1

Retinoic Acid Receptors (RARA, RARB, and RARC),Fig. 2 RAR-mediated RA signaling mediates neurogenesis

and anterior-posterior patterning of central nervous system.

Diagram showing RAR-mediated up- and downregulation of

genes involved in both neurogenesis and anterior-posterior

which are vital in the development of neurons, spinal cord, and

hindbrain

R 1650 Retinoic Acid Receptors (RARA, RARB, and RARC)

but that the receptors required activation by retinoic

acid ligands to mediate their important roles in devel-

opmental signaling programs.

Using combined strategies of selective RAR iso-

form knockouts and mutations in the RA signaling

pathways, much has been learned about the crucial

roles that RARs play in specific stages of organ and

brain development. For instance, RARs are involved in

formation of a number of limb structures and control

the antero-posterior axis of the limbs (Dupe et al.

1999). Moreover, RAR-mediated RA signaling is

responsible for both neurogenesis as well as the

anterior-posterior patterning of the developing central

nervous system through a complex mechanism of gene

activation and repression by RARs (Maden 2002)

(Fig. 2). Several studies have shown that RARs are

required for the formation of a number of eye structures

as well as histogenesis and physiological apoptosis in

the retina. Moreover, it has been discovered that RARs

play important roles in cardiac development,

respiratory system development, as well as the forma-

tion of important structures in the kidneys and urogen-

ital tract.

While much has been learned about the important

roles played by the RARs in embryonic development,

research is now shifting toward elucidating the roles

played by these receptors during the postnatal devel-

opment. To do this, new strategies have emerged to

allow for the selective mutation of the retinoid recep-

tors in specific cell types so as to further understand the

functions of these receptors in the postnatal animal as it

develops and grows (Metzger and Chambon 2001;

Metzger et al. 2003).

Role of RARs in Regulating Cell Proliferationand Cancer

In addition to the effects that retinoic acids and the

RARs have on developmental pathways, a large

Retinoic Acid Receptors (RARA, RARB, and RARC) 1651 R

R

amount of evidence has emerged implicating RARs in

the control of cell-cycle pathways and cellular prolif-

eration. In normal cells, retinoic acids generally inhibit

cell-cycle progression by instituting a block in the G1

phase of the cell cycle (Mongan and Gudas 2007). Of

all the RAR isoforms, these effects are mostly medi-

ated by RARb2 following binding and activation by

retinoic acids (Faria et al. 1999). Several studies have

shown in a number of cell types that activation of

RARb2 leads to the transactivation of several genes

involved in cell-cycle arrest such as p21CIP1 and

p27KIP1 (Li et al. 2004; Suzui et al. 2004). In addition

to activating cell-cycle arrest proteins, RARs also

mediate both the downregulation of mRNA expression

as well as protein ubiquitination and degradation for

both the Cyclin D and E families which prevents pro-

gression of the cell cycle from the G1 to S phase (Tang

and Gudas 2011). Moreover, RARs induce apoptosis

following binding of retinoids in a number of cell types

as a guard against tumor formation. Retinoic acid binds

to RARa and induces apoptosis in both acute lympho-

blastic leukemia cells as well as myeloid leukemia cell

lines (Chikamori et al. 2006; Luo et al. 2009). In

addition, it has been reported that RARg induces apo-

ptosis upon binding to retinoids in both skin

keratinocytes as well as pancreatic adenocarcinoma

cells (Hatoum et al. 2001; Pettersson et al. 2002).

Finally, RARb2 has been implicated in the induction

of apoptosis in breast cells. Taken together, these

observations have provided clear evidence that RARs

play key roles in the regulation of cell-cycle progres-

sion and cell growth as well as apoptosis and therefore

when RAR-mediated signaling pathways are dis-

turbed, there can be major implications for the devel-

opment and progression of cancer.

One of the most well-studied examples of aberrant

RAR signaling leading to cancer is acute promyelocytic

leukemia (APL). It has been shown in a number of

reports that APL is the result of a genetic rearrangement

of the RARa gene that fuses it to the promyelocytic

leukemia gene (PML) or other PML-related genes

(Tang and Gudas 2011). These PML/RARa fusion pro-

teins lead to dramatic increases in expression of both

HDACs and DNA methyltransferases that cause reduc-

tions in gene expression for retinoid-regulated genes

such as those involved in differentiation (Fig. 3). As

previously stated, under normal conditions retinoid-

regulated genes regulate cellular proliferation mainly

through inhibiting cell-cycle progression and promoting

cellular differentiation. When the expression of these

genes is reduced, the regulatory controls on these func-

tions are lost resulting in uncontrolled proliferation. In

addition to inducing epigenetic silencing, the PML/

RARa fusion protein can also repress important RAR

target genes such as RARb2 itself by binding the RAR

response element (RARE) on the RARb2 promoter and

recruiting corepressors such as NuRD that keep RARb2from being expressed.

The loss of retinoic acid signaling is not restricted to

APL and in fact reduction in expression of both RARaand RARb2 has been shown to occur in several types ofcancer such as embryonal carcinomas, acute myeloid

leukemia, and breast cancer (Mongan and Gudas 2007;

Altucci et al. 2007). In contrast to APL where RARa is

mutated into the PML/RARa fusion protein, most can-

cer cell types do not contain mutated RARs but rather

have dramatically downregulated expression of these

receptors. Understanding the underlying mechanisms

behind the silencing of RARs in cancer cells has been

a major focus of recent research and several mecha-

nisms have been uncovered. For example, it was

reported that the RARb2 promoter in many cancer

cell types is silenced by hypermethylation at CpG

regions of its promoter. In addition, corepressors such

as PRAEME, meningioma 1, acinus-S’, HACE1, and

SMRT have all been shown to inhibit expression of

either RARb2 or RAR response genes either due to

overexpression of these corepressors or a greater affin-

ity for the RARb2 response elements concomitant to

changes caused by aberrant AKT signaling in a variety

of cancer cell types (Tang and Gudas 2011). In con-

trast, repression of RAR coactivator expression has

also been observed to downregulate expression of

RARs in neuroblastoma cells. Taken together, these

reports indicate that a multitude of mechanisms are at

work in various cancer cell types that all result in the

repression of RARs and their downstream target genes.

Regardless of mechanism, the end result is an absence

of RAR-mediated balances between differentiation

and proliferation and demonstrates the vital roles

RARs play in the progression of cancer.

Development of RAR Ligands for Use asTherapeutics

Given the correlation between the reduction in RAR-

mediated retinoic acid signaling and the progression of

Corepressors

CoactivatorComplex

Corepressors

RARα/RXR

RARα/RXR

RARα/PML forms dominantnegative repression of

RAREs by displacing RXRand stronger recruitment

of HDACs

Can be overcome withpharmacological doses ofRA and HDAC inhibitors

BasalTranscription

Complex

RARα/PMLHDACs

RARE

+ Retinoid ligands

HATs

HDACsTranscription Off

Transcription On

Retinoic Acid Receptors (RARA, RARB, and RARC),Fig. 3 Model of aberrant repression of RAR gene activation

by the PML/RARa fusion protein. Schematic comparing the

ligand-dependent activation of RAR response genes in normal

tissues expressing the wild-type RARa/RXRa heterodimer

compared to the APL model where that is replaced by the

PML/RARa fusion protein. The PML/RARa fusion protein

forms a dominant negative repressive complex onto the

RAREs due to the lack of RXRa and the enhanced recruitment

of HDACs to the PML portion of the fusion protein. Reversing

this phenomonon requires HDAC inhibitors or excess amounts

of retinoic acids

R 1652 Retinoic Acid Receptors (RARA, RARB, and RARC)

a number of cancers, therapies have been developed to

treat cancer patients with natural retinoids such as all-

trans retinoic acid (ATRA) to induce differentiation

and cell growth arrest. This strategy has been

extremely successful in the treatment of APL as phar-

macological doses of retinoic acid stimulate irrevers-

ible differentiation of leukemic cells into granulocytes.

Moreover, it has been reported that pharmacological

doses of retinoic acid also trigger growth arrest and

differentiation of leukemia stem cells known as

leukemia-inducing cells (LICs) (Tang and Gudas

2011). When combined with other apoptosis-inducing

chemotherapeutic drugs such as anthracyclins, retinoic

acid treatment reverses gene silencing and leads to

induced cell death of the cancer cells curing 70–80%

of APL patients (Altucci et al. 2007). This treatment is

successful since the expression of the PML/RXRa

fusion protein is high and the RARa portion of this

protein contains a functioning ligand-binding domain

and coactivator recruitment site that allows for the

retinoic acid-mediated activation of a number of

RARa genes that stimulate differentiation.

Differentiation therapy involving treatment with

natural retinoids has been developed for many cancers

such as breast, ovarian, renal, head and neck, mela-

noma, and prostate. However, the success of this

approach has been much less successful in these

other types of cancers where the expression of RAR

genes themselves are downregulated by events such as

DNA methylation of their promoters as previously

discussed. Combination therapies have been adopted

with some success to overcome these limitations with

the coadministration of HDAC inhibitors and

DNMTase inhibitors in addition to retinoic acid

+ Retinoic acid

+ Retinoic acid

a

b

c

HATs

HATs5-aza

RAR/RXR

RAR/RXR

BasalTranscription

Complex

BasalTranscription

Complex

Transcription On

Transcription On

Transcription OffCpGIslands

CpGIslands

CH3

CH3

RARβ2/RXR

RARβ2/RXR

Differentiation

Cell-growthInhibition

Apoptosis

Differentiation

Cell-growthInhibition

ApoptosisRARβ2 gene

RARβ2 gene RARβ2 gene

RARβ2 gene

No RARβ2

CoactivatorComplex

CoactivatorComplex

Retinoic Acid Receptors (RARA, RARB, and RARC),Fig. 4 Schematic model of the benefit of combination therapy

to reverse the repression of RARb2 expression in various can-

cers. (a) Normal tissue where RAR/RXRs regulate expression of

RARb2 which is vital for the balance between cell proliferation

and differentiation and inducing apoptosis when necessary (b)Tumor tissue where hypermethylation of CpG islands on the

promoter of RARb2 prevent its expression and in turn repress

genes involved in regulation of cellular proliferation and apo-

ptosis leading to the cancer phenotype. (c) Treatment of tumors

with combinations of retinoic acid and other inhibitors such as

DNA methyltransferase inhibitors (DNMTase inhibitor) that

first remove the detrimental hypermethylation and then restore

normal expression of RARb2 which is activated by retinoic acids

Retinoic Acid Receptors (RARA, RARB, and RARC) 1653 R

R

(Tang et al. 2009). This strategy first reverses the

repressive effects of protein acetylation and DNA

methylation on RAR gene expression and then once

expressed, provides the natural agonist to activate

RAR target genes to induce growth arrest and apopto-

sis (Fig. 4).

While cell differentiation therapies using high

levels of natural retinoids such as ATRA have proven

to be very successful in the treatments of some cancers,

there are significant drawbacks to their therapeutic use.

Retinoids are powerful teratogens that at pharmaco-

logical concentrations can induce congenital defects

and toxicity in all vertebrate species. In addition, there

are some cancers such as prostate cancer where ATRA

and other synthetic retinoid agonists are not effective

in inducing growth arrest and/or apoptosis. Moreover,

a common feature of many cancers is the development

of resistance to the growth inhibitory effects of reti-

noids limiting the utility of these therapies. Even APL,

which responds well to differentiation therapy, has

several variants that display retinoid resistance and

does not respond to this therapy. For these reasons,

efforts have been underway to develop new types of

synthetic ligands for RAR that can promote the posi-

tive effects of retinoids without the detrimental side

effects.

A number of synthetic retinoids have been devel-

oped as potential therapeutics for a variety of cancers.

R 1654 Retinoic Acid Receptors (RARA, RARB, and RARC)

These are often referred to as atypical retinoids or

retinoid-related molecules because they are based on

the retinoic acid structure and have been shown to bind

and transactivate RARs. Many of these compounds

have been approved for the treatment of a number of

diseases such as cancer, acne, and psoriasis (Altucci

et al. 2007). Themajority of these atypical retinoids are

RAR agonists; however, there have been some RAR

antagonists that have also been synthesized. In some

cancers such as prostate cancer, pan-specific antago-

nists of RAR such as AGN194310 demonstrated much

more significant anti-proliferative and pro-apoptotic

effects than any RAR natural or synthetic agonist. In

fact a number of synthetic molecules known as the

retinoid-related molecules such as MX781, AGN

194310, and ST1926 have demonstrated potent anti-

proliferative activities against large panels of human

tumor cells (de Lera et al. 2007). Until recently, all of

the synthetic retinoid-related molecules reported that

directly bind and modulate RAR activity share struc-

tural similarities to the natural agonist retinoic acid.

This means that while some have proven efficacious in

the treatments of a number of important cancers, they

could still be susceptible to the same limitations

regarding retinoid resistance as the natural retinoids.

Interestingly, a recent report has identified the first

synthetic non-retinoid, non-acid RAR modulator that

binds and activates all three isoforms of RAR (Busby

et al. 2011). Synthetic structures such as these may

provide the basis for novel chemical scaffolds of non-

retinoid, non-acid RAR modulators that may be devel-

oped that are potent and efficacious toward restoring

RAR signaling while at the same time overcome the

challenges of toxicity and resistance seen with use of

natural retinoids such as ATRA.

Summary

The retinoic acid receptors (RARs) are ligand-

dependent transcription factors that belong to the

NR1B subtype of the nuclear receptor (NR) super-

family. RARs are ligand-dependent transcription fac-

tors that bind to retinoids, the most potent biologically

active forms of vitamin A, and heterodimerize with

the rexinoid X receptor (RXR) to regulate many genes

involved in the regulation of cellular growth and dif-

ferentiation. RARs play significant roles in a number

of developmental cascades from formation of limbs

and organs to the central nervous system. In addition,

all three RAR isoforms are instrumental in the control

of a cellular growth through the inhibition of the cell

cycle. That combined with the activation of genes

involved in differentiation provides multiple path-

ways that RARs regulate cellular growth. Given

these critical roles in cellular growth, it is not surpris-

ing that a great deal of evidence has emerged that

either mutations or reductions in RAR expression

are correlated with a number of cancers. This has led

to the development of differentiation therapies alone

or in combination with other types of drugs to restore

RAR-mediated retinoic acid signaling in a number of

cancers. Due to the potential toxicity and emergence

of retinoid resistance in some cancers, synthetic reti-

noid-related molecules have been developed includ-

ing one novel non-acid non-retinoid chemical

scaffold that may provide safer, more efficacious

ways to treat cancer by restoring normal RAR-

mediated RA signaling. Further understandings of

the roles of the various RAR isoforms in the progres-

sion of cancer and how to modulate the activities of

RARs may provide important clues to develop novel

therapies to treat cancer.

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RGS 12/Regulator of G-Protein Signaling12 (AGS6)

▶Activators of G-Protein Signaling (AGS)

RGS Protein Family

David P. Siderovski and Adam J. Kimple

Department of Pharmacology, University of North

Carolina at Chapel Hill, Chapel Hill, NC, USA

Synonyms

Ga GAPs; Regulators of G-protein signaling; RGS

proteins

Historical Background

Signal transduction by G protein–coupled receptors

(GPCRs) was considered for many years (Gilman

1987) to be a three-component system: the cell-surface

receptor to receive external input from hormones and

neurotransmitters, the heterotrimeric G protein to

transduce this input to the intracellular compartment

by its structural changes upon the exchange of guano-

sine triphosphate (GTP) for guanosine diphosphate

(GDP), and effector proteins (such as ▶ adenylyl

cyclase, phospholipase C, and ion channels) to propa-

gate the signal forward as changes in cell membrane

potential and/or intracellular second messenger levels.

However, for many physiological responses mediated

by GPCRs, including the visual response controlled by

the photoreceptor, rhodopsin (Arshavsky and Pugh

1998), intracellular signaling was known to be far

shorter in duration than the time observed for the

isolated components to revert to ground state in vitro

(i.e., the time required for the heterotrimeric G-protein

a subunit to hydrolyze GTP and return to its GDP-

bound, inactive state). A critical fourth component to

this system was discovered to be a large family of

“regulators of G-protein signaling” proteins, also

known as RGS proteins (Willard et al. 2008), that

dramatically accelerate GTP hydrolysis by Ga sub-

units and thereby hasten signal termination (Fig. 1).

Signal

Signalα

GTP

αGTP

αGDP

GDPGTP γγ β

γβ

β

Inactive G-proteinheterotrimer

Ligand-activatedG-protein coupled receptor

Exchange activity

GAP activity

PiRGS

RGS

RGS Protein Family, Fig. 1 Role of RGS proteins in GPCRsignaling as negative regulators. Ligand-activated GPCRs act asguanine nucleotide exchange factors for the inactive, GDP- and

Gbg-bound Ga subunit. The resultant release of GDP, and sub-

sequent binding of the more abundant GTP, leads to

a conformational change within Ga, eliminating the high-affinity

binding site for Gbg. The GTP-bound Ga and released Gbgsubunits are then able to bind effector proteins to propagate

intracellular signaling. The intrinsic GTP hydrolysis activity of

Ga subunits is greatly accelerated by the binding of RGS pro-

teins, leading to the release of inorganic phosphate (Pi) and

reassembly of the inactive Ga·GDP/Gbg heterotrimer

R 1656 RGS Protein Family

One of the earliest reports of cloning a human RGS

protein-encoding gene was that of G0S8 (now known

as RGS2) in 1990. Trapped as one of a number of

putative “G0/G1-switch genes” from mitogen-treated,

primary human T-lymphocytes (Siderovski et al.

1990), G0S8/RGS2 was subsequently observed to

encode a protein with sequence similarity to kinases

specific for activated GPCRs (e.g., b-adrenergic recep-tor kinase, ▶ rhodopsin kinase) and to the

yeast “supersensitivity-to-phermone” protein SST2

(Siderovski et al. 1994). Functional complementation

of yeast deficient in SST2 by overexpression of the

human G0S8/RGS2 gene (Siderovski et al. 1996) pro-

vided one of the first clues that this emergent gene

family encoded negative regulators of signal transduc-

tion acting downstream of GPCR activation; other

groups working in disparate systems came to the

same conclusion contemporaneously (Druey et al.

1996; Koelle and Horvitz 1996). These reports were

quickly followed by a definitive demonstration of the

biochemical activity underlying the negative regula-

tory function of RGS proteins: namely, acceleration of

the intrinsic GTPase activity of Ga subunits using

purified proteins and radiolabeled GTP (Berman et al.

1996a).

RGS Protein Activities

The signature enzymatic activity of RGS proteins is

the acceleration of GTP hydrolysis by activated, GTP-

bound Ga subunits. This acceleration can be made on

certain GTPase-deficient Ga mutants as well (e.g.,

point mutation to the arginine, such as Arg-178 of

Gai1, that helps stabilize the g-phosphate leaving

group) but not on classical, GTPase-dead, Gln-to-Leu

Ga mutants (e.g., Gai1 Q204L) (Berman et al. 1996a).

Following discovery of this signature biochemical

activity of RGS proteins, a crystal structure of RGS4

bound to Gai1 in a transition state-mimetic form was

reported (Tesmer et al. 1997). The ability to observe

the interaction between a RGS protein and its Ga targetto high-resolution solidified early speculation that

RGS proteins employ solely their highly conserved,

�120 amino-acid “RGS domain” to stabilize Ga in its

transition state along the path to GTP hydrolysis

(Berman et al. 1996b); this “GTPase-accelerating pro-

tein” or “GAP” mechanism is distinctly different from

that exhibited by the GAPs of Ras-superfamily

GTPases. The GAP activity of certain RGS proteins,

including RGS4, is thought to be modulated in

a cellular context by the binding of phosphatidy-

linositol head-groups or calmodulin to a “B-site,”

within the RGS domain but distinct from the Ga-binding interface (or “A-site”) (Fig. 2); engagement

of the B-site with phosphatidylinositol-3,4,5-

trisphosphate (PIP3) is considered to inhibit A-site

GAP function in an allosteric fashion, whereas the

binding of calmodulin (in a calcium-dependent man-

ner) to the B-site removes the inhibitory influence of

PIP3 (Tu and Wilkie 2004).

Observations of RGS protein overexpression lead-

ing to accelerated on-rates of GPCR signal transduc-

tion without affecting response sensitivity

or amplitude have been presented in the literature

K113K112

K113

K113K112B-site B-site

A-site

Full GAPactivity

PIP3

a

b

PIP3 CaMCa2+

CaM

A A A

B

Full GAPactivity

Reduced GAPactivity

A-site

K99/K100 K99/K100

RGS Protein Family, Fig. 2 Predicted structural determinantsof the allosteric control over RGS protein GAP activity. (a) Forsome RGS proteins, the binding of the RGS domain “B-site”

with phosphatidylinositol-3,4,5-trisphosphate (PIP3) is thought

to allosterically inhibit the GTPase-accelerating activity of the

RGS domain Ga-binding “A-site”; in a calcium-dependent fash-

ion, the binding of calmodulin to the B-site is thought to remove

the inhibitory influence of PIP3 on A-site GAP function.

(b) Visualization of the predicted functional sites within the

RGS domain of RGS4 (Protein Data Bank id 1AGR) responsible

for allosteric control of GAP activity. Highlighted (orange/dark

grey) regions depict lysines thought to be required for PIP3binding, while solid surface (cyan/light grey) areas depict the

proposed A- and B-sites. The alpha-helical secondary structure

that comprises the conserved RGS domain fold is displayed in

red/black as ribbon tracing within the translucent surface ren-

dering of the domain. Rotation about the vertical axis by 90o and

180o are shown in consecutive panels from left to right

RGS Protein Family 1657 R

R

(e.g., Doupnik et al. 1997) as paradoxical findings that

run counter to expectations that RGS protein GAP

activity should only serve to accelerate the off-rate of

GPCR signaling and, thereby, blunt signaling. It is

possible that RGS proteins contribute more than just

GAP activity to the functioning of the GPCR/G–pro-

tein/effector axis, especially since many RGS proteins

contain multiple protein/protein-interaction domains

in addition to the signature RGS domain (see below).

More recently, by combining Gamutations that accel-

erate intrinsic GTPase activity and that eliminate sen-

sitivity to RGS domain GAP activity (while preserving

all other Ga functions), it has been definitively dem-

onstrated that accelerated GTP hydrolysis alone is

sufficient to elicit observations of increased signaling

onset and recovery times (Lambert et al. 2010). This

finding, however, does not exclude the possibility that

conventional (“GAP-active”) RGS proteins and/or

other RGS domain-containing proteins exhibit addi-

tional functional effects on GPCR-initiated signal

transduction in a cellular context.

The Conventional RGS Protein Subfamilies

The “conventional” members of the RGS protein

family (Table 1) exhibit Ga-directed GAP activity

and have been numbered from RGS1 to RGS21

(excluding RGS15, which turned out to be RGS3).

The majority of these proteins target Ga subunits of

the Gai and Gaq subfamilies (Soundararajan et al.

2008), albeit with notable exceptions as described

below. These proteins have been divided into sub-

families based on overall protein architecture and

RGS domain sequence similarity (Willard et al.

2008). The R4-subfamily is the largest by membership,

consisting of RGS1, -2, -3, -4, -5, -8, -13, -16, -18,

and -21, yet the smallest by individual protein size;

most members merely consist of the �120 amino-acid

RGS domain with short N- and C-terminal polypeptide

extensions (e.g., RGS21 is only 152 amino acids

in length). An exception to this small size is the

R4-subfamily member RGS3, given that alterna-

tive isoforms of this protein are expressed that include

RGS Protein Family, Table 1 The conventional RGS proteins

Family Name GenBank locus UniProt id Entrez gene id Distinguishing characteristic(s)

R4 RGS1 NM_002922 Q08116 5996 Implicated in multiple sclerosis

R4 RGS2 NM_002923 P41220 5997 Selective for Gaq; modulator of anxiety and vasoconstrictor

signaling

R4 RGS3 NM_144489 P49796 5998 Isoforms can contain PDZ and C2 domains

R4 RGS4 NM_005613 P49798 5999 Associated with susceptibility to schizophrenia

R4 RGS5 NM_003617 O15539 8490 Expressed in pericytes; associated with neovascularization

R7 RGS6 NM_004296 P49758 9628 Potential modulator of parasympathetic activation in heart

R7 RGS7 NM_002924 P49802 6000 Implicated in CNS opioid and muscarinic acetylcholine signaling

R4 RGS8 NM_033345 P57771 85397 Directly binds GPCR loops (or indirectly via spinophilin); may

control stable cell-surface GPCR expression

R7 RGS9 NM_003835 O75916 8787 Key deactivator of retinal phototransduction cascade

R12 RGS10 NM_002925 O43665 6001 Phosphorylation and palmitoylation control nuclear localization

and Ga substrate selectivity

R7 RGS11 NM_183337 O94810 8786 Modulator of retinal ON-bipolar cell light response

R12 RGS12 NM_198229 O14924 6002 Contains PDZ, PTB, RBD, and GoLoco domains; scaffold for

Ras/Raf/MAPK cascade

R4 RGS13 NM_002927 O14921 6003 Modulator of GPCR signaling in mast cells / allergic responses

R12 RGS14 NM_006480 O43566 10636 Contains RBD and GoLoco domains; scaffold for Ras/Raf/MAPK

cascade

R4 RGS16 NM_002928 O15492 6004 Feeding and fasting controls expression in periportal hepatocytes

RZ RGS17 NM_012419 Q9UGC6 26575 Implicated in lung tumorigenesis

R4 RGS18 NM_130782 Q9NS28 64407 Expressed in leukocytes, megakaryocytes, and platelets

RZ RGS19 NM_005873 P49795 10287 Implicated in Wnt/b-catenin signaling

RZ RGS20 NM_170587 O76081 8601 Modulator of mu-opioid receptor signaling

R4 RGS21 NM_001039152 Q2M5E4 431704 Expressed in lingual taste buds

R 1658 RGS Protein Family

N-terminal PDZ and C2 domains. Within the

R4-subfamily, RGS2 is unique in acting as a potent

GAP solely on Gaq subfamily members (and not Gaisubunits) in vitro (Kimple et al. 2009), although inhi-

bition of Gi-coupled GPCR signaling can be observed

in a cellular context upon RGS2 overexpression

(Ingi et al. 1998). RZ-subfamily members (RGS17, -

19, and -20) are also small polypeptides but are distinct

from the R4-subfamily in containing cysteine-rich

N-termini thought to be reversibly palmitoylated

for differential subcellular trafficking. As the name

suggests, RZ-subfamily members have particular

selectivity for Gaz subunits, although this is

not exclusive, and binding of (and GAP activity on)

Gai subunits is also manifested (e.g., Soundararajan

et al. 2008).

R7-subfamily members (RGS6, -7, -9, and -11) are

known to play key roles in the regulation of various

neuronal processes such as nociception, motor control,

reward behavior, and vision. These four proteins share

an expression pattern biased to neuronal tissues, as

well as a unique multi-domain protein architecture

composed of DEP (Dishevelled/EGL-10/Pleckstrin)

and GGL (G-gamma-like) domains present N-terminal

to a central RGS domain. The DEP domain mediates

interaction with unique membrane anchor proteins

R7BP and R9AP, whereas the GGL domain (as its

name implies) binds a neuronal-specific Gb subunit,

Gb5, to form an obligate dimeric configuration akin to

conventional Gb/Gg subunits (Snow et al. 1998).

While the R12-subfamily member RGS10 consists of

little more than an RGS domain, the other two mem-

bers of this subfamily (RGS12 and RGS14) share elab-

orate multi-domain architectures. C-terminal to their

RGS domains, both RGS12 and RGS14, possess

a tandem repeat of Ras-binding domains (RBDs) and

a single GoLoco motif; the first of the two RBDs binds

selectively to activated H-Ras (Willard et al. 2007),

whereas the GoLoco motif is known to bind Gai sub-units in their GDP-bound inactive state (Kimple et al.

2002). Unlike RGS14, RGS12 also possesses N-

terminal PDZ and PTB domains which play important

RGS Protein Family 1659 R

roles in the functional organization of an H-Ras-Raf-

MAPK signaling cascade required for nerve growth

factor (NGF)-mediated axonogenesis by dorsal root

ganglion neurons (Willard et al. 2007).

Other RGS Domain-Containing Proteins

There are an equivalent number of “nonconventional”

RGS proteins (Table 2) that, while possessing the

highly conserved nine alpha-helical structure of the

RGS domain (Tesmer et al. 1997; Soundararajan

et al. 2008), either have already been identified in

other functional contexts or have yet to be identified

as bona fide Ga-directed GAPs. With respect to

the latter situation, AKAP-10 (also known as

D-AKAP2) and RGS22 possess more than one RGS

domain, but to date neither have been convincingly

shown to bind to (nor accelerate the GTPase activity

of) Ga subunits; this is also true of three sorting nexins

(SNX13, -14, and -25) and the RA-subfamily members

RGS Protein Family, Table 2 Other proteins containing RGS do

Family Name GenBank locus UniProt id Entrez gen

AKAP10 NM_007202 O43572 11216

RGS22 NM_015668 Q9BYZ4 26166

SNX SNX13 NM_015132 Q9Y5W8 23161

SNX SNX14 NM_153816 Q9Y5W7 57231

SNX SNX25 NM_031953 Q9H3E2 83891

RA Axin NM_003502 O15169 8312

RA Axin2 NM_004655 Q9Y2T1 8313

GRK GRK1 NM_002929 Q15835 6011

GRK GRK2 NM_001619 P25098 156

GRK GRK3 NM_005160 P35626 157

GRK GRK4 NM_182982 P32298 2868

GRK GRK5 NM_005308 P34947 2869

GRK GRK6 NM_002082 P43250 2870

GRK GRK7 NM_139209 Q8WTQ7 131890

GEF ARHGEF1 NM_004706 Q92888 9138

GEF ARHGEF11 NM_014784 O15085 9826

GEF ARHGEF12 NM_015313 Q9NZN5 23365

(Axin, Axin2) that each possess a single, central RGS

domain of poorly characterized or controversial Ga-modulatory function.

As previously mentioned, early reports of the dis-

covery of the RGS protein family highlighted the pres-

ence of an N-terminal RGS domain within the known

family of serine/threonine kinases (GRK1 to 7;

Table 2) that are specific for activated GPCRs

(Siderovski et al. 1994; Siderovski et al. 1996); subse-

quent examination of this N-terminal RGS domain

within GRK2 revealed in vitro Gaq binding selectivityand a cellular function in inhibiting Gq-coupled GPCR

signaling, albeit with little (if any) Gaq-directed GAP

activity. N-terminal RGS domains were also identified

in guanine nucleotide exchange factors for the small

GTPase RhoA (i.e., the GEF-subfamily of RGS

proteins; namely, p115-RhoGEF/ARHGEF1, PDZ-

RhoGEF/ARHGEF11, and LARG/ARHGEF12).

This identification helped to explain the ability of

G12/13-coupled GPCRs to activate RhoA in a cellular

context; therefore, the GEF-subfamily of RGS proteins

main(s)

e id Distinguishing characteristic(s)

Contains 2 RGS domains which interact with Rab4 and

Rab11 GTPases

Contains 3 RGS domains; specifically expressed in testes

Also known as RGS-PX1; controversial report of Gas-directed GAP activity

Also known as RGS-PX2

Sorting nexin-25; speculated to bind phosphatidylinositols

with its PX domain

Involved in Wnt signaling; component of b-catenindestruction complex

Also known as conductin; regulator of centrosome

cohesion

Also known as rhodopsin kinase

Also known as b-adrenergic receptor kinase-1 (bARK1);RGS domain binds activated Gaq/11Also known as b-adrenergic receptor kinase-2 (bARK2)Linked to genetic and acquired hypertension

Modulator of NFkB signaling via IkBa interaction

Involved in phosphorylation and desensitization of

CXCR4

Involved in cone phototransduction

Also known as p115 RhoGEF; Ga12/13-dependentexchange factor for RhoA GTPase

Also known as PDZ-RhoGEF

Also known as LARG or “leukemia-associated RhoGEF”

R

R 1660 RGS Proteins

are emblematic of RGS domain-containing proteins

that serve as effectors (i.e., propagating the signal

forward) even while they also serve as GAPs for their

upstream activators (i.e., activated Ga12·GTP and

Ga13·GTP subunits).

Summary

Originally discovered as negative regulators of GPCR

signal transduction owing to their Ga-directed GAP

activity, the RGS proteins are now appreciated to

possess multifaceted functions in cellular signaling

networks. These multiple functions can arise from

elaborate, multiple protein-domain architectures,

unique binding partners, and their individual abilities

to coordinate and/or modulate other signal transduc-

tion components, such as small Ras-superfamily

GTPases. Unique expression patterns and Ga-bindingselectivities of the RGS proteins underlie their individ-

ual involvement in distinct physiological and patho-

physiological phenomena. What remains to be

determined is whether RGS proteins can be selectively

inhibited by small molecules and, even more specula-

tively, whether their activity could be enhanced by

small molecules that usurp normal allosteric control

over the Ga-binding A-site.

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effector complex: emerging importance of RGS proteins.

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Berman DM, Wilkie TM, Gilman AG. GAIP and RGS4 are

GTPase-activating proteins for the Gi subfamily of

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protein RGS4 stabilizes the transition state for nucleotide

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RGS Proteins

▶RGS Protein Family

RGS13 1661 R

RGS13

Zhihui Xie and Kirk M. Druey

Laboratory of Allergic Diseases, National Institute of

Allergy and infectious Diseases, National Institutes of

Health, Bethesda, MD, USA

Synonyms

Regulator of G-protein Signaling 13

R

Historical Background

Regulator of G-protein signaling (RGS) is a protein

superfamily discovered in the mid-1990s (Druey et al.

1996; Watson et al. 1996; Hunt et al. 1996). Since that

time more than 30 members in the family have been

identified. The major physiological function of RGS

molecules is to negatively modulate G-protein

coupled receptor (GPCR)-mediated signaling and

biology (Bansal et al. 2007; Neitzel and Hepler

2006; Thompson et al. 2008). ▶RGS13 was first

identified in 1996 and is one of the smallest molecules

in the family (Druey et al. 1996). Full length human

RGS13 cDNA was cloned and deposited into gene

bank in 1997 by Chatterjee and Fisher. In 2002, the

first functional studies of human RGS13 were

reported. RGS13 was shown to inhibit muscarinic

M1- and M2 receptor-induced ▶MAP kinase activa-

tion (Johnson and Druey 2002). Concurrently, a sep-

arate study characterized mouse RGS13, which was

cloned from B cells. RGS13 expression was

demonstrated in germinal center regions of mouse

spleen and shown to inhibit chemokine receptor

CXCR4 and CXCR5-mediated signaling pathways,

suggesting a function of RGS13 in B cell migration

and trafficking (Shi et al. 2002).

More recently, GPCR-independent functions

of RGS13 were revealed. RGS13 suppressed IgE

receptor-induced mast cell degranulation through

interaction with the p85 subunit of ▶ phosphoi-

nositide 3-kinase (Bansal et al. 2008b). Mice deficient

in RGS13 have enhanced IgE-mediated anaphylaxis

after antigen challenge. RGS13 was also shown to

regulate cAMP-dependent pathways. RGS13 bound

phosphorylated cAMP responsive element binding

protein (▶CREB) in the nucleus and inhibited

CREB-mediated gene transcription (Xie et al. 2008).

Expression Pattern

RGS13 has very restricted tissue distribution expressed

mainly in B cells (Shi et al. 2002), mast cells (Bansal

et al. 2008b), and enteroendocrine cells (Xie et al.,

unpublished observation). Central nervous system

expression of RGS13 mRNA has been observed in rat

brain by in situ hybridization (Grafstein-Dunn et al.

2001), but not in the human brain (Larminie et al.

2004). The expression level of RGS13 may vary con-

siderably under physiological or pathological condi-

tions. For example, although RGS13 is highly

expressed in germinal center B cells and Burkitt lym-

phoma, it is absent in mantle cell lymphoma (Islam

et al. 2003). Upregulated RGS13 mRNA has been

reported in malignant T cells from acute T cell leuke-

mia (ATL) patients by gene expression profiling (Pise-

Masison et al. 2009); however, it was not detected in

normal human tonsil T cells or in Jurkat and MOLT-4

T cell lines (Shi et al. 2002). In BXD2 autoimmune

mice, increased IL-17 production is associated with

elevated RGS13 mRNA expression and the suppres-

sion of B cell chemotactic responses to the chemokine

CXCL12 (Hsu et al. 2008). Restricted tissue distribu-

tion and apparent disease relevance make RGS13 an

excellent target for drug development.

Regulation of RGS13 Expression

Similar to many RGS molecules, RGS13 expression is

relatively low in quiescent cells, which may be neces-

sary for allowing adequate GPCR signaling to main-

tain cell homeostatic functions. Incubation of bone

marrow–derived mast cells (BMMCs) with IgE-

antigen for 24 h results in a four- to fivefold increase

in RGS13 mRNA and protein (Bansal et al. 2008a),

which may have a negative feedback role to amplify

the inhibitory effect of RGS13 on antigen-mediated

mast cell degranulation. In other words RGS13 may

desensitize mast cells to antigen stimulation. Interest-

ingly exposure of human LAD2 mast cells to cAMP

decreases RGS13 mRNA quantities (Xie et al. 2010),

suggesting that RGS13 expression is differentially

regulated by distinct ligands or signaling pathways.

R 1662 RGS13

In human tonsillar B lymphocytes, anti-▶CD40 anti-

body augments RGS13 mRNA expression (Shi et al.

2002), suggesting a role of RGS13 in B cell activation.

Consistent with its expression in splenic germinal cen-

ter B cells, human Burkitt lymphoma cell lines Ramos,

HS-Sultan, and Raji express abundant RGS13, as do

immunized mouse spleen B cells (Shi et al. 2002).

Notably, the tumor suppressor ▶ p53 inhibits RGS13

mRNA transcription in mast cells by binding to

its promoter region (Iwaki et al. 2011).

RGS13 is also regulated at the posttranslational

level. RGS13 protein undergoes proteasome-mediated

degradation. Phosphorylation of RGS13 on Thr41 by

protein kinase A (PKA) protects it from degradation,

resulting in elevated steady-state RGS13 protein levels

(Xie et al. 2010), which could promote the inhibitory

function of RGS13 on transcription factor CREB.

Biological Functions

GPCR-Dependent Functions

RGS proteins interact with a subunit of the

heterotrimeric G-protein in its activated (GTP-bound)

state, leading to increased intrinsic GTPase activity of

Ga. This GTPase activating protein (GAP) activity

increases the rate of GTP hydrolysis, which hastens

deactivation/termination of GPCR signaling and func-

tions (Patel 2004). RGS13 interacts with the a subunit

of Gi and Gq, but not ▶Gs, accelerating the GTPase

activity of Ga (7,8). MAP kinase Erk1/2 activation

stimulated by Gq-coupled muscarinic M2 receptor is

significantly inhibited by overexpression of RGS13 in

human embryonic kidney (HEK) 293 T cells (Johnson

and Druey 2002). Overexpression of RGS13 in Chi-

nese Hamster Ovary (CHO) cells inhibited the Gi-

coupled chemokine receptor CXCR4-mediated cell

migration (Shi et al. 2002).

SiRNA-mediated knockdown of endogenous

RGS13 in HS-Sultan cells results in enhanced Ca2+

flux and chemotaxis induced by the chemokines

CXCL12 and CXCL13, which utilize Gi-coupled

GPCRs CXCR4 and CXCR5, respectively (Han et al.

2006). Depletion of RGS13 in the human mast cell

lines LAD2 and HMC-1 by shRNA increases degran-

ulation evoked by the GPCR ligand sphingosine-1-

phosphate (S1-P), and greater Ca2+ mobilization in

response to several GPCR ligands including C5a,

adenosine, and S1P (Bansal et al. 2008a).

GPCR-Independent Functions

In recent years, GPCR-independent cellular functions

of RGS13 have been characterized, which do not

involve classical RGS13 GAP activity. Cross-linking

of the IgE receptor FceRI by antigen on mast cells

activates signaling molecules including Syk kinase,

phospholipase Cg, ▶LAT, and PI3 Kinase, leading to

mast cell degranulation and cytokine production

(Gilfillan and Tkaczyk 2006). Mice lacking RGS13

displayed enhanced systemic and local cutaneous ana-

phylactic responses when challenged with IgE/antigen

(Bansal et al. 2008b). BMMCs from Rgs13�/� mice

degranulated much more than those from wild-type

littermates. Interestingly cytokine generation by

BMMC after IgE/antigen stimulation were not affected

by RGS13 protein deficiency, indicating that RGS13

specifically regulates IgE-mediated mast cell degranu-

lation. Further analysis revealed that RGS13 binds to

the p85 subunit of PI3 kinase enzyme, which is

a critical downstream effector in the degranulation

pathways. RGS13 inhibited formation of an FceRI-associated p85-▶Gab2-Grb2 signaling complex,

which is required for mast cell degranulation (Bansal

et al. 2008b). Reconstitution of RGS13 deficient

BMMC with a GAP-inactive RGS13 mutant

suppressed antigen-stimulated degranulation similar

to wild-type RGS13, indicating that RGS13 GAP

activity is not required for its inhibition of mast cell

function (Bansal et al. 2008b).

A separate set of studies led to the discovery that

although RGS13 does not interact with Gas, it inhibitsGs-mediated signaling through the b2-adrenergicreceptor downstream of the G protein (Johnson and

Druey 2002). Further studies revealed that PKA acti-

vation induced by Gs-coupled b2-adrenergic receptor

stimulation leads to RGS13 accumulation in the

nucleus. RGS13 binds to the phosphorylated transcrip-

tion factor CREB in the presence of its co-activator

CREB-binding protein (CBP). Binding of RGS13 to

phosphorylated CREB eventually inhibited CREB

transactivation, resulting in decreased CREB target

gene expression (Xie et al. 2008).

Summary

RGS13 acts as a signaling modulator, playing

critical roles in both GPCR-dependent and GPCR-

independent cellular processes. The high expression

Rho-Associated Protein Kinase 1663 R

of RGS13 in murine germinal center B cells and its

suppression of chemokine-induced B cell migration

suggests a potential role of RGS13 in adaptive immune

responses. Altered expression of RGS13 in conditions

of autoimmunity or malignancy suggests disease rele-

vance. In the light of negative regulatory roles of

RGS13 in mast cell functions, upregulated RGS13

expression in mast cells repeatedly exposed to antigen

could increase inhibition of IgE/antigen-mediated

mast cell degranulation and/or anaphylactic responses.

Mast cell degranulation, which leads to release

of granular contents such as histamine, plays a key

role in many diseases including allergy, asthma,

mastocytosis, and anaphylaxis. Therefore, RGS13

could be a potential therapeutic target, especially

given its limited tissue distribution. Since most of the

studies conducted thus far have been in mice or in

murine cell lines, further investigations of human sub-

jects, such as expression patterns of RGS13 in health

and disease, will be of utmost importance.

Acknowledgments This study was supported by the Intramural

Research Program of the NIH, NIAID.

R

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RGS9 Anchor Protein (R9AP)

▶R7BP/R9AP

Rho Guanine Nucleotide ExchangeFactor 25

▶ARHGEF25

Rho Kinase

▶ROCK Kinases

Rho-Associated Coiled Coil Kinase

▶ROCK Kinases

Rho-Associated Kinase

▶ROCK Kinases

Rho-Associated Protein Kinase

▶ROCK Kinases

R 1664 RhoC (RHOC)

RhoC (RHOC)

Nicolas Reymond1, Francisco M. Vega2 and

Anne J. Ridley1

1Randall Division of Cell and Molecular Biophysics,

King’s College London, London, UK2Instituto de Biologıa Molecular y Celular del Cancer,

Centro de Investigacion del Cancer, Consejo Superior

de Investigaciones Cientıficas (CSIC)-Universidad de

Salamanca, Salamanca, Spain

Synonyms

ARH9; ARHC; Ras homolog gene family, member C

Historical Background

RhoC was originally identified, together with its homo-

logues RhoA and RhoB, as a Ras-related small GTPase

(Madaule and Axel 1985). RhoA, RhoB, and RhoC

together comprise the Rho subfamily of small GTPases

characterized by their high homology within the Rho

GTPase family. The human rhoc gene is on chromo-

some 1p13.1-p21. The rhoc gene is proposed to origi-

nate from a duplication of rhoa during evolution

(Boureux et al. 2007). RhoA and RhoC are 93% identical

at the protein level; the divergence is mainly concentrated

in the so-called hypervariable region of the proteins at the

C-terminus. Although multiple Rho subfamily GTPases

exist in many eukaryotic organisms, specific RhoC

orthologs do not exist outside vertebrates (Boureux et al.

2007). This together with the fact that, like RhoA, when

RhoC is overexpressed in cells it induces the formation of

actin stress fibers means that its specific functions were not

investigated until relatively recently. It was originally

described to regulate reorganization of the actin cytoskele-

ton and cell shape, attachment, and motility in a similar

way to RhoA. The focus of attention shifted when it was

discovered thatRhoCbutnotRhoAspecifically contributes

to cancer metastasis (Clark et al. 2000). Since then many

studies have investigated the different functions of RhoA,

RhoB, and RhoC in cell biology and disease processes.

Tools for the Study of RhoC Function

Classic tools for the study of Rho GTPases, including

RhoC, took advantage of some conserved amino acids

essential for GTP hydrolysis to make dominant nega-

tive (T19N substitution) or constitutively active

(G14V or Q63L substitutions) forms of the protein

(Fig. 1; Wheeler and Ridley 2004). However, these

mutations have the disadvantage that they probably

do not allow the functional specificity of closely

related Rho GTPases like RhoA and RhoC to be

explored. Bacterial toxins, like Clostridium Botulinum

exoenzyme C3 transferase or Toxin B, efficiently

inhibit Rho activity by covalently modifying the

protein in key residues, but again they target all Rho

subfamily proteins and, although they were central to

establishing the role of these proteins in actin cytoskel-

etal organization, they cannot be used to study RhoC-

specific functions. More recently, RNA interference

(RNAi) has been used to differentiate the functions

for RhoA, RhoB, and RhoC in cells. This has

shown that RhoA and RhoC regulate cytoskeletal

dynamics, cell morphology, migration, and invasion

in different ways (Bellovin et al. 2006; Wu et al. 2010;

Bravo-Cordero et al. 2011; Vega et al. 2011).

Spatio-temporal regulation of Rho GTPase activity

is crucial to cell migration and invasion. The combined

use of live-cell imaging and fluorescence biosensors is

now helping to decifer how the activity of RhoC and

other Rho GTPases is tightly regulated on a subcellular

level. For example, using a RhoC -specific biosensor in

cancer cells, dynamic RhoC activation in invadopodia

has been visualized (Bravo-Cordero et al. 2011).

Regulation of RhoC Activity

RhoC is a GTPase that act as a molecular switch,

cycling between a GDP-bound inactive state and

a GTP-bound active state. When active it interacts

with effector proteins to regulate a variety of different

processes (Wheeler and Ridley 2004). Its activation is

positively regulated by guanine nucleotide exchange

factors (GEFs), which stimulate the exchange of GDP

for GTP on the protein, and negatively regulated by

GTPase-activating proteins (GAPs), which stimulate

its intrinsic GTPase activity. RhoC is also posttransla-

tionally modified at the C-terminus by prenylation with

the addition of a geranylgeranyl group (Fig. 1). This

modification allows its anchorage to membranes and is

likely to be essential for its biological function. Similar

to several other Rho GTPases, RhoC associates with

Rho GTPase dissociation inhibitor proteins (RhoGDIs),

Effector domain

Rho insert domain

CAAX box

Switch 1

G14T19

Q63

193C-Terminal portion

...QARRGKKKSGCLVL

...QVRKNKRRRGCPIL

GG

RhoC

RhoA

1 Switch 2

Regions involved in GTP orGDP binding

Hypervariable region

RhoC (RHOC), Fig. 1 RhoC domain structure and features.

Schematic of RhoC protein showing the different domains and

residues important for its activity and used to create dominant

negative and constitutively active mutations as described in the

text. The C-terminal 14 amino acids, including the CAAX box,

and comparison with the equivalent region of RhoA is shown.

Red amino acids indicate nonconserved residues. GG depicts the

prenylation site; following geranylgeranlylation the last three

amino acids are cleaved off

RhoC (RHOC) 1665 R

R

which sequester the protein in the cytoplasm by

interacting with the C-terminal geranylgeranyl group.

Thesemodes of regulation are sharedwith the other Rho

proteins, RhoA and RhoB. The different functions of

RhoC compared to RhoA and RhoB are thought to be

achieved throughRhoC-specificGEFs and/orGAPs and

by its binding to specific effector proteins.

Most regulators of Rho subfamily proteins have

only been tested on RhoA, and based on the sequence

similarity with RhoC would be predicted to act on both

proteins. The ones that have been specifically tested on

RhoC are the GEFs Tim and Scambio, the GAPs

p190RhoGAP, GRAF, p50RhoGAP and Myr5 and

the 3 RhoGDI proteins (Bos et al. 2007). Few regula-

tors have been compared for their activity on RhoA

versus RhoC. One of the few examples of a specific

regulator comes from studies of the RhoGEF

ARHGEF3 (also known as XPLN), which acts on

RhoA and RhoB but not RhoC, but no RhoC-selective

GEFs or GAPs have been described so far (Arthur et al.

2002; Bos et al. 2007). A report described enhanced

RhoC activity compared to RhoA in pancreatic carci-

noma cells which correlated with an increase of RhoC

membrane localization (Dietrich et al. 2009); this

might reflect differential regulation of Rho isoform

interaction with RhoGDIs.

RhoC expression is ubiquitous but its levels are var-

iable between different tissues. Overexpression ofRhoC

has been reported in a variety of pathological conditions,

particularly in more aggressive metastatic cancers, and

this indicates that its expression is regulated and plays

a role in its physiological function. A newly discovered

way in which RhoC expression and activity can

be regulated in cells is by the action of noncoding

microRNAs. In breast cancer, for example, the

overexpression of the microRNA miRNA-10b induces

the upregulation of RhoC by inhibiting the translation of

themessenger RNA encodingHOXD10, and this in turn

promotes invasion and metastasis (Ma et al. 2007). In

squamous cell carcinoma, the downregulation of

microRNA-138 induces metastasis by reducing direct

RhoC mRNA degradation (Jiang et al. 2010).

Some Rho proteins are subject to posttranslational

modifications that regulate their stability or activity.

Notably, the residue Ser188 present in RhoA that is

subject to protein kinase A and protein kinase

G phosphorylation is not conserved inRhoC (Ellerbroek

et al. 2003). RhoA is also ubiquitinated and thereby

targeted for proteasomal degradation, but RhoC does

not appear to be regulated similarly (Chen et al. 2009). It

is possible that as-yet-identified residues in RhoC could

be subject to phosphorylation, ubiquitination, or other

posttranslational modifications.

RhoC Effectors

Because RhoA, B, and C possess a high level of iden-

tity at the protein level, they share many common

downstream effectors (Wheeler and Ridley 2004).

R 1666 RhoC (RHOC)

The affinity of these interactions may vary due to their

amino acid sequence differences. These interactions

all involve the Rho switch I and II regions (see

Fig. 1) and Rho-binding domains (RBDs) of their

effectors, which include Rho-associated kinase

(▶ROCK-1 and-2), Protein kinase N (PKN1-3, also

known as PRKs), Citron kinase, mDia1-3, Rhotekin,

and Rhophilin-1. RhoC and RhoA also interact with

Phospholipase C e (PLC-e) via its catalytic core.

RhoC, but not RhoA, has recently been reported to

bind to the Formin-like family members FMNL2 and

FMNL3 (Kitzing et al. 2010; Vega et al. 2011). RhoC

also binds to IQGAP1 (Wu et al. 2011).

RhoC Functions in Tumorigenesis

Formation of Metastases

RhoC expression and activity is often increased in can-

cer and correlates with progression, metastasis

formation and therefore a poor prognosis for patients

(Vega and Ridley 2008). Increased RhoC expression in

cancer was first identified in a screen for genes

upregulated in melanoma metastases (Clark et al.

2000). RhoC expression was subsequently found to be

upregulated in a variety of cancers including prostate

cancer, breast cancer, gastric cancer, ovarian cancer,

bladder cancer, hepatocellular cancer, pancreatic ductal

adenocarcinoma, non-small cell lung carcinoma

(NLCLC), oesophageal squamous cell carcinoma,

head and neck squamous cell carcinoma, and skin

squamous cell carcinoma (Karlsson et al. 2009). RhoC

has been shown to play a causal role in metastasis in

animal models. Initial studies found that overexpression

of dominant and negative forms ofRhoC correlatedwith

the formation or the inhibition of experimental

lung metastases, respectively (Clark et al. 2000).

Subsequently, it was shown using RhoC-null mice that

RhoC was dispensable for breast cancer initiation and

growth but confirmed that RhoC is critical for formation

of metastases (Hakem et al. 2005). The inhibition of

RhoC has since been described to reduce cancer cell

invasion and metastasis in several in vitro and in vivo

cancer models. RhoC is now proposed to be a marker

for poor prognosis in many different cancers.

No genomic or somatic mutations in human cells

have been described for RhoC. Somatic mutations

have been reported in the RhoA and RhoC downstream

effector ▶ROCK-1 (Lochhead et al. 2010).

Migration and Invasion

Recent studies show that RhoC has a unique role in cell

migration, distinct from RhoA, which could underlie

its specific contribution to cancer cell invasion and

metastasis (Fig. 2). For example, RhoC expression is

increases during colon carcinoma cell epithelial-

mesenchymal transition (EMT) and regulates

EMT-induced migration (Bellovin et al. 2006). RhoC

promotes polarized cell migration and invasion by

controlling cell spreading and Rac1 activation around

the cell periphery hence restricting lamellipodial

broadening (Vega et al. 2011). RhoC regulates breast

cancer cell adhesion to the extracellular matrix and

motility and invasion by modulating the expression

and co-localization of a2 and b1 integrins on collagen

I (Wu et al. 2011). RhoC is also implicated in the

degradation of extracellular matrix as it is involved in

the formation of matrix-degrading invadopodia in can-

cer cells: an active ring of RhoC restricts Cofilin activ-

ity and focuses invadopodial protrusion and matrix

degradation (Bravo-Cordero et al. 2011). In addition,

RhoC coordinates prostate cancer cell invasion in vitro

by activating the protein kinases Pyk2, FAK, MAPK,

and AKT, which results in activation of the matrix-

degrading metalloproteinases 2 and 9 (MMP2 and

MMP9; Fig. 2) (Iiizumi et al. 2008). RhoC is also

involved in the transcriptional program that controls

the TGFß1-induced switch from cohesive to single-

cell motility in breast cancer cells (Giampieri et al.

2009).

Angiogenesis

RhoC can stimulate the production of pro-angiogenic

factors by breast cancer cells (Merajver and Usmani

2005). RhoC is a downstream effector of vascular

endothelial growth factor (VEGF) in endothelial cells

and cancer cells. RhoC is thus essential for VEGF-

mediated angiogenesis induced by hepatocellular car-

cinoma cells (Wang et al. 2008). These pro-tumoral

functions could potentiate the vascularization of

tumors that express RhoC and also may facilitate can-

cer cell intravasation and extravasation during tumor

metastasis.

Proliferation and Apoptosis Resistance

Contradictory studies have been reported concerning

the role of RhoC in cancer cell proliferation and apo-

ptosis resistance, which remain to be clarified. On the

one hand, RNAi-mediated suppression of RhoC in

ActinMyosinll

MMP2MMP9Activity

AP1 familyTranscription

Factor

AngiogenesisFactor

ActinMicrotubules

Rhokinases

mDia FMNL3

Rac1 Cofilin

RhoC

Rhokinases

FMNL2

MIGRATION AND INVASION

LamellipodialBroadeningRestriction

InvadapodialProtrusions

Amoeboid vsMesenchymal

ShapeEMT

α2β1Integrin

Localisation

METASTASIS FORMATION

IQGAP1???

Tumour cell

Pyk2FAK

MAPKAKT

RhoC (RHOC), Fig. 2 RhoC functions in cancer. Schematic of

RhoC protein showing the different functions of RhoC in cancer

and its downstream effectors. Noncolored functions and

interactors are shared with RhoA while orange-colored

functions and interactors are RhoC-specific. Note the arrowbetween migration/invasion and metastasis formation is

a dotted line because not all of the RhoC effectors have been

tested in vivo

RhoC (RHOC) 1667 R

R

hepatocellular carcinoma cells showed that RhoC does

not regulate cancer cell proliferation in mice and that

depletion of RhoC in endothelial cells does not affect

their apoptosis (Wang et al. 2008). On the other hand,

RhoC depletion in human gastric carcinoma cells was

reported to inhibit proliferation and increase apoptosis

in vitro (Sun et al. 2007); and RhoC promoted human

oesophageal squamous cell carcinoma and breast can-

cer cell proliferation in mice in vivo (Faried et al.

2006).

RhoC Regulates Transcription Factors

RhoA is well known to regulate transcription through

actin-dependent and actin-independent effects on a

variety of transcription factors (Jaffe and Hall 2005).

Recent evidence indicates that RhoC also plays a role

in transcriptional regulation (Fig. 2). RhoC is induced

in melanoma cells by the transcriptional regulator

ETS-1. RhoC then indirectly stabilizes the AP-1 fam-

ily transcription factor c-Jun through the actin cyto-

skeleton (Spangler et al. 2011). c-Jun is an oncogene

which is a critical mediator of tumor development.

Summary

RhoC is a member of the small family of Rho GTPases

that is very closely related to RhoA and RhoB. It is best

known for its role in cell migration regulation and

control of actin cytoskeleton dynamics. Although

RhoC was originally considered to act similarly to

RhoA and share the same partners and functions,

there is now good evidence that RhoC has unique

functions in cells and is probably regulated by specific

R 1668 Rhodopsin Kinase

partners. Recently, some RhoC-specific downstream

effectors have been described and the analysis of

these interactions gave new insights into RhoC func-

tion in cells. This is reflected by studies in mouse

models, where RhoC has distinct functions to RhoA

in cancer metastasis. RhoC is upregulated in many

types of cancer and is a critical regulator of cancer

progression and metastasis formation. It is likely to

be useful as a prognostic marker in many types of

cancer and could be a possible target in cancer therapy.

The understanding of RhoC function in cells and

organisms will be advanced in the future by the use

of biosensors and fluorescence resonance energy trans-

fer methods to decipher where and when RhoC is

active and interacts with its partners.

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Bellovin DI, Simpson KJ, et al. Reciprocal regulation of RhoA

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Bos JL, Rehmann H, et al. GEFs and GAPs: critical elements in

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Boureux A, Vignal E, et al. Evolution of the Rho family of

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203–16.

Bravo-Cordero JJ, Oser M, et al. A novel spatiotemporal RhoC

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Chen Y, Yang Z, et al. Cullin mediates degradation of RhoA

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actin cytoskeleton structure and cell movement. Mol Cell.

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Clark EA, Golub TR, et al. Genomic analysis of metastasis

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Dietrich KA, Schwarz R, et al. Specific induction of migration

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TGFbeta signalling switches breast cancer cells from cohe-

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Rhodopsin Kinase

▶G-Protein-Coupled Receptor Kinase 1 (GRK1)

Ric-8 1669 R

RhoK

▶G-Protein-Coupled Receptor Kinase 1 (GRK1)

Rh-Related Antigen

▶CD47

Ribosomal Protein S6 Kinase, 90kD,Polypeptide 5

▶MSK1

Ribosomal Protein S6 Kinase,Polypeptide 5

▶MSK1

RIBP

▶ SH2D2A

R

Ric-8

Gregory G. Tall

Department of Pharmacology and Physiology,

University of Rochester Medical Center,

Rochester, NY, USA

Synonyms

Resistance to inhibitors of cholinesterase 8; Ric-8A;

Ric-8B; Synembryn

Historical Background

Discovery of Ric-8 proteins: The Caenorhabditiselegans RIC-8 gene and a homologous mouse gene

that was later termed Ric-8A (or a synembryn) were

discovered by Miller and Rand using a genetic screen

to obtain C. elegans mutants that were resistant to the

inhibitor of cholinesterase, aldicarb (Miller et al.

1996). Aldicarb treatment of wild-type worms leads

to neurotoxic accumulation of postsynaptic acetylcho-

line and subsequent death. Ric mutants lived in the

presence of aldicarb because they contained gene

defects that restored normal acetylcholine levels, pri-

marily by decreasing neurotransmitter secretion or

release. Through epistasis analyses, the gene

complementing the ric-8 mutant allele was predicted

to elicit action upstream of or parallel to the gene

encoding G protein a q in a diacylglycerol-dependent

synaptic-vesicle-priming pathway (Miller et al.

2000a). Miller and Rand also first showed that centro-

some (spindle pole) movements in the dividing

C. elegans zygote were perturbed in ric-8 mutants,

a phenotype shared by G protein ao(i) mutants (Miller

and Rand 2000b).

Ric-8 proteins were first linked physically to

▶G protein a subunits when two mammalian Ric-8

homologues were identified by the ability to bind Gaoand Gas directly in yeast two-hybrid screens and in

purified protein–protein binding and functional

assays (Tall et al. 2003). The two homologues are

the products of separate genes and were named mam-

malian Ric-8A and Ric-8B. Ric-8A binds Gai, Gaq,and Ga12/13 class G protein a subunits, whereas

Ric-8B binds the Gas, Gaq, and Ga12/13 classes.

Preliminary experiments demonstrating a preferred

interaction of Ric-8A with the GDP-bound form of

G protein a led to the hypothesis that Ric-8A might

alter G protein catalysis by serving as an alternative

guanine nucleotide dissociation inhibitor (GDI), or a

non-receptor guanine nucleotide exchange factor

(GEF). Purified Ric-8A stimulated intrinsic G protein

a GDP release dramatically leading to accelerated,

observed GTP(gS) binding kinetics (G protein activa-

tion) (Fig. 1). The mechanism of Ric-8A-stimulated

guanine nucleotide exchange was elucidated. Ric-8A

initially interacted with GDP-bound G alpha and stim-

ulated rapid GDP release. In the absence of GTP,

Ric-8A formed a stable nucleotide-free transition state

complex with Ga. Addition of GTP and magnesium

dissociated this complex to produce free Ric-8A and

activated Ga-GTP. Currently, a biochemical character-

ization of Ric-8B has not been made. Ric-8B is

predicted to be a Gas and Gaq-class GEF based on

Gαq100

a b

80

60

40

20

0

100

80

60

40

20

00 20 40 60 80 100 1200 10 20 30

+ Ric-8A

No addition

+ Ric-8A

No additionGαi1

Frac

tiona

l GT

PγS

bin

ding

Frac

tiona

l GT

PγS

bin

ding

Time (min) Time (min)

Ric-8, Fig. 1 Ric-8A is a Ga subunit GEF and accelerates the

kinetics of (a) Gaq and (b) Gai1 GTPgS binding. Purified Gasubunits (200 nM) were incubated in reactions containing

radiolabeled GTPgS with (squares), or without (circles) purifiedRic-8A (200 nM). At the indicated times, Ga and its bound

nucleotide were trapped on nitrocellulose filters and the amount

bound GTPgS was measured by scintillation counting. This

research was originally published in the Journal of BiologicalChemistry (4) # the American Society for Biochemistry and

Molecular Biology

R 1670 Ric-8

its Ga binding preferences. Since these initial observa-

tions of Ric-8 function, the field has endeavored

to understand how Ric-8 GEF activity regulates

G protein function in cells. Many of the predicted

Ric-8 physiological functions have been acquired from

genetic studies in model organisms. These include func-

tional interactions with varied G protein species to reg-

ulate neurotransmission, asymmetric cell division,

olfaction, and G protein residence at the plasma

membrane.

C. elegans RIC-8 and Gaq/Gas/Gao modulate neu-rotransmission. The original function ascribed to the

C. elegans RIC-8 gene was made by its identification

in the ric mutant screen designed to isolate mutants of

genes whose products positively regulated neurotrans-

mission (Miller et al. 1996). Gaq is one other such

gene. Various genetic and biochemical tests confirmed

that ric-8 mutants acted epistatically (upstream) to

other Gaq-pathway-dependent synaptic-vesicle-

priming defects. Gaq gain of function alleles,

diacylglycerol (DAG) kinase mutants (deregulates

DAG by phosphorylation), or application of phorbol

esters (DAG analogs) all suppressed the ric-8 mutant

neurotransmission defect (Miller et al. 2000a). These

and other collective observations indicated that

the RIC-8 protein likely acted upstream of Gaq to

promote neurotransmission through phospholipase

Cb-dependent DAG production and subsequent

UNC13 stimulation (see Fig. 2). UNC13 is a DAG

binding protein/sensor that interacts with the

membrane in a DAG-dependent manner. UNC13 also

interacts with syntaxin proteins and may regulate

SNARE-dependent vesicle fusion.

Ric-8 was later shown to be epistatic to Gas throughgenetic suppression experiments. Gain of function

mutants in Gas, ▶ adenylyl cyclase, or protein kinase

A individually suppressed the ric-8 paralysis defect

(Reynolds et al. 2005; Schade et al. 2005). This

showed that C. elegans RIC-8 gene action intersected

with a second G protein, Gas, to provide a cAMP

second messenger input into the neurotransmitter

release process. Collectively the C. elegans RIC-8

gene acts upstream of at least Gaq and Gas and may

maintain both G protein signaling pathways in acti-

vated states. It is not understood precisely how RIC-8

acted upon these divergent G proteins. The proposed

role was gathered from the observation that mamma-

lian Ric-8A acted as a Gaq GEF and directly activated

these G proteins in a manner apart from the action of

G protein–coupled receptors (GPCRs).

Ric-8 and G protein ai control of (asymmetric) celldivision: Asymmetric cell division (ACD) is a process

by which stem cells or progenitor cells divide such that

the two daughter cells adopt unique characteristics and

cell fate determinants during division (intrinsic ACD),

or acquire these characteristics after division by influ-

ence of the surrounding tissue/niche (extrinsic ACD).

ACD allows one daughter cell to retain stem/progeni-

tor potential and the other to adopt a committed fate.

Ric-8 and members of the G protein ai class of

RIC-8Gαq

(EGL30)Phospholipase Cβ

(EGL8)GPCR / serotonin

Diacylglycerol (DAG)Gαs (GSA1)

Adenylyl cyclase

Protein kinase ASynaptic vesicle priming /syntaxin-dependent fusion

DAG

UNC13 DAG kinase

Gαo (GOA1)

..

Ric-8, Fig. 2 Proposed pathways depicting C. elegans RIC-8control of Gaq- and Gas-dependent neurotransmission (Arrows:activation, Bars: inhibition). Genetic experiments showed that

the requirement of the RIC-8 gene for neurotransmission was

manifested before the action of the Gaq and Gas genes.

G proteins aq and as provide divergent inputs to regulate neu-

rotransmitter release through the activation of phospholipase

Cb and adenylyl cyclase, respectively. These two pathways

converge to modulate UNC13 function/localization and regula-

tion of synaptic vesicle priming. G protein ao provides an

inhibitory input into the pathway, perhaps through

diacylglycerol kinase that is regulated by a serotonin responsive

GPCR. Gao regulation was not necessarily thought to involve

RIC-8 (5–7)

Ric-8 1671 R

R

G proteins have an essential role in directing intrinsic

asymmetric-, and perhaps normal-cell division. Gairegulation in this context is thought to be GPCR-inde-

pendent, as Gai-family members reside not only on the

plasma membrane, but on intracellular mitotic struc-

tures including spindle poles (centrosomes), spindle

microtubules, and the cytokinesis midbody (Blumer

et al. 2006).

The protein machinery that works with Gai to con-

trol cell division is conserved and consists of compo-

nents discovered in worms, flies, and mammals. Gai,Ric-8(A), and orthologous GoLoco- or GPR-domain

containing proteins, GPR1/2, PINS, or LGN/AGS3 in

mammals are required for asymmetric cell division

(ACD) of multiple cell types, including human adult

progenitor cells. These and other conserved proteins in

the pathway, including NuMA/Lin5/MUDs receive

signals from cell polarity determinants to differentially

regulate the strength of pulling forces on the two sets of

aster microtubules during mitosis (for review and

a comprehensive account of primary references, see

Siderovski and Willard (2005), Siller and Doe (2009),

Wilkie and Kinch (2005), and Yu et al. (2006)). This

force differential pulls the entire mitotic spindle and

metaphase plate as a unit toward one side of the cell.

The asymmetric location of the metaphase plate marks

the position of the cleavage plane for cytokinesis.

Figure 3 depicts in cartoon format, the original obser-

vations demonstrating that the Ric-8, Gai/o, and

GPR1/2 genes were required for C. elegans zygote

ACD. When these genes were mutated individually,

zygotic cell division proceeded symmetrically, leading

to developmental catastrophe and death at gastrulation.

The majority of the genetic and cell biological work

delineating the influence of this nontraditional G protein

pathway on mitotic spindle dynamics has been learned

from ACD model systems including the C. eleganszygote, Drosophila neuroblast, and sensory organ pre-

cursor cell. It is clear that the network also operates in

mammals to direct both symmetric and ACD because

homologous and orthologous components of the path-

way influence keratinocyte (skin) progenitor cell ACD

(LGN, NuMA), neural precursor development (LGN,

AGS3, G protein subunits) (for review of primary liter-

ature see Siller and Doe (2009)), and division of COS

and HeLa cells (Gai, AGS3) (Blumer et al. 2006; Cho

and Kehrl 2007). Recently, Ric-8A andGai were shownto recruit LGN, NuMA, and the dynein microtubule

motor to the plasma membrane of mitotic HeLa cells.

Perturbation of Ric-8A expression, or its interaction

with Gai resulted in defective orientation of the HeLa

mitotic spindle to the substratum and aberrant cell divi-

sion (Woodard et al. 2010).

A biochemical demonstration of Ric-8A, Gai, andGoLoco protein concerted function accounts for how

Ric-8 GEF activity could supplant the lack of apparent

GPCR-mediated G protein activation in ACD and pro-

vides one cellular context for the GoLoco component

A P

A P

C. elegans wildtype zygote asymmetric cell division

ric-8, gαo, or gpr1 / 2 mutant zygote symmetric-like cell division

Ric-8, Fig. 3 C. elegans zygote asymmetric cell division is

perturbed in ric-8, gao, or gpr1/2 mutants. Zygote polarity is

determined by the site of sperm entry and designates the poste-

rior (P), and anterior (A) sides of the zygote. During prophase themitotic spindle (black microtubule bundles) rotates 90� (not

depicted) to orient the spindle poles (red color) along the ante-

rior and posterior axis of the zygote. Spindle rotation is an aster

microtubule (brown color)-dependent process and is slowed in

ric-8, gao, or gpr1/2 mutants. Chromosomes (blue color) areshown aligned at the metaphase plate with intact mitotic spin-

dles. The distance between spindle poles is reduced in ric-8, gao,or gpr1/2 mutants. During meta/anaphase, the posterior spindle

pole flattens and adopts a violent up and down rocking motion in

relation to the A-P axis (green arrow) as it and the entire mitotic

spindle is pulled toward the posterior side of the zygote by forces

generated from the posterior aster microtubules. Overall aster

microtubule force and the force differential between the anterior

and posterior aster microtubules are considerably weaker in

ric-8, gao, or gpr1/2 mutants. The mutants exhibit a very weak

rocking motion of the posterior spindle pole and reduced move-

ment of either spindle pole toward the cell cortex. As chromo-

some segregation occurs, the cytokinetic cleavage plane appears

asymmetrically in wild-type zygotes, closer to the posterior side.

Ric-8, gao, and gpr1/2 mutants lack asymmetrically oriented

spindles and subsequently divide symmetrically

R 1672 Ric-8

as an atypical guanine nucleotide dissociation inhibitor

(GDI). In conditions where traditional GPCR signaling

is turned off, Gbg subunits serve as a Ga protein GDI

and prevent GDP release. During nontraditional

G protein signaling in mitosis and ACD, LGN

(GoLoco) binds plasma membrane-bound Gai-GDPsubunits as a GDI. The nuclear mitotic apparatus pro-

tein (NuMA) is recruited in allosteric fashion to

a binding site on LGN that becomes available when

LGN is bound to Gai (Du and Macara 2004). NuMA is

not active in this complex with respect to its incapacity

to participate in direct interactions with microtubules

(Du et al. 2002). However, complexed NuMA may

interact with the dynein microtubule motor complex

(Siller and Doe 2009). Ric-8A dissociated a purified

Gai/GoLoco/NuMA complex by removing Gai-GDPfrom LGN or AGS3 (GoLoco) through stimulation of

nucleotide exchange and production of free Gai-GTP(Tall and Gilman 2005; Thomas et al. 2008). Once Gaiwas dissociated from LGN, the ability of NuMA to

bind GoLoco domains was decreased and NuMA was

released. Whether the intact GoLoco protein complex

or any of the released species (NuMA, GoLoco

ortholog, and/or Gai-GTP) regulate aster microtubule

pulling forces to direct spindle asymmetry is not

completely clear and is an area of considerable interest.

It is unlikely that a static, plasma membrane–bound

Ga-GDP:GoLoco complex is the sole form of the

G protein responsible for force generation, as rounds

of nontraditional G protein guanine nucleotide

consumption seem to be required (Wilkie and

Kinch 2005). Gai-GTP is inactivated by RGS

GTPase-activating-proteins, which presumably resets

the system for another cycle of Ric-8-mediated activa-

tion (Hess et al. 2004). Important unresolved questions

remain. Does Ric-8(A) actually perform this function

in cells and dissociate Gai/GoLoco/NuMA complexes

in the context of aster microtubule force regulation?

What is the precise ordering of molecular events that

occur downstream of Ric-8A, the intact GoLoco com-

plex, or the dissociated Gai-GTP, GoLoco, and/or

NuMA species that directly signal to the dynein

motor complex force generator (Fig. 4)?

Ric-8B and Gaolf regulation of olfaction: The sec-ond mammalian Ric-8 homologue was named Ric-8B

after it was discovered in yeast two-hybrid screens

intended to uncover Gas-interacting proteins (Tall

et al. 2003; Klattenhoff et al. 2003). Mammalian Ric-

8/synembryn was renamed Ric-8A at this time. Ric-8A

and Ric-8B share �40% overall amino acid identity.

Traditional G protein signaling:

Gβ

Gγ

GDP

GDP release GTP binding Signaling

Effectorenzymes

Gα

Gβ

Gγ GαGβ

Gγ

GTP

Gα

Alternative Gα signaling during asymmetric cell division:

GTP hydrolysis (C.e. RGS7)

Tetratricopeptide repeats

Ric-8A Ric-8A

GDP

GoLoco GoLoco GoLoco GoLoco

GoLoco GoLoco GoLoco GoLoco

GDP GDP GDP

GTP

GTP binding

?

?

??

GDP release

LGN / AGS3

NuMA

AsterMT forces

(Dynein / Dynactin)

Gα Gα Gα GαGα

Gα

Tetratricopeptide repeats

Ric-8, Fig. 4 Biochemical model comparing traditional GPCR-

mediated activation of Gabg trimers and alternative Ric-8(A)

activation of Gai/GoLoco/NuMA complexes. In the traditional

G protein signaling paradigm, inactive G protein trimers are

bound to GDP. Gbg serves as a GDI and prevents Ga subunit

GDP release in the absence of GPCR activating ligand (●). Gbgis also obligate for ligand-receptor complex stimulation of

G protein GTP for GDP nucleotide exchange of the Ga subunit.

Activated Ga-GTP and Gbg transduce signals to downstream

effector enzymes. In the proposed alternative Ga signaling

model that regulates aster microtubule force generation during

ACD, Ga-GDP is bound to the alternative GDI GoLoco.

A mammalian GoLoco ortholog, LGN, contains four carboxyl-

terminal GoLoco domains, and an amino-terminal

tetratricopeptide repeat region that binds NuMA. Ric-8A binds

Gai-GDP, and in the process dissociates GDP fromGai, and Gaiprocessively from GoLoco domains. GTP then rapidly binds the

Ric-8A:Ga nucleotide-free complex producing free Ric-8A and

Ga-GTP. As a consequence of Ga:GoLoco dissociation, NuMA

is released from LGN and could be available to participate in

enhanced interactions with microtubules or microtubule motors.

Elucidation of the complete repertoire of molecules, or direct

events that result in aster microtubule force generation is not

entirely clear (10). Evidence in favor of G protein a i catalytic

cycle involvement in this process comes from the finding that

mutants of C. elegans RGS7 (an activator of G protein GTP

hydrolysis) have many opposed phenotypes to ric-8, gao, andgpr1/2 mutants during ACD (19)

Ric-8 1673 R

R

The amino-terminal�400 amino acids are more diver-

gent (34% identity) and have been predicted to be

highly a-helical in content and consist of weakly scor-ing Armadillo repeats (Wilkie and Kinch 2005).

Ric-8A and Ric-8B share greater amino acid identity

between the carboxyl-terminal 130–160 amino acids

(�56% identity). No obvious protein sub-domains

are present in the predicted a-helical Ric-8 carboxyl-

termini, but the region in Ric-8B is alternatively

spliced.

Ric-8B binds all G protein alpha classes in vitro

with the exception of the Gai-class, and unlike Ric-8A,uniquely binds members of the Gas-family (Tall et al.

2003; Klattenhoff et al. 2003). Evidence supports

a role for Ric-8B in the regulation of Gaolf duringolfaction (Von Dannecker et al. 2006). Gaolf is the

olfactory/brain-specific Gas homologue that activates

adenylyl cyclase to stimulate olfactory nerve firing.

Malnic and colleagues portrayed functional differ-

ences between two expressed Ric-8B splice variants

Wildtype ric-8 mutant

a b

Ric-8, Fig. 5 Drosophila Gai was mis-localized and not

expressed on the plasma membrane when RIC-8 was P-element

disrupted. Epithelial cells from (a) wild-type or (b) ric-8 mutant

Drosophila embryos were stained with an anti-Gai antibody(Green) and a DNA stain (Red) (Adapted by permission from

Macmillan Publishers Ltd: Nature Cell Biology, (28). 2005)

R 1674 Ric-8

(full length, FL and deleted exon 9, D9).Overexpression of Ric-8BFL, but not Ric-8BD9enhanced Gaolf-dependent adenylyl cyclase activa-

tion (Von Dannecker et al. 2006). When Gaolf, Gb,Gg, odorant receptors, receptor co-factors, and Ric-

8BFL, but not Ric-8BD9, were co-transfected into

a heterologous system (HEK cells), functional odorant

receptor coupling was achieved. Application of odor-

ants activated Gaolf-dependent signaling (Von

Dannecker et al. 2006; Zhuang and Matsunami

2007). The unresolved question raised from these stud-

ies is: Does Ric-8B promote odorant receptor signaling

because it activates Gaolf/Gas as a guanine nucleotideexchange factor, or because it facilitates functional

Golf membrane expression in the heterologous system,

thereby enhancing Golf coupling to odorant receptors?

Overexpression of Ric-8BFL did increase the amount

of overexpressed Golf in a crude membrane fraction

(Kerr et al. 2008). It was later reported that the single

copy of RIC-8 in Xenopus appeared to stimulate mam-

malian Gas GTPgS binding, although the intrinsic rate

of Gas GTPgS binding reported in this study was

negligible (Romo et al. 2008). A positive demonstra-

tion of mammalian Ric-8B protein function as a Gas-class GEF has not been made as of yet (Nagai et al.

2010). Ric-8B is hypothesized to be a Gas and Gaq-class GEF since it interacts with these subunits in vitro,

and by analogy (and homology) to the described activ-

ities of Ric-8A. Ric-8A is a GEF for all Ga subunits

that it can bind.

Ric-8 proteins support heterotrimeric G protein

membrane expression: An alternative hypothesis of

Ric-8 protein function was proposed from work

performed on the sole copy of the Drosophila and

C. elegans RIC-8 genes. Attenuation of Drosophila

RIC-8 expression caused nearly complete and

pleotropic loss of multiple Ga subunits and the major

Gb (and presumably Gg) subunit from the plasma

membrane (for review and primary literature refer-

ences see Matsuzaki (2005)). Drosophila Gai(Fig. 5), Gao, and Gb13F subunits were expressed in

the cytosol or vesiculated compartments ofDrosophilaembryonic epithelial cells derived from larvae

possessing both a maternal and zygotic P-element dis-

ruption of RIC-8. Overall steady-state expression

levels of the mis-localized Gai and Gb were reduced

in the absence of RIC-8. These findings were corrobo-

rated at the same time in C. elegans when cortical

Gpa16 (a Gai homologue) localization was attenuated

in mitotic ric-8 mutant embryos (Afshar et al. 2005).

More recently, RNAi-mediated reduction of mamma-

lian Ric-8B resulted in reduced Gas expression, and

Ric-8B overexpression protected Gas from ubiquitin-

mediated degradation (Nagai et al. 2010). The provoc-

ative hypothesis arising from these findings is that the

function of Ric-8 protein(s) may be to serve as key

factors required for G protein subunit biosynthesis, or

trafficking to, or retention at the plasma membrane. It

is known that cytosolic G proteins are less stable than

membrane-bound G proteins, so a shift of G proteins

from the membrane to the cytosol in the absence of

Ric-8 would be realized as a reduction in G protein

steady-state expression levels. Aside from the finding

that Ric-8B overexpression reduced levels of

ubiquitinated Gas, there is little understanding of the

mechanism by which Ric-8-proteins mediate G protein

membrane localization.

Summary

The hypothesis that Ric-8 proteins are required for

efficient G protein membrane expression provides

a potential explanation, in part, for the findings that

Ric-8 proteins seem to exert affects upstream of diver-

gent G protein signaling pathways (i.e., Gaq, synaptictransmission, Gai, cell division, Gaolf olfaction, etc.).The one observation that cannot be explained intui-

tively by the idea that Ric-8 proteins are simply

factors required for G protein expression is that

Ric-8(A) is a mitotic spindle (pole) binding protein

(Fig. 6 and Woodard et al. 2010; Hess et al. 2004).

This result suggests that Ric-8A might participate as

Ric-8, Fig. 6 Ric-8A localizes proximally to the mitotic spindle

poles. HeLa cells were transfected with YFP-Ric-8A (Yellow-green) and stained with DAPI (Blue) and by immunofluores-

cence with anti-a-tubulin monoclonal antibody (Sigma) and an

anti-mouse Alexafluor-568 antibody. The tricolor images were

captured using a Zeiss epifluorescence microscope with appro-

priate filters and subjected to nearest-neighbors deconvolution

using Slidebook 4.0 software (unpublished observations)

Ric-8 1675 R

R

a non-receptor GEF to activate intracellular Gai-classG proteins that are localized on mitotic structures,

including the mitotic spindle.

There are multiple regulatory points where Ric-8

proteins could act to promote or stabilize G protein

membrane expression. Ric-8 proteins might act during

G protein biosynthesis in positive fashion to promote

stable membrane expression, or they could exert

a protective role upon mature membrane–bound

G proteins as these G proteins cycle between the

plasma membrane and endo-membranes. Biosynthetic

(forward) trafficking of G proteins to the plasma mem-

brane requires two basic events: (1) G protein

heterotrimer assembly on the endoplasmic reticulum

(ER) and/or Golgi and (2) covalent attachment of

lipid moieties to Ga and Gg subunits (Marrari et al.

2007). Chaperone proteins including phosducin-like

protein-1 (PhLP) and DRIP78 bind nascent Gb and

Gg proteins, respectively, prior to Gbg dimer forma-

tion (Dupre et al. 2007; Lukov et al. 2005). No com-

ponent is known that binds nascent Ga after its

synthesis, and aids its assembly into G protein trimers.

Ric-8 proteins could fulfill such a role. In the absence

of Ric-8 expression, Ga subunits not assembled into

trimers would not reach the plasma membrane and

would be more susceptible to degradation.

Ric-8 proteins could also potentially function in

a capacity to counteract G protein downregulation by

promoting G protein subunit recycling to the mem-

brane. Debate exists whether G protein subunits

become “solubilized” from membranes or clustered

into membrane micro-domains upon activation by hor-

mone receptors. Ric-8A and Ric-8B reside predomi-

nantly in the cytosol. Perhaps Ric-8 proteins function

to “scavenge” cytosolic Ga subunits to restore mem-

brane association. Upon experimental perturbation of

Ric-8 protein expression, Ga subunits might slowly

“leach off” the membrane over time due to tonic

GPCR stimulation. If the pace of Ga biosynthesis

does not keep up, this would be realized as

a reduction in steady-state G protein expression.

G proteins also cycle between the plasma membrane

and Golgi membranes, perhaps as a means to receive

reversible palmitoylation at the Golgi. Heterotrimeric

G protein retrograde movement to the Golgi occurs at

a rate faster than that predicted of a vesicular-trans-

port-mediated event (t1/2 <1 min) (Chisari et al. 2007;

Tsutsumi et al. 2009). As such, G proteins were

predicted to transit diffusively through the cytosol to

the Golgi. It is not entirely clear whether G proteins

transit as heterotrimers, or as free Ga or Gbg species. Ifthe latter, one could easily envision that an escort

factor would be required to transit Ga (and Gbg) tothe proper intracellular membranes lest it signal inap-

propriately during transport. Small G proteins includ-

ing Ras and Rab homologues share this type of

membrane cycling principle. Rab GTPases utilize

a combination of soluble GDI, GEF, and “escort”

factors to shuttle unlipidated Rabs throughout the

cell, present Rabs to the lipidation machinery, and

target the lipidated Rabs to the correct cellular mem-

brane compartment (Ali and Seabra 2005).

Given the known in vitro biochemical function of

Ric-8 proteins as soluble G protein a subunit GEFs, it

is a challenge, although conceivable, to envision

a role for Ric-8 protein involvement in any one of

these G protein subunit trafficking or biosynthetic

processes. In this context, Ric-8 “GEF activity”

could serve simply as a mechanism to regulate Ga:Ric-8 binding/dissociation. In this highly prospective

role, Ric-8 would bind Ga subunits (either nascent

chains, or mature membrane Ga) and traffic/escort theRic-8:Ga nucleotide-free complex to the proper des-

tination (the Golgi, or Gbg on the ER or plasma

membrane). At the destination, Ric-8 would be

R 1676 Ric-8

invoked to stimulate GTP binding to Ga and release

an activated-Ga species. Ga would hydrolyze its

bound GTP, return to the inactive state, and

reassociate with Gbg or GoLoco.

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Ric-8A

▶Ric-8

Ric-8B

▶Ric-8

Rin (Ras-Like Protein in Neurons)

Weikang Cai, Jennifer L. Rudolph and

Douglas A. Andres

Department of Molecular and Cellular Biochemistry,

University of Kentucky College of Medicine,

Lexington, KY, USA

R

Synonyms

Small G-protein; Small GTPase; Small GTP-binding

protein

Historical Background

Ras superfamily small GTP (guanosine triphosphate)-

binding proteins function as molecular switches,

responding to extra- and intracellular stimuli to control

the activity of diverse signaling cascades. To date, over

150 different small GTPases have been identified and

are classified into six distinct subfamilies: Ras (Rat

sarcoma), Rho (Ras homolog gene family), Rab (Ras-

related GTP-binding protein), ARF (ADP-ribosylation

factor), Ran (Ras-related nuclear protein), and RGK

(Rad/Gem/Kir family), based upon both sequence

homology and the regulation of common cellular func-

tions (Colicelli 2004). Rin (Ras-like protein in

neurons), along with Rit (Ras-like protein in many

tissues) and Drosophila Ric (Ras-related protein

which interacted with calmodulin), comprise the Rit

subfamily of Ras-related small GTPases (Lee et al.

1996). Rin is expressed exclusively within neurons

and has been characterized as regulating neuronal dif-

ferentiation by controlling distinct signaling cascades

(Lee et al. 1996; Shi et al. 2005, 2008; Spencer et al.

2002). Rin contributes to nerve growth factor (NGF)-

mediated neurite outgrowth via ERK (extracellular

signal regulated MAP kinase) and p38 MAP

kinase pathways (Shi et al. 2005), whereas in

pituitary adenylate cyclase-activating polypeptide 38

(PACAP38)-mediated neuronal differentiation, Rin

controls the cAMP (cyclic adenosine monophosphate)

/PKA (protein kinase A) signaling cascade (Shi et al.

2008). In addition, Rin is involved in Brn-3a and

Plexin signaling (Calissano and Latchman 2003;

Hartwig et al. 2005). Rin also associates with the

polarity protein PAR6 and has been found to display

modest transforming ability (Hoshino et al. 2005).

Biochemical Characterization of Rin GTPase

Rin was originally isolated from mouse retina, and

both Northern analysis and in situ hybridization studies

indicated that it is expressed solely in neurons (Lee

et al. 1996). Rin possesses most of the common char-

acteristics of Ras subfamily members, including a high

degree of amino acid conservation within the five Ras

core regions (the G1-G5 domains) which are essential

for GTP binding and hydrolysis (Colicelli 2004).

However, Rin also contains a number of unique fea-

tures including calmodulin (CaM) binding (Harrison

et al. 2005), the lack of prenylation consensus sites or

other lipidation signals within the C-terminus, and an

effector domain that differs from other Ras subfamily

proteins (Lee et al. 1996). These features have gener-

ated speculation that Rin might regulate important

aspects of neuronal growth and differentiation

(Hoshino and Nakamura 2003; Shi et al. 2005, 2008;

Spencer et al. 2002).

Recombinant Rin has been shown to specifically

bind guanine nucleotides (guanosine triphosphate or

guanosine diphosphate, GTP/GDP) in the presence of

2+

R 1678 Rin (Ras-Like Protein in Neurons)

Mg and to exhibit low intrinsic GTPase activity

(Shao et al. 1999). Surprisingly, the guanine nucleotide

dissociation rates for purified Rin protein are signifi-

cantly different when compared with the majority of

Ras-related GTPases. Rin displays higher koff values

for GTP than GDP (Shao et al. 1999). These GTP

dissociation rates are five- to tenfold faster than most

Ras-like GTPases. Since the cellular concentration of

GTP is much higher than that of GDP, these biochem-

ical studies suggest that a relatively high percentage of

Rin may remain in the GTP-bound state under basal

conditions. Despite these unique biochemical proper-

ties, the available data indicate that Rin functions as

a nucleotide-dependent molecular switch, and as

predicted, only a very low in vivo level of GTP-

bound Rin is found in unstimulated cells (Shi et al.

2008; Spencer et al. 2002). Therefore, these in vitro

studies do not appear to accurately represent Rin activ-

ity in the normal cellular environment. It is possible

that the presence of cellular regulatory proteins con-

tribute to these differences.

Rin Cellular Distribution and Trafficking

For the majority of Ras-related GTPases, association

with specific cellular membranes is essential for their

biological activities (Colicelli 2004). Conserved

C-terminal cysteine-rich motifs are used to direct

covalent modification by isoprenoid lipids

(prenylation). Prenylation is the initial step in the

attachment of these proteins to the cytoplasmic leaflets

of a variety of cellular organelles. However, specific

membrane localization often requires additional

targeting signals, provided either by a cluster of basic

amino acids or the palmitoylation of internal cysteine

residues (Heo et al. 2006). For example, adjacent to the

CAAX prenylation motif (“C” is Cysteine, “A” is an

aliphatic amino acid, and “X” is variable), H-Ras and

N-Ras have a cysteine residue for palmitoylation,

directing the protein to the plasma membrane via the

Golgi apparatus. In contrast, K-Ras4A has a lysine-

rich polybasic motif, which leads the protein to the

plasma membrane more rapidly through a Golgi-

independent route (Colicelli 2004).

Surprisingly, analysis of an over-expressed GFP

(green fluorescent protein)-tagged protein indicates

that Rin primarily localizes to the nucleus, although

a detectable amount of GFP protein is also found at the

plasma membrane in these studies (Heo et al. 2006).

Investigation of the Rin C-terminus has provided key

insights into this unique subcellular distribution. Dis-

tinct from the majority of G-proteins, the Rit and RGK

family GTPases lack a CAAX motif, and instead con-

tain a polybasic cluster at the C-terminus (Heo et al.

2006). Interestingly, sequence analysis of the Rin

polybasic motif reveals a canonical nuclear localiza-

tion signal (NLS) (K-K/R-x-K/R). Additionally, Heo

et al. have demonstrated the importance of the

polybasic C-terminal region in targeting the protein

to the plasma membrane via interactions with

phosphatidylinositol 4,5-bisphosphate (PI4,5P2) and

phosphatidylinositol 3,4,5-trisphosphate (PI3,4,5P3)

lipids (Heo et al. 2006). Thus, Rin appears capable of

shuttling between the nucleus and plasmamembrane in

a PIP-lipid dependent fashion, allowing for a novel

cellular trafficking pattern, which may contribute to

the physiological function of Rin.

The C-terminus of Rin has also been reported to

direct Ca2+-dependent interactions with calmodulin

(CaM) (Hoshino and Nakamura 2002; Lee et al.

1996). Although CaM binding has been shown to be

important for neurite outgrowth mediated by Rin

(Hoshino and Nakamura 2003), the exact signaling

mechanism as well as the physiological significance

of CaM binding still needs to be elucidated. The Rin

homolog Drosophila Ric was originally identified as

a CaM binding protein, and genetic studies suggest that

CaM association contributes to the regulation of Ric

signaling (Harrison et al. 2005).

Regulation of Rin Activity

Like most Ras GTPases, Rin is activated by exchang-

ing GDP with GTP in the nucleotide binding pocket,

which results in conformational changes that expose

the effector domain and promote the recruitment of

proteins responsible for signal transduction cascade

activation. This classic GTPase cycle is regulated by

guanine nucleotide exchange factors (GEFs) and

GTPase-activating proteins (GAPs), which are respon-

sible for the release of bound GDP and the induction of

intrinsic GTPase activity, respectively. However, to

date no Rin-specific GEFs or GAPs have been

identified.

Several Rin point mutations have been reported to

block intrinsic GTPase activity or GTP/GDP

Rin (Ras-Like Protein in Neurons) 1679 R

exchange, leading to a protein locked in the active or

inactive state, respectively. Mutation of Gln78 to Leu

(RinQ78L), equivalent to the oncogenic RasQ61L

mutation, causes complete inhibition of GTP hydroly-

sis, resulting in a constitutively active Rin mutant.

Meanwhile, the RinS34N mutation, equivalent to dom-

inant-negative RasS17N, is predominantly GDP-bound

(Shao et al. 1999), and disrupts signal transduction

when over-expressed (Shi et al. 2005). Both of these

mutations have proved useful in the analysis of Rin

GTPase function.

R

Rin Effectors

The G2 domain of Ras GTPases is known as the

“effector domain” and serves as the primary region of

interaction responsible for directing downstream

effector protein binding following G-protein activa-

tion. Rin shares a conserved effector domain

(HDPTIEDAY) with Rit, and this domain is evolution-

arily conserved within the subfamily, as there is only

a single amino acid substitution in the Drosophila Ric

effector loop (HDPTIEDSY). In addition, the Rin

effector domain shares seven out of nine residues

with the Ras effector loop (YDPTIEDSY) (Shao

et al. 1999). This high rate of conservation has led to

the suggestion that Rin is likely to control distinct, but

perhaps partially overlapping, downstream signaling

pathways compared to Ras.

A variety of proteins have been implicated as can-

didate Rin effectors using yeast two-hybrid screens

(Shao et al. 1999). These studies first identified the

Ras-binding domain (RBD) from the Raf kinases as

binding partners for constitutively active RinQ78L.

Recent in vivo co-immunoprecipitation analyses in

pheochromocytoma cells (PC6) support these initial

findings by demonstrating that active Rin preferen-

tially associates with B-Raf, suggesting that B-Raf

functions as a valid downstream target of Rin (Shi

et al. 2005). However, whether B-Raf directly binds

Rin in vivo, thus acting as a true effector, has yet to be

determined. Rin also interacts with the Ras interacting

domains (RID) of two Ral exchange factors, RalGDS

and RLF, in yeast two-hybrid screens suggesting that

Rin may regulate Ral GTPase signaling (Shao et al.

1999), although this hypothesis has not been formally

tested. While Rin appears capable of associating with

a number of known Ras effectors, in agreement with

Rin’s distinct G2 domain sequence, not all known Ras

effectors demonstrate Rin binding. For example, active

Rin does not bind to RIN1 or p110, the catalytic

subunit of PI3K (Shao et al. 1999). More interestingly,

Rin directly associates with the PDZ domain of PAR6,

a cell polarity-regulating molecule, in a GTP-

dependent fashion. This interaction requires an intact

Rin G2 effector domain, suggesting that PAR6 may

represent the first authentic Rin effector that does not

also associate with Ras (Hoshino et al. 2005).

The identification of effectors for Ras-like small

GTPases is not only important for understanding their

physiological functions, but the minimum GTPase

binding domain of select effector proteins have been

adapted for use in pull-down activity assays. In the

case of Rin, both the Raf-RBD and the RalGDS-RID

have been used to examine in vivo Rin activity

(Hoshino and Nakamura 2002; Spencer et al. 2002).

Functions of Rin GTPase

Rin can be activated by a variety of extracellular stim-

uli, including NGF, epidermal growth factor (EGF),

ionomycin, and tocopherol acetate (Hoshino and

Nakamura 2002; Spencer et al. 2002), suggesting that

Rin controls the activation of a variety of signaling

pathways and cellular functions. Indeed, a significant

amount of work has been performed to understand the

physiological role of Rin GTPase-mediated signaling.

Hoshino et al. first demonstrated that GTP-bound

Rin associated with the PDZ domain of PAR6

(Hoshino et al. 2005), which has previously been

shown to bind the GTP-bound active forms of the

Rho family GTPases Rac and Cdc42 (Qiu et al.

2000). In contrast, despite the high homology to Rin,

Rit associates in a GTP-independent fashion with

PAR6C, one of the three PAR6 isoforms, which is

preferentially expressed in the brain. Unlike Rin/

PAR6 binding, the Rit/PAR6C interaction does not

require an intact PAR6C PDZ domain (Rudolph et al.

2007). These differences suggest distinct mechanisms

in Rit/Rin-mediated PAR6 signaling. More impor-

tantly, these same studies suggested that Rin can pro-

mote the formation of a larger Rin-PAR6-Rac/Cdc42

ternary complex scaffolded by PAR6 (Hoshino et al.

2005). Enlightened by the role of Rac/Cdc42 in tumor-

igenesis, Hoshino and colleagues have further

explored the role of Rin in cell transformation and

Q78L

R 1680 Rin (Ras-Like Protein in Neurons)

demonstrated that the Rin-PAR6-Rac/Cdc42 complex

was capable of enhancing the formation of foci in NIH-

3T3 cells (Hoshino et al. 2005). Although the lack of

endogenous Rin expression in fibroblasts complicates

the interpretation of this study, these data suggest that

Rin may function in tumorigenesis. Moreover, since

PAR6 is known to contribute to the regulation of axon

specification (Shi et al. 2003), it is possible that the

Rin-PAR6-Rac/Cdc42 complex plays a pivotal role in

axonal/dendritic growth.

The exclusive expression of Rin in neurons indi-

cates a critical role for Rin in the central nervous

system. Indeed, over-expression of a constitutively

active Rin mutant alone is capable of inducing neurite

outgrowth in PC6 cells (Hoshino and Nakamura 2003;

Shi et al. 2005), suggesting a physiological role for Rin

signaling in neuronal differentiation and development.

shRNA-mediated knockdown of Rin dramatically

reduces NGF-mediated neurite outgrowth in PC6

cells, suggesting that Rin is a pivotal component of

NGF signaling by regulating both ERK and p38

MAPK pathways (Shi et al. 2005). Further detailed

mechanistic studies have revealed that Rin activates

ERK through B-Raf. Rin selectively regulates the

p38a MAPK isoform in vitro; however, the mecha-

nism of Rin-mediated p38 activation has yet to be

determined (Shi et al. 2005). The identification of

a Rin-PAR6-Rac/Cdc42 complex suggests a putative

pathway (Hoshino et al. 2005), since Rac and Cdc42

are known to serve as upstream regulators of p38

MAPK signaling (Shin et al. 2005).

PACAP38 potently induces neuronal differentia-

tion through the activation of PACR1, a heterotrimeric

G-protein-coupled receptor (GPCR). Rin is activated

downstream of PACAP38 and appears to play a critical

role in PACAP38-mediated neuronal differentiation.

Typically, PACAP38-mediated PACR1 activation

results in stimulation of a Gas-adenylate cyclase

(AC)-cAMP signaling cascade. However, PACAP38-

dependent Rin activation is mediated by Src down-

stream of Gas/i and functions upstream of the cAMP/

PKA signaling cascade, which in turn regulates

heat shock protein 27 (HSP27) phosphorylation

(Shi et al. 2008). HSP27 serves as a chaperone to

facilitate correct protein folding, has been reported

to regulate cytoskeleton stability, and has known

roles in neuronal morphology and survival signaling

(Stetler et al. 2009). Indeed, RNAi-mediated silencing

of HSP27 is sufficient to block PACAP38- and

Rin -induced neurite outgrowth (Shi et al. 2008),

further supporting the notion that HSP27 serves

as a critical downstream target of Rin in neuronal

differentiation.

Rin has also been found to associate with the intra-

cellular domain of Plexin B3, a member of the Plexin

family of semaphorin receptors (Hartwig et al. 2005).

Semaphorins are secreted or membrane-bound pro-

teins that provide critical guidance signals to redirect

or inhibit axonal growth (Vanderhaeghen and Cheng

2010). Given the ability of Rin to promote neurite

outgrowth, an intriguing possibility is that Rin signal-

ing contributes to Plexin B3-mediated axonal exten-

sion/retraction. Indeed, Plexin B1 has been shown to

function as a GAP, down-regulating R-Ras GTPase

activity to induce axonal retraction (Oinuma et al.

2004). Despite the report of the direct interaction of

Rin with Plexin B3, it remains to be determined

whether Plexin B3 functions as a RinGAP.

Calissano et al. used a yeast two-hybrid screen to

identify a putative interaction between Rin and the

N-terminus of Brn-3a, a transcriptional factor widely

expressed in the peripheral nervous system. Surpris-

ingly, Brn-3a preferentially associated with GDP-

bound Rin (Calissano and Latchman 2003), and only

GDP-bound Rin was shown to induce Brn-3a-mediated

gene transcription. However, unstimulated neurons

contain high basal levels of GDP-Rin, and additional

studies are needed to determine whether transient

in vivo fluctuations in GDP-Rin levels contribute to

Brn-3a-mediated neuronal gene transcription.

Summary

To our knowledge, Rin is the only Ras subfamily

GTPase to be expressed exclusively in neurons. In keep-

ing with this unique expression pattern, in vitro studies

indicate that Rin serves as a critical mediator of pheo-

chromocytoma cell neurite outgrowth, acting to couple

diverse extracellular stimuli (e.g., NGF, PACAP38) to

the activation of ERKand p38MAPKpathways, cAMP/

PKA cascade signaling, and the PAR6-Rac/Cdc42

polarity pathway. In addition, preliminary reports sug-

gest that Rin contributes to both Plexin- and Brn-3a-

mediated neuronal differentiation. Despite this progress,

much remains to be elucidated concerning themolecular

mechanisms that govern these diverse actions. To

date, our understanding of Rin function comes from

RIN Family Proteins (RIN1, RIN2, and RIN3) 1681 R

over-expression and RNAi silencing studies in mamma-

lian cell lines, and future studies must investigate the

neuronal effects of Rin ablation making use of geneti-

cally engineered Rin null animals. A deeper under-

standing of in vivo Rin regulation, together with the

identification of additional Rin effector proteins, will

be critical to define the physiological function of this

novel neuronal regulatory protein.

Acknowledgments This work was supported by Public Health

Service grant NS045103 from the National Institute of Neuro-

logical Disorders and Stroke and 2P20 RR020171 from the

National Center for Research Resources.

R

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RIN = Ras and Rab INteractor

▶RIN Family Proteins (RIN1, RIN2, and RIN3)

RIN Family Proteins (RIN1, RIN2,and RIN3)

John Colicelli, Pamela Y. Ting and Christine Janson

Department of Biological Chemistry, David Geffen

School of Medicine at UCLA, Los Angeles, CA, USA

Synonyms

RIN ¼ Ras and Rab INteractor

Historical Background

The RIN1 and RIN2 genes were first identified in

a selection for expressed human cDNAs that suppress

R 1682 RIN Family Proteins (RIN1, RIN2, and RIN3)

phenotypes associated with oncogenic RAS mutations

in a yeast model system (Colicelli et al. 1991). Subse-

quent analysis demonstrated that RIN1 binds specifi-

cally to activated (GTP-bound) human HRAS (Han

and Colicelli 1995; Han et al. 1997), suggesting that

RIN1 is a downstream effector of RAS family proteins.

RIN2 and RIN3 also bind to activated HRAS

(Rodriguez-Viciana et al. 2004). All three RIN pro-

teins contain a carboxy terminal RAS association (RA)

domain that mediates this interaction (Colicelli

(2004)).

It was subsequently reported that RIN1 encodes

a VPS9-related guanine nucleotide exchange factor

(GEF) function specific for the RAB5 family of early

endocytosis GTPase (Tall et al. 2001). RIN2 and RIN3

also encode VPS9-type GEF domains upstream of

the RA domain. This suggests that RIN family proteins

are RAS effectors that promote RAB5 function,

connecting RAS activation to receptor endocytosis.

Another defining feature of RIN proteins is their

amino terminal SRC homology 2 (SH2) domain. The

RIN1 SH2 domain mediates binding to activated

receptor tyrosine kinases (Barbieri et al. 2003), which

likely facilitates endocytosis and downregulation of

these receptors. The RIN2 and RIN3 SH2 domains

show some species variation (discussed below).

RIN1 also binds and activates ABL1 and ABL2

tyrosine kinases (Cao et al. 2008; Hu et al. 2005).

This capacity requires an amino terminal fragment of

RIN1, which may not be functionally conserved in

RIN2 and RIN3.

RIN Family: Evolution and GeneralProperties

The RIN family of genes likely evolved from

a progenitor represented in arthropods by the fruit fly

Drosophila melanogaster gene Sprint (Szabo et al.

2001) and in echinoderms by the sea urchin Strongylo-

centrotus purpuratus gene annotated as Rin2L (www.

spbase.org). The fruit fly and sea urchin gene products

show strong conservation with vertebrate RIN proteins

in the amino terminal SH2 domain and the carboxy

terminal GEF domain. However, the fruit fly and sea

urchin RIN proteins show no significant alignment

with vertebrate RIN proteins in the central region or

in the RA domain. This suggests that the gene expan-

sion that gave rise to a RIN family in vertebrates was

accompanied by the development of RAS effector

capability. A recently described vertebrate RINL

gene (Woller et al. 2011) encodes a protein that aligns

with RIN1-3 but has no RA domain (Fig. 1), and may

represent the vestige of a pre-vertebrate RIN gene.

Specific properties of each are described in the

following sections.

RIN1

RIN1, the defining member of the RIN protein family,

is a RAS effector that couples cell signaling with

receptor trafficking and cytoskeletal remodeling. It is

localized to the cytoplasm and the plasma membrane.

Binding of RIN1 to RAS is GTP-dependent and medi-

ated by the carboxy-terminal region of RIN1 (Han

et al. 1997). RIN1 overexpression suppresses fibroblast

transformation by HRASG12V, likely by competing

with RAS effectors that promote mitosis and suppress

apoptosis (Wang et al. 2002). RIN1 localizes in cyto-

plasmic and membrane compartments, and this parti-

tion is regulated through a 14-3-3 interaction.

Phosphorylation of RIN1-Ser351 by ▶ PKD enhances

14-3-3 binding, and mutation of this residue shifts

RIN1 localization to the plasma membrane (Wang

et al. 2002). This result suggests that 14-3-3 binding

reduces access to RAS proteins, which are membrane

tethered.

RIN1 encodes a VPS9 subfamily GEF domain that

mediates endosome fusion and receptor endocytosis

through activation of RAB5 family proteins (Tall

et al. 2001). RIN1 preferential associates with and

activates the RAB5A isoform (Chen et al. 2009). The

SH2 domain of RIN1 binds to activated (tyrosine

phosphorylated) EGFR to promote receptor

downregulation (Barbieri et al. 2003). Through

a proline-rich domain, RIN1 may recruit STAM2,

a component of the ESCRT (endosomal sorting com-

plex required for transport) machinery, and facilitate

trafficking of ubiquitinated EGFR to lysosomes (Kong

et al. 2007). RIN1 also promotes internalization of the

TGF-b receptor through RAB5 activation. In this case,

however, the result is increased signaling through

SMAD2/3 and the transcription repressor SNAI1 (Hu

et al. 2008). The contribution of RIN1 to growth factor

receptor internalization and signaling may account for

the observed silencing of RIN1 in breast tumor cells

(Milstein et al. 2007) and enhanced expression in non-

small cell lung adenocarcinoma cells (Tomshine et al.

2009).

RIN1

RIN2

RIN3

RA

RA

RA

SH2

SH2

SH2

RINL

RAB5GEF

RAB5GEF

RAB5GEF

RAB5GEF

RIN Family Proteins (RIN1, RIN2, and RIN3), Fig 1 RIN

Protein Family in Vertebrates. Conserved domains include

SRC Homology 2 (SH2), Guanine nucleotide Exchange Factor

(RAB5 GEF) and RAS Association (RA). Regions of sequence

conservation are shown in green and blue. Lines indicate gaps in

alignment. Red bars are used to denote proline-rich motifs com-

mon to SH3 ligands ([RKHYFW]xxPxxP and PxxPx[RK]) and

WW domain ligands (PPxY and PPPPP)

RIN Family Proteins (RIN1, RIN2, and RIN3) 1683 R

R

Other downstream effectors of RIN1 are the ABL

family non-receptor tyrosine kinases. The ABL SH3

domain mediates binding to a proline-rich motif in

RIN1, leading to ABL-mediated phosphorylation of

RIN1-Y36 and subsequent association with the ABL

SH2 domain (Han et al. 1997; Afar et al. 1997). Bind-

ing of RIN1 to ABL relieves autoinhibition and stim-

ulates ABL tyrosine kinase activity (Cao et al. 2008;

Hu et al. 2005). As a positive regulator of ABL activ-

ity, RIN1 regulates actin remodeling. Mammary epi-

thelial cells from Rin1�/� mice show extensive

peripheral-actin networks, enhanced attachment to

fibronectin, and increased cell motility (Hu et al.

2005). RIN1 potentiates the catalytic and transforming

activity of the BCR-ABL1 fusion oncogene, and RIN1

over-expression increases BCR-ABL1 mediated leu-

kemogenesis in a mouse model system (Afar et al.

1997). Importantly, the ABL1T315I mutant resistant to

therapeutic kinase inhibitors remains responsive to

positive regulation by RIN1 (Cao et al. 2008), and

Rin1�/� bone marrow cells were refractory to trans-

formation by BCR-ABL1T315I (Thai et al. 2011).

These results suggest that the RIN1::ABL1 interaction

may be a drugable vulnerability of oncogenic ABL

fusion proteins.

Rin1 is most strongly expressed in mature forebrain

neurons, with moderate expression in hematopoietic

and epithelial cells (Dzudzor et al. 2010). This

restricted expression of Rin1 is mediated in part by

SNAI1. The Rin1 promoter sequence also contains

a consensus recognition site for the transcription

repressor REST. Deletion of this region surprisingly

led to a reduction in reporter gene expression,

suggesting that elements in this region enhance

expression and may be positively regulating Rin1

expression in neuronal cells (Dzudzor et al. 2010).

Rin1�/� mice are viable and fertile and show no

gross morphological abnormalities. However, they

have elevated amygdala LTP (long-term potentiation)

and enhanced fear conditioning, suggesting that Rin1

normally acts as a negative regulator of synaptic plas-

ticity in this region (Dhaka et al. 2003). In addition,

Rin1�/� mice are deficient in conditioned fear extinc-

tion and latent inhibition (Bliss et al. 2010). Rin1�/�

mice have normal hippocampal-dependent learning, as

well as normal motor learning, anxiety, and explor-

atory behavior, suggesting that Rin1�/� mice may be

a useful model for studying neuropsychiatric condi-

tions such as PTSD (post-traumatic stress disorder).

RIN2

The RIN2 gene is widely expressed in mouse, based on

analysis of mRNA levels ((Kajiho et al. 2003),

BioGPS.gnf.org). The SH2, GEF, and RA domains

first characterized in RIN1 are well conserved in

RIN2, which has demonstrable guanine nucleotide

exchange activity on RAB5 (Saito et al. 2002) and

RAS interaction properties (Rodriguez-Viciana et al.

2004). However, RIN2 gene products in primates

(human and chimpanzee) differ from their orthologs

in other vertebrates (cow, dog, mouse, opossum,

chicken, frog, and fish) in two notable aspects. First,

the amino termini of primate RIN2 proteins extend

about 50 residues beyond other vertebrate RIN2 gene

products. Second, an arginine residue critical for the

phosphotyrosine binding function of SH2 domains

(mouse Rin2: FLVR122) is instead a histidine in pri-

mate RIN2 (human RIN2: FLVH171).

R 1684 RIN Family Proteins (RIN1, RIN2, and RIN3)

Loss of function mutations in RIN2 are associated

with two related human connective tissue disorders

referred to as MACS (Basel-Vanagaite et al. 2009)

and RIN2 Syndrome (Syx et al. 2010).

RIN3

RIN3 was first described as a novel RAB5 guanine

nucleotide exchange factor, isolated from a human

leukocyte cDNA library based on a yeast two-hybrid

screen for RAB5BQ79L interacting proteins (Kajiho

et al. 2003).

RIN3 contains the GEF, RA, and SH2 domains

conserved throughout the RIN gene family. It demon-

strates guanine nucleotide exchange activity for RAB5

(Kajiho et al. 2003) and associates with activated RAS

(Rodriguez-Viciana et al. 2004). The SH2 domain

sequence of RIN3, like that of RIN2, shows a curious

divergence at the FLVR motif. The sequences of most

vertebrates, including human, encode the arginine res-

idue critical for phosphotyrosine binding. But several

species (pig, cow, cat, rat, and mouse) have a cysteine

substitution at this key position, implying that RIN3 in

these organisms may function somewhat differently.

RIN3 also associates with BIN1 (a.k.a.

amphyphisin II), a membrane-bending protein

involved in endocytosis (Kajiho et al. 2003). RIN3

can translocate to RAB5 positive endosomes and dele-

tion of the RA domain caused RIN3 to be constitu-

tively located to endocytic vesicles, suggesting that the

RA domain has an autoinhibitory effect on RIN3’s

endosomal localization (Yoshikawa et al. 2008).

In mice, RIN3 expression is highly enriched in mast

cells with lower expression levels in other hematopoi-

etic tissues including lymph node, bone, and T cells

(http://symatlas.gnf.org). Protein levels in established

human cell lines also indicate enrichment in mast cells

(CJ and JC, 2011).

Summary

RIN1-3 proteins connect RAS signal transduction with

RAB5 activation and receptor endocytosis. RIN1 has

a special role in ABL tyrosine kinase regulation, with

a likely contribution to cytoskeleton remodeling.

A mouse knockout model suggests RIN1 involvement

in stimulation-induced signal transduction in multiple

cell types. Mouse models of RIN2 and RIN3 deficien-

cies have not yet been described.

There are several outstanding questions to be

answered regarding the biochemistry of RIN proteins.

Particularly curious is a species-specific divergence in

the SH2 domain arginine residue required for

phosphotyrosine binding. Does this imply an alternate

function for these SH2 domains? Further study is also

needed to identify the full range of protein partners,

and to determine the location and consequence of these

interactions.

Evidence for the involvement of RIN proteins in

human pathologies is just beginning to emerge. RIN1

appears to collaborate in tumorigenesis at multiple

levels in ways that are complex and cell type

specific. In addition, the fear learning and extinction

phenotypes of Rin1�/� mice suggest that reduced

RIN1 function in forebrain neurons could contribute

to post-traumatic stress disorder. The correlation of

RIN2 deficiency with connective tissue disorders pro-

vides another clear indication that RIN proteins play

diverse and essential roles in human physiology.

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RING Finger Protein 85 (RNF85)

▶TRAF6

RIP140 (Receptor-InteractingProtein 140)

▶ nrip1 (Nuclear Receptor-Interacting Protein 1)

RK

▶G-Protein-Coupled Receptor Kinase 1 (GRK1)

RLPK

▶MSK1

RLSK

▶MSK1

ROCK I

▶ROCK Kinases

ROCK II

▶ROCK Kinases

Rho-GTP

ROCK 1&2

R 1686 ROCK Kinases

ROCK Kinases

Michael S. Samuel1 and Michael F. Olson2

1Centre for Cancer Biology, SA Pathology, Adelaide,

SA, Australia2Molecular Cell Biology Laboratory, Beatson Institute

for Cancer Research, Glasgow, UK

MYPT1 LIMK1&2

MLC Cofilin

Synonyms

p160ROCK; Rho-associated coiled coil kinase; Rho-

associated kinase; Rho-associated protein kinase;

Rho kinase; ROCK I; ROCK II; ROCK: ROK kinase;

ROCK1; ROCK2; ROKa; ROKb

Actin-myosin interactionsContractile force generation

ROCK Kinases, Fig. 1 ROCK pathways leading to increased

actin–myosin contractility. Active GTP-bound Rho associates

with ROCK and increases specific activity with the consequence

of increased phosphorylation of proteins including the MYPT1

myosin-binding subunit of the myosin light chain phosphatase.

ROCK phosphorylation of MYPT1 affects both its substrate

binding and catalytic activity, resulting in inhibition of myosin

light chain dephosphorylation. MLC has also been reported to be

a directly phosphorylated and activated by ROCK. ROCK phos-

phorylation of LIMK1 and LIMK2 increases their specific activ-

ity, resulting in phosphorylation and inactivation of cofilin

family proteins. Cofilin phosphorylation inhibits its filamentous

actin-severing activity. The sum total of these events is stabili-

zation of filamentous actin and increased actin–myosin

contractility

Historical Background

The small molecular weight GTP-binding proteins of

the Rho family, which comprises 22 proteins including

RhoA, Rac1, and Cdc42, were originally isolated

based on their high degree of homology with the Ras

proto-oncogenes (Rho ¼ Ras homologue). Following

their identification, a variety of approaches were used

to isolate interacting proteins that might convey sig-

nals downstream to regulate numerous biological pro-

cesses. One approach was to use GTP-loaded RhoA as

a high-affinity reagent to fish for interacting proteins,

and the ROCK kinases were identified in this way

(Leung et al. 1995; Ishizaki et al. 1996; Matsui et al.

1996). A number of different names have been used to

describe the two kinases and early reports were some-

times not careful in accurately describing which

isoforms were actually used in the studies. However,

the official gene names have been set as ROCK1 and

ROCK2. The function of ROCK kinases downstream

of active RhoA was quickly determined to be the

regulation of actin-myosin contractile force generation

(Fig. 1), mediated via the phosphorylation of a number

of substrates including ▶LIMK (which in turn phos-

phorylates cofilin and inactivates its actin-severing

function), myosin light chain (MLC), and the myo-

sin-binding subunit of the MLC phosphatase

(MYPT1). In cultured cells, ROCK activation leads

to the formation of actin stress fibers and focal adhe-

sions. The discovery of the selective ROCK inhibitor

Y27632 (Uehata et al. 1997) rapidly advanced the

understanding of ROCK contributions to numerous

biological functions.

Structure and Function of ROCK Kinases

The ROCK kinases are both 160 kDa serine-threonine

kinases with identical domain organization. The pri-

mary sequence of the kinase domains places ROCK in

the AGC family, their closest relatives being the myo-

tonic dystrophy kinase (DMPK) and the myotonic

dystrophy kinase-related CDC42-binding kinases

(MRCK). As depicted in Fig. 2, the ROCK kinases

are composed of an N-terminal kinase domain,

hROCK1

64 % 89 % 55 % 80 %

Kinase domain PH PHC1

PH PHC1Kinase domain

Coiled coil

RB

DR

BD

Coiled coilhROCK2

399

75 414

948

1,01

4

1,09

6

1,12

0

1,20

31,

229

1,27

51,

292

1,31

8

1,35

41,

388

415

91

11

428

978

1,04

6

1,12

5

1,15

2

1,23

51,

262

1,30

71,

324

1,35

0

ROCK Kinases, Fig. 2 ROCK functional domains. Common

functional domains in human ROCK1 and ROCK1 with the

positions of starting and ending residues. The percentage iden-

tities between matched regions were determined by pairwise

BLAST comparisons. RBD Rho-Binding Domain; PHPleckstrin Homology domain; C1 Protein kinase C conserved

region 1 (Not to scale)

ROCK Kinases 1687 R

R

a coiled-coil region that contains the Rho-binding

domain (RBD), and a pleckstrin homology (PH)

domain that is split into two portions on either side of

a cysteine-rich C1 domain. The high degree of homol-

ogy between the ROCK1 and ROCK2 kinase domains

suggests that they probably phosphorylate the same

substrates and experimental data bears out this conclu-

sion. Differences in substrate phosphorylation or

biological functions observed in vivo likely result

from different sub cellular localization and/or pro-

tein/protein interactions. Although there are no three

dimensional structures of either entire protein, crystal

structures of the kinase domains alone or liganded with

various small molecule inhibitors have been reported.

These structural studies have revealed some novel

aspects of the kinase domain. In comparison to other

related kinases, there are extensions to both N-terminal

and C-terminal ends that promote dimerization. Also,

unlike most other AGC kinases, phosphorylation in the

kinase activation loop or hydrophobic motifs does not

appear to be required for ROCK activation. Instead,

dimerization appears to allow the kinase to adopt an

active conformation without the need for phosphoryla-

tion. NMR structural information for the PH and C1

domains of ROCK2 has also been reported (Wen et al.

2008). Activity of the kinase domain is autoinhibited

by the C-terminus, which can be relieved through

binding to RhoA, B, or C (Leung et al. 1995; Ishizaki

et al. 1996; Matsui et al. 1996) or through proteolytic

cleavage (Coleman et al. 2001; Sebbagh et al. 2005).

Multiple sites in the C-terminus appear to interact with

the kinase domain to regulate activity (Lochhead et al.

2010). Interestingly, a single Proline to Serine amino

acid substitution within the first portion of the split PH

domain was sufficient to activate ROCK1, suggesting

that the conformation of the C-terminal region is

important for kinase regulation (Lochhead et al.

2010). ROCK1 activity may be inhibited by another

Rho family member RhoE (Riento et al. 2003), which

in turn can be antagonized by the kinase PDK1 in

a manner independent of its catalytic activity (Pinner

and Sahai 2008). Given the key role that ROCK kinases

play in promoting contractile force generation, it is not

surprising that they have been found to make important

contributions to fundamental processes including:

motility, adhesion, cytokinesis, apoptosis, phagocyto-

sis, smooth muscle contraction, and neurite retraction.

ROCK in Development

Genetic deletion of either ROCK1 (Shimizu et al.

2005) or ROCK2 (Thumkeo et al. 2003) in mice

resulted in similar phenotypes, which helped reveal

that a major function of the ROCK kinases in vivo is

the regulation of epithelial cell motility. The homozy-

gous deletion of ROCK1 still allowed for the birth of

mice at the expected Mendelian ratios, indicating that

R 1688 ROCK Kinases

there were no major problems during growth and

development in utero but newborns had defects in

eyelid and ventral body wall closure that gave rise to

eyes-open at birth (EOB) and omphalocele (organs

such as the liver and gut not being contained with the

abdomen) phenotypes, respectively. EOB and

omphalocele were also observed in homozygous

ROCK2 knockout mice, but in this case a sub-

Mendelian incidence of ROCK2�/� mice resulted

from defects in the placental labyrinth layer, causing

decreased blood flow to developing ROCK2�/�

embryos (Thumkeo et al. 2003, 2005). Given these

results, it seems consistent that ROCK1þ/�;ROCK2þ/� double heterozygous mice also exhibited

EOB and omphalocele, indicating that both kinases

contribute to the same actin-driven movement and

reorganization of epithelial sheets for eyelid and ven-

tral body wall closure (Thumkeo et al. 2005). That

being said, homozygous ROCK1 knockout mice were

also independently generated, showing no obvious

phenotypic differences from wild-type littermates,

which suggests that strain background differences

may contribute to the penetrance of the ROCK1 defi-

cient phenotype (Zhang et al. 2006).

ROCK in Disease

The ready availability of potent and selective ROCK

inhibitors has made it possible to examine whether

ROCK kinases are involved in a wide variety of patho-

logical conditions, including cancer, hypertension and

cardiovascular disease, neuronal degeneration, kidney

failure, asthma, glaucoma, osteoporosis, erectile dys-

function, and insulin resistance. However, the areas

that have attracted the most research effort are cancer,

hypertension/cardiovascular disease, and glaucoma.

Interest in ROCK as a cancer target stems from its

wide range of activities that contribute to the growth

and progression of tumors including proliferation, sur-

vival, and metastasis (reviewed in [Wickman et al.

2010]). Early studies revealed that Rho GTPs are

over-expressed in a variety of tumors, consistent with

increased signaling through the ROCK pathway being

a contributory factor. In addition, elevated ROCK

expression has been reported in bladder (Kamai et al.

2003) and testicular cancers (Kamai et al. 2002) and

correlates with poor survival (Kamai et al. 2003).

Large scale sequencing efforts directed at the

identification of genetic alterations in human cancers

revealed a number of activating somatic ROCK1muta-

tions in human tumors and tumor cell lines (Lochhead

et al. 2010). A current concept is that it would be

advantageous to develop ROCK2 selective inhibitors

for the treatment of cancer to avoid the pronounced

hypotension that is a side effect of ROCK1 inhibition.

The connection between ROCK and hypertension

was revealed with the development of ROCK selective

inhibitors. Inhibition of ROCK with Y-27632 and

related compounds was shown to relieve hypertension

in rats by inhibiting the calcium sensitization of

smooth muscle contraction. Since that time, numerous

studies have built on these observations to show that

ROCK activity mediates increased smooth muscle

contraction principally via modulation of MLC phos-

phorylation. In particular, ROCK appears to contribute

to aberrant vascular contraction, for example, during

coronary vasospasm, cerebral vasospasm following

subarachnoid hemorrhage, pulmonary hypertension,

and Raynaud’s phenomenon, a condition in which the

blood supply to distal extremities such as fingers and

toes is decreased to the point of numbness or pain and

which is the result of vasospasms.

Glaucoma is a disease in which damage to the optic

nerve progressively leads to impaired vision and pos-

sibly blindness. One way that optic nerve damage may

occur is through elevated intraocular pressure. ROCK

inhibition helps to relieve this pressure by increasing

aqueous outflow by reducing MLC phosphorylation in

cells lining the trabecular meshwork. Significant

research by pharmaceutical companies in this area

has resulted in several promising clinical trials.

Within the nervous system, ROCK has been shown

to be an important trigger of neuronal growth cone

collapse and neurite withdrawal. As a result, there has

been considerable interest in the possibility that ROCK

inhibition would actually promote neurite outgrowth

and dendrite formation. Possible applications for this

include recovery from spinal cord injury by assisting

the re-establishment of neural connections across the

lesion and Alzheimer’s disease treatment through the

decreased production of amyloid precursor protein.

ROCK and Stem Cell Survival

When human embryonic stem cells (hESC) are disso-

ciated or plated at low density they often undergo

ROCK Kinases 1689 R

apoptotic death. Chemical biology screens to identify

agents that would promote survival identified the

ROCK selective inhibitor Y-27632 as a particularly

potent agent. Mechanistic studies revealed that

ROCK-mediated actin–myosin contraction makes epi-

blast derived hESCs die during low density growth and

validate the use of inhibitors of ROCK or actin–myosin

contractility for the propagation and genetic manipu-

lation of hESCs for eventual therapeutic use (Samuel

and Olson 2010).

R

ROCK Inhibitors

1. HA-1077 (fasudil) and hydroxyfasudil have been in

clinical use in Japan for cerebral vasospasm since

1995 (Olson 2008). Since it has been used for such

a long period, there is positive post-marketing

safety data, which has encouraged trials for

a number of indications, including angina, acute

ischemic stroke, cerebral blood flow, stable angina

pectoris, coronary artery spasm, heart failure asso-

ciated vascular resistance and constriction, pulmo-

nary arterial hypertension, essential hypertension,

atherosclerosis, and aortic stiffness (Olson 2008).

2. Y-27632 was the first published selective ROCK

inhibitor (Uehata et al. 1997) and its ready availabil-

ity has made it the inhibitor of choice. Although this

inhibitor is not strictly specific for ROCK kinases,

ROCK1 and ROCK2 siRNA experiments have not

revealed significant off-target effects in cells.

3. H-1152 was developed as an improved version of

HA-1077 with greater ROCK selectivity over PKA

and PKC. Although also readily available from

commercial sources, H-1152 is less often used

than HA-1077 to corroborate results from experi-

ments in which Y-27632 was used, despite the

improved selectivity.

4. SLx-2119 was developed as a potential cancer ther-

apeutic initially, but more recently has gone into

preclinical development for the treatment of meta-

bolic disease.

A number of studies have highlighted the fact that no

inhibitor is entirely specific. Therefore, greater robust-

ness can be built into studies that make use of ROCK

inhibitors if a number of additional conditions were

satisfied including:

1. Structurally unrelated inhibitors should produce the

same biological effects at concentrations that

produce equivalent kinase inhibition. The lowest

effective doses should be used to reduce off-target

effects.

2. Dose-response experiments to establish rank order

of potency for a set of inhibitors, i.e., the most

potent ROCK inhibitors should be the most effec-

tive if a biological response is mediated by ROCK.

3. Examination of the relationship between ROCK

inhibitor dose, substrate phosphorylation and bio-

logical endpoint.

4. Where possible additional methods should be used

to inhibit ROCK function, such as RNAi-mediated

knockdown.

Although there is no doubt that inhibitors are useful

and convenient research tools, care should be taken in

interpreting the results. The substantial knowledge

base of the biological functions of ROCK has been

made possible due to the ready availability of such

inhibitors. Their greatest utility is actually in excluding

a possible involvement of ROCK in specific biological

responses when there are adequate positive controls

in place.

Summary

The most important and central cellular function of

ROCK kinases is to regulate morphology, largely

through actin–myosin contractility. Due to its pro-

found influence on morphology and contractility,

ROCK directly influences numerous activities, such

as cytokinesis, adhesion, motility, endothelial barrier

function, and membrane blebbing. In addition, via

direct or indirect pathways, the ROCK kinases also

influence biological processes including gene tran-

scription, proliferation, regulation of cell size, and

survival. There has been considerable interest in

ROCK kinases as potential therapeutic targets for can-

cer, hypertension/cardiovascular disease, and glau-

coma, and a number of potent and selective inhibitors

have been discovered. In fact, one ROCK inhibitor

fasudil has been used clinically in Japan for a number

of years for the treatment of cerebral vasospasm.

Although there is a substantial literature on ROCK

function, largely generated due to the availability of

pharmacological inhibitors, greater knowledge at the

tissue and organismal levels will result from condi-

tional knock-out and conditional-activation mouse

models (Samuel et al. 2009). In vivo experiments

R 1690 ROCK: ROK kinase

using these types of genetically modified models will

validate the role of ROCK in various pathological

conditions, and will highlight additional indications

for the use of ROCK inhibitors. Although quite

a few ROCK substrates have been identified and

well-characterized, recent phosphoproteomic studies

have identified a large number of previously unknown

ROCK substrates. As a result, a significant opportunity

awaits to characterize the biological outcomes of

ROCK-mediated phosphorylation on these novel

substrates and ultimately to determine their possible

contributions to human disease.

References

ColemanML, Sahai EA, Yeo M, Bosch M, Dewar A, Olson MF.

Membrane blebbing during apoptosis results from caspase-

mediated activation of ROCK I. Nat Cell Biol. 2001;3:

339–45.

Ishizaki T, Maekawa M, Fujisawa K, Okawa K, Iwamatsu A,

Fujita A, et al. The small GTP-binding protein Rho binds

to and activates a 160 kDa Ser/Thr protein kinase homolo-

gous to myotonic dystrophy kinase. EMBO J. 1996;15:

1885–93.

Kamai T, Arai K, Sumi S, Tsujii T, HondaM, Yamanishi T, et al.

The rho/rho-kinase pathway is involved in the progression of

testicular germ cell tumour. BJU Int. 2002;89:449–53.

Kamai T, Tsujii T, Arai K, Takagi K, Asami H, Ito Y, et al.

Significant association of Rho/ROCK pathway with invasion

and metastasis of bladder cancer. Clin Cancer Res.

2003;9:2632–41.

Leung T, Manser E, Tan L, Lim L. A novel serine/threonine

kinase binding the Ras-related RhoA GTPase which trans-

locates the kinase to peripheral membranes. J Biol Chem.

1995;270:29051–4.

Lochhead PA, Wickman G, Mezna M, Olson MF. Activating

ROCK1 somatic mutations in human cancer. Oncogene.

2010;29:2591–8.

Matsui T, Amano M, Yamamoto T, Chihara K, Nakafuku M, Ito

M, et al. Rho-associated kinase, a novel serine/threonine

kinase, as a putative target for small GTP binding protein

Rho. EMBO J. 1996;15:2208–16.

Olson MF. Applications for ROCK kinase inhibition. Curr Opin

Cell Biol. 2008;20:242–8.

Pinner S, Sahai E. PDK1 regulates cancer cell motility by

antagonising inhibition of ROCK1 by RhoE. Nat Cell Biol.

2008;10:127–37.

Riento K, Guasch RM, Garg R, Jin B, Ridley AJ. RhoE binds to

ROCK I and inhibits downstream signaling. Mol Cell Biol.

2003;23:4219–29.

Samuel MS, Olson MF. Dying alone: a tale of Rho. Cell Stem

Cell. 2010;7:135–6.

Samuel MS, Munro J, Bryson S, Forrow S, Stevenson D, Olson

MF. Tissue selective expression of conditionally-regulated

ROCK by gene targeting to a defined locus. Genesis.

2009;47:440–6.

Sebbagh M, Hamelin J, Bertoglio J, Solary E, Breard J. Direct

cleavage of ROCK II by granzyme B induces target cell

membrane blebbing in a caspase-independent manner.

J Exp Med. 2005;201:465–71.

Shimizu Y, Thumkeo D, Keel J, Ishizaki T, Oshima H,

Oshima M, et al. ROCK-I regulates closure of the eyelids

and ventral body wall by inducing assembly of actomyosin

bundles. J Cell Biol. 2005;168:941–53.

Thumkeo D, Keel J, Ishizaki T, Hirose M, Nonomura K,

Oshima H, et al. Targeted disruption of the mouse

rho-associated kinase 2 gene results in intrauterine growth

retardation and fetal death. Mol Cell Biol. 2003;23:5043–55.

Thumkeo D, Shimizu Y, Sakamoto S, Yamada S, Narumiya S.

ROCK-I and ROCK-II cooperatively regulate closure of

eyelid and ventral body wall in mouse embryo. Genes

Cells. 2005;10:825–34.

UehataM, Ishizaki T, Satoh H, Ono T, Kawahara T,Morishita T,

et al. Calcium sensitization of smooth muscle mediated by

a Rho-associated protein kinase in hypertension. Nature.

1997;389:990–4.

Wen W, Liu W, Yan J, Zhang M. Structure basis and unconven-

tional lipid membrane binding properties of the PH-C1 tan-

dem of Rho kinases. J Biol Chem. 2008;283:26263–73.

Wickman GR, Samuel MS, Lochhead PA, Olson MF. The Rho-

regulated ROCK kinases in cancer. In: van Golen K, editor.

The Rho GTPases in cancer. New York: Springer; 2010.

p. 163–92.

Zhang YM, Bo J, Taffet GE, Chang J, Shi J, Reddy AK, et al.

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2006;20:916–25.

ROCK: ROK kinase

▶ROCK Kinases

ROCK1

▶ROCK Kinases

ROCK2

▶ROCK Kinases

ROKa

▶ROCK Kinases

RPN8 1691 R

ROKb

▶ROCK Kinases

ROS (Reactive Oxygen Species)

▶ p38 MAPK Family of Signal Transduction Proteins

Rotamase Pin1

▶ Pin1

RP11-175O19.1

▶LCoR

RPN8

James P. Brody

Department of Biomedical Engineering, University of

California, Irvine, CA, USA

R

Synonyms

PSMD7; S12

Background

The gene PSMD7 encodes the protein RPN8, also

known as S12. RPN8, the human homologue of Mov-

34, is a non-ATPase component of the 19S regulatory

complex (Dubiel et al. 1995). Two 19S regulatory

complexes bind to each end of the 20S proteasome to

form the 26S proteasome.

The Proteasome

The proteasome plays an important role in the cell, but

its mechanism of action is not well understood.

Although the proteasome was first isolated in 1979

(DeMartino and Goldberg 1979), the essential role

the proteasome plays within a cell was not realized

until 1990 (Fujiwara et al. 1990). The proteasome is

the main component in the intracellular protein degra-

dation pathway. This pathway was once thought to be

a relatively unimportant part of the cell, but is now

recognized to play an important role in regulating the

lifetime of cellular proteins (Spataro et al. 1998).

Degradation is an important process within the cell

(Glickman and Ciechanover 2002). The ultimate state

of a cell is governed by the set of cellular proteins

that exist within it. Cellular proteins are regulated

both through creation (transcription/translation) and

through degradation (ubiquitin–proteasome pathway).

The degradation of proteins is just as important as

the creation of them when studying regulatory pro-

cesses within the cell. Since the understanding of

the proteasome is immature, fundamental studies on

assembly, activity, and control within the proteasome

may well reveal important information about the com-

plex and, in turn, the regulation of cellular proteins.

The proteasome plays an important role in regulating

protein concentrations inside eukaryotic cells through

the ubiquitin–proteasome pathway (Hochstrasser 1995).

Ubiquitin is a small (76 amino acid) protein that may be

specifically conjugated to lysine residues on a protein.

This ubiquitin conjugation signals the proteasome to

degrade the protein into short polypeptides. Proteins

are ubiquitinated through a process that involves three

classes of enzymes called E1, E2, and E3 (Weissman

2001). First an E1 enzyme activates the ubiquitin by

modifying the C-terminal glycine residue. Simulta-

neously, an E3 enzyme binds to the target protein.

Finally, an E2 enzyme transfers the activated ubiquitin

to the E3/target complex, resulting in a ubiquitin tagged

target protein that will be degraded by the 26S

proteasome in eukaryotic cells. There aremany (dozens)

of each class of enzyme, and the combination of E2/E3

enzyme is thought to give specificity to the degradation

(Glickman and Ciechanover 2002).

The eukaryotic proteasome consists of several com-

ponents. The entire complex is known as the 26S

proteasome. The 26S complex is composed of a single

20S proteasome (total mass 700 kDa, composed of 28

subunits) and two 19S multi-subunit complexes. The

20S proteasome has a cylindrical shape, and the 19S

complexes form caps for the two ends of the 20S

proteasome (Orlowski and Wilk 2000). The activity of

R 1692 RPN8

the proteasome can be modified by the substitution of

different subunits. In cultured cells, stimulation by

gamma interferon changes the composition of the

proteasome (Tanka 1994). A sub-complex known as

the 11S regulator replaces the entire 19S sub-complex.

Three of the 28 proteins in the 20S proteasome are

replacedwith completely different proteins. It is thought

that these changes enhance the production of polypep-

tides suitable for MHC class I antigen presentation. This

form of the proteasome is called the immunoproteasome

(Van den Eynde and Morel 2001).

A heterogeneous population of proteasomes exists

within cells. A structural study using transmission

electron microscopy of individual 26S proteasomes

isolated from both Drosophila melanogasterand Xenopus laevis revealed that the 19S cap has an

extraordinary flexible attachment to the 20S

proteasome core (Walz et al. 1998). This study also

revealed that one portion of the 19S cap has at least

four different conformations. The heterogeneous pop-

ulation implies that the proteasome is used as a general

framework for protein degradation machinery.

The standard model of the proteasome’s protein deg-

radation mechanism works like this: a protein binds to

the 19S subunit at one end of the proteasome, the protein

is unfolded and fed into the 20S proteasome, the protein

is cleaved inside the 20S barrel, and the cleavage prod-

ucts pass out the other end of the proteasome. Themodel

is based upon structural (Walz et al. 1998; Unno et al.

2002) and kinetic measurements (Stein et al. 1996;

Akopian et al. 1997; Kisselev et al. 1998; Nussbaum

et al. 1998; Peters et al. 2002) of the proteasome. How-

ever, there are other models consistent with this data.

For instance, some (Nussbaum et al. 1998) suggest that

degradation products may exit through the sides of the

20S proteasome. The structural data (Walz et al. 1998;

Unno et al. 2002) makes it clear that the 20S proteasome

is symmetric, with no preferred direction.

Polyubiquitinated proteins can be degraded by the

proteasome. The 26S proteasome is a large (approxi-

mately 2,000 kDa) complex composed of three differ-

ent components. The core component, called the 20S

proteasome is a barrel-shaped protease, with the active

sites protected inside the barrel. The 20S core is regu-

lated through several mechanisms. A complex termed

the 19S binds to the top and bottom of the 20S core to

form the 26S proteasome (Fukunaga et al. 2010; Isono

et al. 2004). The 19S complex is thought to recognize,

bind, and unfold poly ubiquitinated proteins.

These proteins are then fed into the barrel of the 20S

proteasome, where they are degraded into polypeptide

chains. Other complexes, such as the PA28 complex,

can also bind to the 20S core, and these are thought to

regulate the proteasome in different ways (Bochtler

et al. 1999; Wigley et al. 1999).

Structure

Several domains and motifs for RPN8 have been iden-

tified. These include the C-terminal KEKE motif,

a putative site of protein–protein interaction (Realini

et al. 1994), the Jun activation-domain binding protein

(JAB) domain, originally described as a regulator of

transcription, the MPR1p and PAD1p N-terminal

(MPN) domain which, along with surrounding

sequence, has been shown to be important for pairing

with S13 (Rpn11/POH1) (Fu et al. 2001), and possibly

even weak homology to a MAPKK activation loop

motif (Seeger et al. 1998).

The RPN8 MPN domain has been crystallized and

the crystal structure has been solved. This structure

showed that the MPN domain contains a

metalloprotease fold, as expected, but this

metalloprotease fold is surprisingly unable to coordi-

nate a metal ion (Sanches et al. 2007).

Localization

RPN8 is cytosolic and localized around the nucleus. This

was determined using an antibody raised against amino

acids 1–205 of recombinant RPN8 to localize the 26S

proteasome in human JU77 mesothelioma cells (Seeger

et al. 1998). Furthermore, phosphorylated RPN8 was

found by using an anti-RPN8 antibody that reacted

with both the phosphorylated and unphosphorylated

forms from 26S proteasomes immunoprecipitated from

human L-132 cells (Mason et al. 1998). These data

identified phosphorylated RPN8 migrating above

50 kDa, whereas unphosphorylated RPN8 migrates at

40 kDa (Braun et al. 1999).

Roles in Human Disease

The proteasome has been implicated in multiple

diseases. Defects in proteasome activity are associated

RPN8 1693 R

R

with chronic human neurodegenerative diseases. Pro-

tein aggregates are found in some patients with

Alzheimer’s (Ciechanover and Brundin 2003),

Parkinson’s (Dauer and Przedborski 2003), and

Huntington’s (Rubinsztein 2006) diseases. These

aggregates are thought to form when the proteasome

is unable to keep up with the amount of ubiquitinated

protein being produced by the cell. The ubiquitin–

proteasome pathway of protein degradation is also

the target of cancer-related deregulation. Proteasome

inhibitors form a new class of anticancer drugs and are

under study for use in pancreatic, colon, lung, breast,

prostate, and ovarian cancers (Voorhees and Orlowski

2006). One proteasome inhibitor, Velcade, has been

approved by the FDA for treatment of multiple

myeloma.

Certain mutations in the gene BRCA1 are known to

lead to a predisposition to developing breast cancer.

These cases account for less than 10% of all breast

cancers. The protein product of BRCA1 exhibits E3

(ubiquitin protein ligase) activity. Furthermore, it was

shown that cancer-predisposing mutations in BRCA1

also lead to a loss of this E3 activity (Ruffner et al.

2001). This clearly implicates the ubiquitin–

proteasome pathway as a potential mechanism in the

development of breast cancer. Other defects leading to

the aberrant regulation of the ubiquitin–proteasome

pathway (e.g., E1, E2, E3, or proteasome subunits)

may be responsible for a greater percentage of breast

carcinomas.

RPN8/PSMD7 is one of a cohort of 231 genes

whose expression levels were significantly associated

with clinical outcome in breast cancer patients (van’t

Veer et al. 2002).

In cell line studies, RPN8 appears to be functionally

related to transformed cells. RPN8 was posttransla-

tionally modified in six normal breast epithelial

cell lines, but not four transformed cell lines. Rpn8

was unique among proteasome subunits in this

characteristic. Modified RPN8 has identical mass, but

different isoelectric points then unmodified RPN8, see

Fig. 3 in (Thompson et al. 2004). Modified RPN8 does

not associate with the 26S proteasome, but does associ-

ate with a separate high molecular weight complex, see

Fig. 5 in (Thompson et al. 2004). Finally, modified

RPN8 is localized to the nuclei of normal cells, whereas

unmodified RPN8 is only present in the cytoplasm.

The differential RPN8 protein expression and

nuclear localization observed between the normal and

cancer cell lines suggests that RPN8 has multiple func-

tions associated with normal and cancer phenotypes.

For instance, while mRNA levels are essentially the

same between normal and cancer cell lines, the differ-

ence in levels of modified RPN8 is great. Thus, mech-

anisms that modulate posttranslational modifications

of RPN8 in normal cells are disrupted in each of the

cancer cell lines studied. Furthermore, no differences

exist in the protein expression pattern of six other

proteasome subunits between the ten cell lines, with

the exception of S10a, lending significance to the dif-

ferences observed with RPN8.

Summary

In summary, RPN8 exists in normal cell lines with at

least two different posttranslationalmodifications, but in

transformed cell lines with only one. The modified form

of RPN8 does not associate with the 26S proteasome,

but does associate with the immunoproteasome. These

data suggest a differential nuclear function of modified

and unmodified RPN8 in cancer cells.

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R 1694 Rps6ka5

Fujiwara T, Tanaka K, Orino E, Yoshimura T, Kumatori A,

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Proteasomes are essential for yeast proliferation. cDNA clon-

ing and gene disruption of two major subunits. J Biol Chem.

1990;265(27):16604–13.

Fukunaga K, Kudo T, Toh-e A, Tanaka K, Saeki Y. Dissection of

the assembly pathway of the proteasome lid in Saccharomy-ces cerevisiae. Biochem Biophys Res Commun.

2010;396(4):1048–53.

Glickman MH, Ciechanover A. The ubiquitin-proteasome pro-

teolytic pathway: destruction for the sake of construction.

Physiol Rev. 2002;82(2):373–428.

Hochstrasser M. Ubiquitin, proteasomes, and the regulation of

intracellular protein degradation. Curr Opin Cell Biol.

1995;7:215–23.

Isono E, Saeki Y, YokosawaH, Toh-e A. Rpn7 is required for the

structural integrity of the 26 s proteasome of Saccharomycescerevisiae. J Biol Chem. 2004;279(26):27168–76.

Kisselev AF, Akopian TN, Goldberg AL. Range of sizes of

peptide products generated during degradation of different

proteins by archaeal proteasomes. J Biol Chem.

1998;273(4):1982–9.

Mason GG,Murray RZ, Pappin D, Rivett AJ. Phosphorylation of

ATPase subunits of the 26S proteasome. FEBS Lett.

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Rps6ka5

▶MSK1

RPT

Julie A. Maupin-Furlow and Hugo V. Miranda

Department of Microbiology and Cell Science,

University of Florida, Gainesville, FL, USA

Synonyms

Rpt, Regulatory particle triple-A (Rpt) subunits of 26S

proteasomes; triple-A (or AAA) is a family of proteins

within the AAA+ superfamily of ATPases associated

with various cellular activities

RPT 1695 R

R

Historical Background

Regulatory particle triple-A (Rpt) subunits are associ-

ated with the ubiquitin-proteasome system, a central

mechanism of protein breakdown with a rich historical

background. Aaron Ciechanover, Avram Hershko, and

Irwin Rose were awarded the 2004 Nobel Prize in

Chemistry for their “discovery of ubiquitin-mediated

protein degradation” by proteasomes and have

outlined some of this history in reviews (e.g.,

Ciechanover 2010). The term “proteasome” was first

coined by Arrigo et al. (1988) for a “large alkaline

multifunctional protease” known to mediate energy-

dependent intracellular protein breakdown, once it was

clear that this protease was related to the ring-shaped

“prosomes” of unknown function commonly found in

eukaryotic cells. The Rpt nomenclature was later pro-

posed by Finley et al. (1998) to designate the subunits

of the yeast 26S proteasome that cluster to the ATPasesassociated with various cellular activities (AAA) fam-

ily of the AAA+ superfamily (Wollenberg

and Swaffield 2001). AAA+ proteins are required

for the degradation of folded proteins by self-

compartmentalized proteases such as proteasomes.

Proteasomes and Rpt Subunits

Protein degradation by proteasomes is a major regula-

tory mechanism in eukarya, archaea, and

actinobacteria. Proteasomes can degrade short-lived

proteins that control cell division, apoptosis, DNA

repair, information processing (transcription and trans-

lation), and other cellular processes (Navon and

Ciechanover 2009). Proteasomes are also important

in maintaining protein quality by destroying improp-

erly synthesized, foreign, and damaged proteins

(Schrader et al. 2009).

All proteasomes are composed of a 20S core parti-

cle (CP) lined on its interior with proteolytic active

sites that are sequestered away from the cytosol

(Stadtmueller and Hill 2011) (Fig. 1). CPs are barrel-

like and formed from four-stacked heptameric rings of

a- and b-type subunits. The a-type subunits form the

outermost rings, while the b-type subunits form the

inner two rings. The N-terminal tails of a-type subunitscan gate the pores on each end of CPs and limit sub-

strate access to the proteolytic active sites formed by

b-type subunits.CPs associate with various regulators (Stadtmueller

and Hill 2011). In eukarya, CPs bind 19S regulatory

particles (RPs) to form an energy-dependent protease

known as the 26S proteasome (Fig. 2). Substrates of

26S proteasomes include proteins covalently

conjugated to chains of ubiquitin (Ub) through a pro-

cess termed ubiquitylation (Ravid and Hochstrasser

2008). The primary roles of 19S RPs are to recognize,

unfold, and translocate substrate proteins to the cata-

lytic central chamber of CPs for protein degradation. In

yeast, the 19S RP can be separated into base and lid

subcomplexes by deletion of the gene encoding the

regulatory particle non-ATPase Rpn10 subunit

(Tomko and Hochstrasser 2011). The base subcomplex

is composed of three Rpn subunits (Rpn1, Rpn2, and

Rpn13) and six proteins termed regulatory particle

triple-A subunits (Rpt1-6) that cluster to the AAA

family of the AAA+ superfamily. Rpt1-6 form

a heterohexameric ring that directly interacts with

20S CPs and are arranged in Rpt1-Rpt2-Rpt6-Rpt3-

Rpt4-Rpt5 order in 26S proteasomes (Tomko and

Hochstrasser 2011). The lid subcomplex is composed

of nine other Rpn subunits and harbors the deubiqui-

tylating enzyme Rpn11 that removes polyubiquitin

chains from substrate proteins.

Archaea and actinobacteria synthesize proteasomes

that are relatively simple in subunit complexity, yet

these proteases share many basic structural and func-

tional features with eukaryal 26S proteasomes

(Bar-Nun and Glickman 2011). Like eukarya, the

CPs and proteasomal AAA+ proteins of archaea and

actinobacteria can associate together in vitro and cat-

alyze the energy-dependent degradation of folded pro-

teins (Fig. 2). The prokaryotic AAA+ proteins are

often hexameric rings formed from a single protein

including the archaeal proteasome-associated nucleo-

tidase (PAN) and actinobacterial AAA ATPase

forming ring-shaped complexes (ARC) or mycobacte-

rium proteasome ATPase (Mpa). The archaeal PAN is

closely related to the eukaryal Rpt subunits, while the

actinobacterial ARC/Mpa proteins are divergent mem-

bers of the AAA family. It has been proposed that the

six different types of eukaryal Rpt proteins (Rpt1-6)

evolved from a single archaeal PAN ancestor, which

over time was duplicated and diversified (Wollenberg

and Swaffield 2001).

Rpt and Related AAA+ Proteins in Proteolysis

While the hydrolysis of peptide bonds is exergonic,

ATP binding and hydrolysis by AAA+ proteins (or

domains) is needed to fuel the regulated degradation

activesite

ante-chamber

Gate

cutawayside-view

α1-7

β1-7

β1-7

α1-7

20S

CP

ante-chamber

catalyticchamber

RPT, Fig. 1 20S proteasomes. The central proteolytic compo-

nent of all proteasomes is a cylindrical 20S core particle (CP)

formed from four-stacked heptameric rings. The outer rings are

of a-type subunits and inner rings are of b-type subunits. The

N-terminal tails of a-type subunits gate substrate access to the

central channel that connects three chambers (two antechambers

and one catalytic chamber). The proteolytic active sites are

sequestered from the cytosol and formed by the N-terminal

threonine residues of b-type subunits that are exposed by auto-

catalytic processing of the b-type proteins during complex

assembly (Modified with permission from Stadtmueller and

Hill (2011))

19S RP

20S CP

19S RP

lidba

se

Rpn3, 5-9,11-12, 15

Archaeal PAN orActinobacterial Mpa/ARC

Rpn1-2, 13

Rpt1-6 ringRpn10

LID

LID

Eukaryotic26S proteasome

Prokaryoticproteasome

α1-7

β1-7

β1-7

α1-7

α

β

β

α

RPT, Fig. 2 ATP-dependent

proteasomes. Proteasomal CPs

can associate on each end with

heptameric rings of AAA+

proteins including eukaryal

Rpt1-6, archaeal PAN, and

actinobacterial ARC (or Mpa).

In eukaryotes, Rpt1-6 are the

ATPase subunits of the 19S

regulatory particles (RPs) that

together with CPs form 26S

proteasomes. In yeast, the 19S

RP can be dissociated into lid

and base subcomplexes by

deletion of the RPN10 gene

R 1696 RPT

of folded proteins by self-compartmentalized prote-

ases such as 26S proteasomes. The AAA+ rings can

bind substrate proteins, open the gates of the protease

chamber (closed-gates limit substrate access to the

proteolytic active sites), unfold substrate proteins,

and facilitate the translocation of protein substrates

into the proteolytic center of the protease.

Subdomain X-ray crystal and cryo-electron micros-

copy structures of proteasomal AAA+ proteins are

now available and provide valuable insight into how

this group of ATPases might function at the atomic

level (Bar-Nun and Glickman 2011). Details on the

related bacterial AAA+ proteases such as ClpXP and

HslUV have also guided proteasomal models (Sauer

and Baker 2011). In general, proteasomal ATPases

have a central channel that is coaxial to the 20S CP

channel (Fig. 3). On the distal face of the proteasomal

ATPase ring are six protruding and paired N-terminal

coiled-coil (CC) domains that are required for sub-

strate binding. Moving down the ATPase channel,

one finds a conserved interdomain region with an oli-

gonucleotide-binding (OB)-fold domain that is

substrate

ATP ADP

Substraterecognition

Unfolding andtranslocation

Degradation

CC-domainOB-fold

Ar-philoop

α7

β7

β7

α7

PANAAA+

20S CP

RPT, Fig. 3 Model of proteasome-mediated degradation of

folded proteins. Model is based on archaeal PAN, a close relative

of the Rpt1-6 subunits of eukaryal 26S proteasomes. Substrate

proteins are thought to be unfolded on the distal face of the

proteasomal ATPase, translocated through the central ATPase

channel, and ultimately reach the central chamber of CPs for

destruction. The N-terminal coiled-coil (CC) domain, oligonu-

cleotide-binding (OB)-fold domain, and aromatic-hydrophobic

(Ar-phi) loop discussed in text are indicated (Modified with

permission from Zhang et al. (2009))

RPT 1697 R

R

important for AAA+ ring formation. The OB-fold

domain also appears to serve as a rigid entryway for

substrates that traverse the ATPase channel. The

archaeal PAN and eukaryal Rpt1-6 proteins have

a single OB-fold (as depicted in Fig. 3), while the

actinobacterial ARC/Mpa has a double OB-fold that

forms two separate ring-like structures within the

ATPase channel. The C-terminal AAA+ domain medi-

ates cycles of ATP hydrolysis and harbors a highly

conserved aromatic-hydrophobic (Ar-phi) loop within

its channel. The Ar-phi loop is common to energy-

dependent AAA+ proteases and is thought to grab

onto hydrophobic tails of substrates that may extend

into the ATPase channel. Once bound, cycles of ATP

hydrolysis around the ATPase ring are thought to

mediate conformational changes in the Ar-phi loop

that result in the tugging and unfolding of the substrate

protein. The rigid OB-fold pore is believed to serve as

a platform for this unfolding process. Thus, the protein

substrate is thought to be unfolded and translocated

through the ATPase and into the CP for destruction.

Many of the proteasomal ATPases (e.g., archaeal

PAN and eukaryal Rpt2, 3 and 5) have C-terminal

hydrophobic-tyrosine-any residue (HbYX) motifs

that bind to pockets between the a-type subunits of

CPs and induce CP gate opening. The proteasome-

associated regulator Blm10 and a-ring assembly

factor Pba1-Pba2 also have penultimate tyrosine

(or phenylalanine) residues that appear important

for binding within the a/a-intersubunit interface

(Stadtmueller and Hill 2011; Kusmierczyk et al. 2011).

Substrate Recognition by Proteasomes

Protein degradation is a highly regulated process. In

order to ensure that only the desired proteins are

degraded, eukaryotes and prokaryotes have evolved

various strategies for identifying proteins for degrada-

tion. Specific degradation signals or “degrons” within

the protein often initiate the process of proteolysis.

Degrons range from the phosphorylation or glycosyl-

ation status of a protein to the exposure of destabilizing

N- or C-termini (Ravid and Hochstrasser 2008). In

eukaryotic cells, the ubiquitylation system often rec-

ognizes these degrons and results in the covalent

attachment of polyubiquitin (Ub) chains. While all

seven lysine residues of Ub can form chains, Ub chains

linked through Lys48 commonly target proteins for

destruction by 26S proteasomes (Xu et al. 2009).

These ubiquitylated proteins are thought to first bind

the Rpn10 and Rpn13 subunits of 26S proteasomes by

a mechanism stimulated by ATP binding to the Rpt

subunits (Peth et al. 2010) (Fig. 4). Next, Rpt-mediated

ATP hydrolysis is thought to unfold and expose

unstructured portions of the substrate protein for

tighter binding to 26S proteasomes. Ultimately, this

is thought to ready the substrate for the editing or

RPT, Fig. 4 26S proteasome-mediated degradation of

ubiquitylated proteins. Initially, the 26S proteasomal subunits

Rpn10 and 13 (Ub receptors) bind the Ub chain of ubiquitylated

substrate proteins by a mechanism that is stimulated by ATP

binding. In the next phase, ATP is hydrolyzed by the Rpt

subunits and an unfolded domain of the substrate protein is

exposed, resulting in tighter binding to 26S proteasomes. After

these binding events, deubiquitylation, Ub-chain editing, sub-

strate unfolding, CP gate opening, translocation, and proteolysis

can occur (Modified with permission from Peth et al. 2010)

R 1698 RPT

removal of polyUb chains by deubiquitylases. The Rpt

subunits would unfold and translocate the protein

while opening the CP gates for protein degradation.

In actinobacteria and archaea, the covalent attach-

ment of small protein modifiers to substrate proteins

appears to be linked to proteasome-mediated

proteolysis– much like the process of ubiquitylation

in eukaryotic cells. It has recently been shown in

Mycobacterium tuberculosis that a small protein

termed Pup can covalently modify proteins at lysine

residues (Burns and Darwin 2010). While the mecha-

nism of pupylation differs from ubiquitylation and the

disordered structure of Pup is not like the highly

ordered beta-grasp fold of Ub, Pup can target proteins

for proteasome-mediated degradation (Burns and

Darwin 2010). Interestingly, the mycobacterial

proteasomal AAA+ Mpa (required for cellular resis-

tance to reactive nitrogen intermediates) binds and

induces formation of an alpha helix within Pup and is

required for degradation of pupylated substrates by

proteasomes (Burns and Darwin 2010). The

haloarchaeon Haloferax volcanii can also attach

small archaeal protein modifiers or SAMPs to substrate

proteins (Humbard et al. 2010). However, unlike Pup,

the archaeal SAMPs have a predicted beta-grasp fold

structure similar to Ub and require the presence of an

E1 Ub activating homolog (UbaA) for attachment

(Miranda et al. 2011). Other enzyme (E2 Ub conjugat-

ing and E3 Ub ligase) homologs for the attachment of

SAMPs to protein targets have yet to be identified.

Sampylation has not been directly linked to targeting

proteins for proteasome-mediated degradation. How-

ever, the sampylome accumulates in strains deficient in

the synthesis of the proteasomal Rpt-like PAN-A and

CP a1 subunits, suggesting sampylation may trigger

proteins for degradation by proteasomes. However,

green fluorescent protein (GFP) derivatives with

hydrophobic C-terminal tails that are not modified by

sampylation or other types of posttranslational modifi-

cation are degraded in vitro by proteasomal CPs in the

presence of archaeal PAN and hydrolyzable ATP

(Navon and Goldberg 2001). Therefore, by an ATP-

dependent mechanism, Rpt-like proteins can bind and

unfold non-sampylated protein substrates with

exposed hydrophobic regions. Interestingly, in eukary-

otic cells, the chaperonin-like activities of the

proteasomal Rpt proteins appear to also serve non-

proteolytic roles (Kodadek 2010).

Summary

Proteins of the AAA+ superfamily that include

eukaryal Rpt, archaeal PAN, and actinobacterial

RRAD 1699 R

Mpa/ARC are structurally related and important in

protein degradation. These proteasomal ATPases can

unfold proteins, open CP gates, and translocate pro-

teins for ultimate destruction within the self-

compartmentalized 20S CP structure that protects

cells from uncontrolled proteolysis. In eukarya, Rpt1-

6 associate with non-ATPase subunits in complexes

such as the 19S RP to form 26S proteasomes but also

appear to serve non-proteolytic roles. Whether the

archaeal PAN and actinobacterial Mpa/ARC are simi-

lar to eukaryal Rpt in their association with

proteasomal CPs and other non-ATPase factors

in vivo remains an exciting area of research.

Acknowledgments This work was funded in part by grants

from the National Institutes of Health (GM57498) and the

Department of Energy Office of Basic Energy Sciences

(DE-FG02-05ER15650).

R

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Rpt, Regulatory particle triple-A (Rpt)Subunits of 26S Proteasomes

▶RPT

RPTPe

▶ PTPe (RPTPe and Cyt-PTPe)

RRAD

▶Ras-Related Associated with Diabetes

R 1700 RSK (p90 Ribosomal S6 Kinase)

RSK (p90 Ribosomal S6 Kinase)

Philippe P. Roux

Department of Pathology and Cell Biology, Institute

for Research in Immunology and Cancer (IRIC),

Universite de Montreal, Montreal, QC, Canada

Synonyms

90 kDa ribosomal S6 kinase; p90RSK

Historical Background

The p90 ribosomal S6 kinase (RSK) family comprises

four mammalian Ser/Thr kinases (RSK1-4) (Anjum and

Blenis 2008; Carriere et al. 2008). The first RSK family

member was identified as a kinase activity inmaturating

Xenopus laevis oocytes that phosphorylated the 40S

ribosomal subunit protein S6 (rpS6) (Erikson andMaller

1985, 1986). Although the p70 ribosomal S6 kinases 1

and 2 (S6K1 and S6K2) were later shown to be the

predominant S6 kinases operating in somatic cells

(Blenis et al. 1991; Chung et al. 1992), RSK1 and

RSK2 were found to phosphorylate rpS6 in response to

▶MAP kinase pathway activation (Cohen et al. 2007;

Roux et al. 2007). Interestingly, whereas S6K1/2 were

found to phosphorylate all sites on rpS6 (Ser235,

Ser236, Ser240, and Ser244), RSK1 and RSK2 were

shown to specifically phosphorylate Ser235 and

Ser236 (Roux et al. 2007). The role of this specific

regulation is unknown, but some evidence suggests

that rpS6 phosphorylation may be involved in fine-

tuning the cellular response elicited by some growth

factors (Meyuhas 2008). At the moment, little is

known about specific and overlapping functions of the

RSK isoforms, but the recent identification of specific

RSK inhibitors (Cohen et al. 2005; Smith et al. 2005;

Sapkota et al. 2007), the use of RNA interference, and

the generation of mouse knockouts should help shed

light on the contribution of each RSK isoform.

Structure of the RSK Isoforms

The RSK isoforms are 73–80% identical to each

other and are mostly divergent in their amino- and

carboxyl-termini sequences. The structure of RSK is

complex and comprises two functionally distinct

kinase domains, a linker region, and N- and C-terminal

tails. While the N-terminal-kinase domain (NTKD)

shares homology with kinases of the AGC family

(PKA, PKG, PKC), the C-terminal kinase domain

(CTKD) is homologous to the calcium/calmodulin-

dependent protein kinases (CaMKs). It is thought

that, during evolution, the genes for two different

protein kinases have fused, generating a single poly-

peptide capable of receiving an upstream activating

signal from the Ras/MAPK pathway to its CTKD and

transmitting, with high efficiency and fidelity, an

activating input to its NTKD. Thus, the CTKD appears

to be only involved in autophosphorylation of RSK,

resulting in its activation, and the NTKD is responsible

for downstream substrate phosphorylation (Bjorbaek

et al. 1995). The C-terminal tail contains an ERK1/2

docking motif, known as the D domain, and interaction

of RSK with ERK1/2 was shown to depend on a short

motif consisting of Leu-Arg-Gln-Arg-Arg (Smith et al.

1999; Roux et al. 2003). Finally, the C-terminal tail of

all RSK isoforms contains a type 1 PDZ domain-

binding motif, consisting of Thr-Xaa-Leu, where Xaa

is any amino acid. This motif was shown to be func-

tional with at least some PDZ domain-containing

proteins (Thomas et al. 2005), but more will need to

be done to fully determine its role and biological

significance.

Activation Mechanisms

The RSK isoforms contain six phosphorylation sites

that are responsive to mitogenic stimulation. Muta-

tional analysis revealed that four of these sites

(Ser221, Ser352, Ser369, and Thr562 in mouse

RSK1) are essential for RSK activation (Dalby et al.

1998). Of these, Ser221 (located in the NTKD

activation loop), Ser352 (turn motif), and Ser369

(hydrophobic motif) are located within sequences

highly conserved in other AGC kinases (Newton

2003). The current model of RSK activation suggests

that ERK and RSK form an inactive complex in quies-

cent cells that is mediated by the D domain on RSK

(Hsiao et al. 1994; Zhao et al. 1996). After mitogenic

stimulation, ERK1/2 phosphorylates Thr562 in the

activation loop of the CTKD (Sutherland et al. 1993)

and possibly Thr348 and Ser352 in the linker region

RSK (p90 Ribosomal S6 Kinase) 1701 R

R

between the two kinase domains (Dalby et al. 1998).

Activation of the CTKD leads to autophosphorylation

at Ser369 (Vik and Ryder 1997), which creates

a docking site for phosphoinositide-dependent protein

kinase 1 (PDK1) (Frodin et al. 2000). In turn, PDK1

phosphorylates Ser221 in the activation loop of the

NTKD (Jensen et al. 1999; Richards et al. 1999)

and, along with phosphorylated Ser352 and Ser369,

promotes an intramolecular allosteric mechanism

that allows the NTKD to phosphorylate downstream

substrates (Frodin et al. 2002). RSK also

autophosphorylates at a C-terminal residue that

releases ERK1/2 binding, presumably to allow these

kinases to find their respective substrates throughout

the cell (Roux et al. 2003).

The process of RSK activation is closely linked to

ERK1/2 activity, and MEK1/2 inhibitors (U0126,

PD98059, PD184352) have been widely used to

study RSK function. Recently, three different classes

of RSK inhibitors targeting the NTKD (SL0101

and BI-D1870) or the CTKD (FMK) have been

identified (Cohen et al. 2005; Smith et al. 2005; Sapkota

et al. 2007). While BI-D1870 and SL-0101 are compet-

itive inhibitors with respect to ATP, FMK

(fluoromethylketone) is an irreversible inhibitor that

covalently modifies the CTKD of RSK1, RSK2, and

RSK4. These compounds have been tested against a

panel of protein kinases and found to be relatively

specific for the RSK isoforms (Bain et al. 2007). There

are alternate mechanisms of activation for RSK, but

these appear to be cell type- and context-specific. One

of these involves tyrosine phosphorylation by ▶Src,

which was shown to stabilize the interaction between

ERK and RSK, and thereby increase the rate at which

RSK becomes activated (Kang et al. 2008). Another

mechanism involves the regulation of the hydrophobic

motif of RSK by the related enzymes, MAPK-activated

protein kinases 2 and 3 (▶MK2/3), which can facilitate

RSK activation upon stimulation of the stress-

responsive p38 MAPK pathway (Zaru et al. 2007).

Biological Functions

RSK seems to be a multifunctional ERK effector

because it participates in various cellular processes

(Cargnello and Roux 2011). Although a number of

RSK functions can be deduced from the nature of its

substrates, data from many groups point toward roles

for the RSKs in nuclear signaling, cell cycle progres-

sion and cell proliferation, cell growth and protein

synthesis, and cell migration and cell survival. RSK

was found to regulate several transcription factors,

including SRF, c-Fos, and Nur77. On the basis of its

substrates, RSK seems to have important functions in

cellular growth control and proliferation. RSK may

stimulate cell cycle progression through the regulation

of immediate early gene products, such as c-Fos, which

promotes the expression of cyclin D1 during the G0/

G1 transition to S phase. RSK may also promote pro-

liferation by regulating cell-growth-related protein

synthesis. Indeed, RSK was found to regulate the

tumor suppressor TSC2 (Roux et al. 2004), and thereby

promote▶mTOR signaling. RSK has also been shown

to regulate cell survival. RSK phosphorylates and

inhibits the pro-apoptotic proteins Bad and ▶DAPK,

thereby promoting survival in response to mitogenic

stimulation (Shimamura et al. 2000; Anjum et al.

2005). Many additional RSK substrates have been

identified through the years, but at this point, very little

is known regarding isoform specificity. Whereas more

substrates have been identified for RSK2 than any

other RSK isoforms, most studies have not determined

isoform selectivity. Therefore, many known substrates

of RSK2 may be shared by different RSK family mem-

bers and more effort will be necessary to assess poten-

tial overlapping functions.

Physiological Functions

An important clue into the physiological roles of the

RSK isoforms came from the finding that inactivating

mutations in the Rps6ka3 gene (which encodes RSK2)

were the cause of Coffin-Lowry Syndrome (CLS)

(Trivier et al. 1996). CLS is an X-linked mental retar-

dation syndrome characterized in male patients by

psychomotor retardation and facial, hand, and skeletal

malformations (Pereira et al. 2010). Rps6ka3 muta-

tions are extremely heterogeneous and lead to loss of

phosphotransferase activity in the RSK2 kinase, most

often because of premature termination of translation.

It was shown that individuals with CLS consistently

presented markedly reduced total brain volume, with

cerebellum and hippocampus volumes being particu-

larly impacted in CLS patients. The physiological role

of RSK2 was also studied in the mouse through the

generation of a deletion model. These mice were

R 1702 RSK (p90 Ribosomal S6 Kinase)

shown to have deficiencies in learning and cognitive

functions, as well as having poor coordination

compared to wild-type littermates (Dufresne et al.

2001; Poirier et al. 2007). The exact cause for these

phenotypes remains unknown, but a recent study dem-

onstrated that shRNA-mediated RSK2 depletion

perturbs the differentiation of neural precursors into

neurons and maintains them as proliferating radial

precursor cells (Cargnello and Roux 2011). Evidently,

more experimentation using RSK2-deficient animals

will be required to fully understand the developmental

role of RSK2 in the nervous system.

Mice deficient in RSK2 expression also develop

a progressive skeletal disease, called osteopenia, due

to cell-autonomous defects in osteoblast activity

(David et al. 2005). Both c-Fos and ATF4

transcription factors were shown to be critical RSK2

substrates involved in these effects in osteoblasts

(Cargnello and Roux 2011). In addition, RSK2 knock-

out mice are approximately 15% smaller than their

wild-type littermates, with a specific loss of white

adipose tissue that is accompanied by reduced serum

levels of the adipocyte-derived peptide, leptin. RSK1/

RSK2/RSK3 triple knockout mice are viable, but no

other information regarding their phenotype has yet

been reported (Cargnello and Roux 2011). The

Rps6ka6 gene (that codes for RSK4) is located on

chromosome X and was suggested to be involved in

nonspecific X-linked mental retardation, but definitive

evidence remains to be provided. Interestingly, dele-

tion of Drosophila RSK was found to result in defects

in learning and conditioning (Putz et al. 2004), but

whether these deficits result from the specific loss of

RSK activity will require further investigation.

Summary

Many studies have now clearly established RSK as an

important effector of the Ras/MAPK pathway, and it is

becoming increasingly evident that deregulation ofRSK

signaling has a significant role in several human diseases

(Romeo and Roux 2011). Recent studies have expanded

the repertoire of biological functions linked to the RSK

family of protein kinases, ranging from the regulation of

transcription, translation, and protein stability to the

control of cell survival, cell motility, cell growth, and

proliferation. It has become clear that other AGC family

members such as Akt and S6K have similar functions

and often share similar protein targets. These findings

emphasize the importance of a tight and intricate regu-

lation by more than one pathway of cellular processes

that are important for cell growth and cell survival.

Ongoing systems approaches that aim to identify RSK

phosphorylation targets usingmass spectrometry should

expand our understanding of RSK signaling and func-

tion. The generation of isoform-specific mouse knock-

outs will similarly help resolve the distinct functionality

of these protein kinases. Combined, these studies

will help to identify additional targets for therapeutic

intervention in diseases that are associated with inap-

propriate signaling downstream of Ras and Raf, and

reveal novel targets for biomarker development for dis-

ease detection.

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Rsk-Like (RSKL)

▶MSK1

R 1704 Rsk-Like Protein Kinase (RLPK)

Rsk-Like Protein Kinase (RLPK)

▶MSK1

Rtef-1

▶Tead

R-Type

▶Voltage-Gated Calcium Channels: Structure and

Function (CACNA)

RXFP1: LGR7

▶Relaxin Family Peptide Receptors (RXFP) 1 and 2

RXFP2: LGR8

▶Relaxin Family Peptide Receptors (RXFP) 1 and 2

RXFP3: GPCR135, SALPR

▶Relaxin Family Peptide Receptors (RXFP) 3 and 4

RXFP4: GPCR142

▶Relaxin Family Peptide Receptors (RXFP) 3 and 4

Ryanodine Receptor

▶Ryanodine Receptor (RyR)

Ryanodine Receptor (RyR)

Filip Van Petegem and Kelvin Lau

Department of Biochemistry and Molecular Biology,

University of British Columbia, Vancouver,

BC, Canada

Synonyms

Calcium Release Channel; Ryanodine Receptor;

Ryanodine-sensitive Ca2+ Release Channels; Sarco-

plasmic Reticulum Calcium Release Channels

Historical Background

The contraction of cardiac and skeletal muscles

requires the release of Ca2+ from the sarcoplasmic

reticulum (SR) through specialized membrane

proteins. The ion channels responsible for the release

were not identified until the late 1980s. Ryanodine is

an alkaloid found in the South American plant

Ryania speciosa, and has insecticidal activity. This

compound was found to bind to the elusive Ca2+

release channel, which was subsequently named the

Ryanodine Receptor (RyR) (Meissner 1986). Early

biochemical evidence first showed that the channel

exists as a homotetramer, with each monomer having

a mass of �550 kDa. This makes RyRs the largest

known ion channels at �2.2 MDa (Lai et al. 1988).

They were shown to be located in the SR with a “foot”

region that spans the gap between the transverse tubule

and the SR. Work on SR vesicles showed that Ca2+

release can be observed from these channels and that

they could be modulated by various small molecules

like ATP, Mg2+, and Ca2+. Recordings made in planar

lipid bilayers showed that RyRs are permeable only to

small inorganic and organic cations and are completely

impermeable to anions.

Mammalian organisms contain three known RyR

isoforms. In 1989, Takeshima et al. reported the

first primary sequence and cloning of cDNA of

RyR derived from rabbit skeletal muscle (RyR1)

(Takeshima et al. 1989). The following year,

the group of MacLennan published the cloning of the

human skeletal muscle RyR and rabbit cardiac muscle

isoform (RyR2) (Otsu et al. 1990). A third isoform

2+

Ryanodine Receptor (RyR) 1705 R

(RyR3), distinct from both the cardiac and skeletal

muscle isoforms, was originally cloned from brain

tissue (Hakamata et al. 1992). The genes of all

the isoforms are located on different chromosomes in

humans. RyR1 is found on chromosome 19q13.2

spanning 104 exons. The gene encoding RyR2 is on

chromosome 1q43 (102 exons) and RyR3 is encoded

on 15q13.3-14 (103 exons). In addition to mammalian

organisms, RyRs have also been identified in

nonmammalian vertebrates with two isoforms, RyRa(homologous to RyR1) and RyRb (similar to RyR3).

Moreover, RyR genes have also been identified in

invertebrates including Caenorhabditis elegans and

Drosophila melanogaster.

R

Overall Function

RyRs are large (�2.2 MDa) ion channels that release

Ca2+ from the endoplasmic (ER) or sarcoplasmic retic-

ulum (SR) upon opening. In doing so, they play amajor

role in the contraction of both cardiac and skeletal

muscle. Their expression in many cell types suggests

they are involved in many diverse processes that are

Ca2+ dependent. Three different isoforms have been

found in mammalian organisms (RyR1, RyR2, RyR3).

They are highly similar with amino acid sequence

identities of �70%. There are several divergent

regions between the RyRs, with the greatest dissimi-

larity found near the C-terminus. RyR1 is known as the

skeletal muscle form, whereas RyR2 is often referred

to as the cardiac isoform. However, all three isoforms

are expressed in many diverse cell types. RyRs

display significant homology to another intracellular

Ca2+ release channel, the inositol-1,4,5-trisphosphate

receptor.

The primary signal to trigger the opening of RyRs

is Ca2+. This is especially relevant in cardiac

myocytes, in a process known as Ca2+-induced Ca2+

release (CICR) (Fig. 1). In this scheme, depolariza-

tion of the plasma membrane down the T-tubules

leads to opening of L-type voltage-gated Ca2+ chan-

nels, resulting in the influx of Ca2+ from the extracel-

lular space. This signal is detected by RyR2, which

binds Ca2+, opens and releases more Ca2+ from the

SR. The RyR thus acts as a signal amplifier, by

detecting small elevations in Ca2+ concentrations

and increasing the signal. In addition, RyRs can be

stimulated by rising Ca2+ levels in the ER lumen, in

a process known as store overload induced Ca

release (SOICR) (Jiang et al. 2004). When Ca2+ levels

in the cytoplasm rise too high, or when the lumenal

Ca2+ levels drop too low, several mechanisms cause

the channel to shut down.

A different situation occurs in skeletal muscle.

Whereas RyR1 is also affected by Ca2+ levels, it can,

in addition, detect conformational changes in the skel-

etal muscle variant of the voltage-gated Ca2+ channel

(CaV1.1). It is thought that there is a direct link

between an intracellular loop of CaV1.1, and the cyto-

plasmic face of RyR1(Block et al. 1988; Tanabe et al.

1990). Conformational changes in CaV1.1 can then

directly cause structural changes in RyR1 in the

absence of an initial Ca2+ signal.

The importance of RyRs is evident from several

knockout (KO) studies. RyR1 double KO mice

die immediately after birth (Takeshima et al. 1994),

whereas an RyR2 KO is embryonically lethal

(Takeshima et al. 1998). RyR3 KO mice survive, but

have impaired learning abilities (Balschun et al.

1999).

Structure

RyRs have been studied using both low- and high-

resolution structural methods. Most notably, several

cryo-electron microscopy (cryo-EM) studies have

yielded high-quality images of the entire RyR1 iso-

form in the closed state, with a maximum reported

resolution of 9.6 A (Ludtke et al. 2005). This study, as

well as many others on RyR2 and RyR3, revealed that

RyRs display a mushroom-like shape (Fig. 2). The

bulk of the protein (�90%) is located in the cyto-

plasm, with the remainder forming the transmem-

brane segments and ER lumenal portions. The

cytoplasmic portion is often referred to as a “foot,”

and consists of several tubes and holes. This creates

a maximum amount of exposed surface for the bind-

ing of auxiliary proteins and ligands. At least five ahelices can be observed per monomer in the cryo-EM

maps, but there is more volume to accommodate

additional helices or possibly even b strands (Ludtke

et al. 2005). In addition to the closed state architec-

ture, cryo-EM studies have revealed the structure of

RyR1 in the open state at 10.2 A (Samso et al. 2009).

Upon opening, the channel seems to undergo large

conformational changes in the cytoplasmic region,

Ryanodine Receptor (RyR), Fig. 1 An overview of processes

involved in RyR activation in cardiac myocytes. Particular volt-

age-gated calcium channels known as dihydropyridine receptors

(DHPR) allow an influx of Ca2+ upon the arrival of an action

potential. Through CICR, the RyR releases Ca2+ from the sar-

coplasmic reticulum, triggering muscle contraction. Also shown

is a PKA-dependent phosphorylation pathway which activates

RyR upon b-adrenergic stimulation. The Sodium-Calcium

Exchanger pumps remove one Ca2+ ion for 3 Na+ in return.

The Sarco/endoplasmic Reticulum Ca2+-ATPase (SERCA)allows for reuptake of Ca2+ from the cytosol. It is regulated by

phospholamban (PLB) which is also a substrate target for PKA

R 1706 Ryanodine Receptor (RyR)

Ryanodine Receptor (RyR), Fig. 2 (Top) A cryo-EM struc-

ture of RyR1 at 9.6 A (Ludtke et al. 2005), as viewed from the

side. A clear majority of the channel is located in the cytoplasm

while only a fraction is embedded within the sarcoplasmic retic-

ulum membrane. (Bottom) Top view of the RyR1 cryo-EMmap,

also showing the crystal structure of the first three N-terminal

domains of RyR1, docked into the map (Tung et al. 2010).

Shown are domains A (blue), B (green), and C (red). The

domains form a four-fold symmetric vestibule of the channel

that faces the cytoplasm. The RyR displays many tubes and holes

for the binding of modulatory ligands and proteins

Ryanodine Receptor (RyR) 1707 R

R

along with motions in the pore-forming part of the

transmembrane region. The RyR is therefore a bona

fide allosteric protein, whereby motions in the pore

are coupled to regulatory domains in the cytoplasmic

portion.

High resolution studies have been limited to

individual domains of the RyR. The largest portion

includes�550 amino acids at the N-terminus of RyR1

(Fig. 2). This region folds up as three individual

domains, including two b trefoil domains (domains

A and B), and a bundle of five a helices (domain C).

The three domains are located near the center at the

cytoplasmic face, and connect to the corresponding

domains in the neighboring subunits (Tung et al.

2010). These domains are predicted to undergo rela-

tive motions during opening and closing of the chan-

nel, thus being allosterically coupled to the pore

region.

Protein and Ligand Interactions

As structural studies have shown, the majority of the

volume of RyRs is exposed in the cytoplasm. The large

surface area serves as a docking platform for numerous

proteins and small molecules that are able to bind and

modulate RyR function (Fig. 3). In addition, proteins

in the ER lumen can also associate with the RyR. The

precise sites of action for many of these interactions

are still to be discovered.

Among the notable binding partners of RyRs is

Calmodulin (CaM), a Ca2+-binding protein that can

provide Ca2+-dependent feedback to RyRs and either

stimulate or inhibit the channel depending on Ca2+

levels and the precise isoform. FK506-binding proteins

(FKBPs) are known to bind tightly to RyRs and stabi-

lize their closed-states. FKBP12 primarily associates

with RyR1, while FKBP12.6 has the highest affinity for

RyR2. Experimental evidence suggests that removal of

FKBP results in subconductance states (Ahern et al.

1997). The ER lumen contains calsequestrin (CASQ),

a Ca2+-binding protein that can bind multiple Ca2+ ions

and oligomerize. Together with the integral membrane

proteins junctin and triadin, CASQ is thought to report

on luminal Ca2+-levels by providing feedback to the

channel (Gyorke and Terentyev 2008). A peculiar

interaction is present in skeletal muscle, where RyR1

is thought to interact directly with a protein in the

plasma membrane, the voltage-gated calcium channel

(CaV1.1) (Block et al. 1988; Tanabe et al. 1990). This

allows for a direct link that couples electrical signals in

the plasma membrane to Ca2+ release from the SR.

RyRs are the target of phosphorylation and dephos-

phorylation events. Enzymes implicated in this cycle

include Protein Kinase A (PKA), which is anchored via

an A kinase anchoring protein (AKAP), Protein

Kinase G, protein phosphatases PP1 and PP2A, and

the kinase CaMKII. Phosphorylation by both PKA and

CaMKII has been found to increase the open probabil-

ity of the channels, although a lot of controversy exists

around the exact effect and mechanism (Bers 2004).

Because RyRs are phosphorylated by PKA, they are

under the control of b-adrenergic receptors, through

a pathway involving G proteins, adenylate cyclase,

cAMP, and PKA (Fig. 1). In addition to the proteins

mentioned here, RyRs have been found to be modu-

lated by many other binding partners (Fig. 3) (Kushnir

and Marks 2010).

Ryanodine Receptor (RyR),Fig. 3 Multiple effectors

modulate RyRs. Multiple

agonists (blue) and antagonists(red) are known to affect both

RyR isoforms. Some

modulators have dual effects,

depending on their

concentration or specific RyR

isoform. Modulation of RyRs

can occur from either the

cytoplasmic or lumenal side

R 1708 Ryanodine Receptor (RyR)

Several small molecule ligands are also known to

affect RyRs (Fig. 3). The receptor is sensitive to Ca2+,

in addition to other small cations. Notably, caffeine has

a low-affinity binding site on the RyR and stimulates

Ca2+ release. Ryanodine, a plant alkaloid that was

originally found to affect the Ca2+ release channel,

has dual effects on the channel. It stabilizes

subconductance states at lower concentrations,

but becomes inhibitory at higher levels (Lai et al.

1989). Various adenosine nucleotides are able to

activate RyRs with ATP being the most potent.

In addition, RyRs are sensitive to molecules present

during oxidative stress such as NO.

Disease

Because Ca2+ is a very potent intracellular second mes-

senger, it is not surprising that mutations in RyR genes

can cause serious conditions. So far, only mutations in

RyR1 and RyR2 have been associated with disease

phenotypes (Betzenhauser and Marks 2010). The most

commonly associated phenotypes are shown below.

Point mutations in RyR1 can lead to Malignant

Hyperthermia (MH), Central Core Disease (CCD), and

Multi Mini Core Disease (mmCD). MHmanifests itself

as severe rises in body temperatures upon the adminis-

tration of volatile anesthetics, or under conditions of

heat stress. CCD and mmCD are disorders with variable

pathologies, but they are usually associated with cores

of metabolically inactive tissue in the center of muscle

fibers. Very often this results in muscle weakness.

In cardiac muscle, mutations in RyR2 are a cause of

sudden cardiac death, mostly associated with two

types of cardiac arrhythmias, catecholaminergic

polymorphic ventricular tachycardia (CPVT) and

arrhythmogenic right ventricular dysplasia type 2

(ARVD2). CPVT is associated with bidirectional VT,

usually triggered by emotional or physical stress.

ARVD2 results in the replacement of ventricular tissue

with fibrofatty deposits. In addition, idiopathic ventric-

ular fibrillation can be the result of loss of RyR2

function.

The bulk of the mutations characterized so far cause

a gain of function, resulting in increased or prolonged

Ca2+ signals in the cytoplasm. In cardiac myocytes,

this may activate the Na+/Ca2+ exchanger, situated in

the plasma membrane, which exchanges one Ca2+ for 3

Na+ ions. The net influx of additional positive charges

causes a delayed after depolarization (DAD), underly-

ing the arrhythmogenic nature of the mutations.

In skeletal muscle, the activation of RyR1 by

halogenated anesthetics can cause a massive release

of Ca2+, leading to activation of Ca2+ ATPases. This

depletes the ATP reserve, driving the cell in

a hypermetabolic state which causes acidosis and a

lethal increase in temperature (Betzenhauser

and Marks 2010).

Disease mutations in RyR1 and RyR2 seem to

cluster in three different regions of the receptor genes

(hot spots). These three locations in both receptors

match, suggesting important functional roles for

those portions. They are located near the N-terminus,

in a central domain region, and a C-terminal

Ryanodine-Sensitive Ca2+ Release Channels 1709 R

region covering the transmembrane segments. A high-

resolution structure of the N-terminal hot spot shows

that most mutations affect domain–domain interac-

tions (Tung et al. 2010). As these domains likely

move during opening of the channel, weakening

their interfaces is thought to facilitate the opening,

causing inadvertent leak of Ca2+ into the cytoplasm.

Summary

Ryanodine Receptors (RyRs) are large ion channels,

located in the membrane of the ER, that release Ca2+

upon stimulation. Three isoforms are found in

mammalian organisms. All form homotetrameric

assemblies of up to 2.2 MDa in size, and have

a very large cytoplasmic portion that contains phos-

phorylation targets and docking sites for multiple small

molecules and protein binding partners. Ca2+ is the

primary ligand that triggers opening, and the channel

can sense both cytoplasmic and lumenal Ca2+ levels.

They play a prime role in excitation-contraction

coupling, whereby they amplify the signal generated

by voltage-gated calcium channels. Mutations are

known to cause devastating diseases originating

either in skeletal or cardiac muscle, and most of them

are responsible for a gain of function, leading to

increased Ca2+ release.

R

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Ryanodine-Sensitive Ca2+ ReleaseChannels

▶Ryanodine Receptor (RyR)