Benita SjögrenDepartment of Pharmacology, University of Michigan, Ann Arbor, Michigan, USA
Regulator of G Protein SignalingProteins as Drug Targets: CurrentState and Future Possibilities
Abstract ___________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________Regulators of G protein signaling (RGS) proteins have emerged in the
past two decades as novel drug targets in many areas of research. Theirimportance in regulating signaling via G protein-coupled receptors has be-come evident as numerous studies have been published on the structure andfunction of RGS proteins. A number of genetic models have also beendeveloped, demonstrating the potential clinical importance of RGS proteinsin various disease states, including central nervous system disorders, cardio-vascular disease, diabetes, and several types of cancer. Apart from theirclassical mechanism of action as GTPase-activating proteins (GAPs), RGSproteins can also serve other noncanonical functions. This opens up a newapproach to targeting RGS proteins in drug discovery as the view on thefunction of these proteins is constantly evolving. This chapter summarizes thelatest development in RGS protein drug discovery with special emphasis onnoncanonical functions and regulatory mechanisms of RGS protein expres-sion. As more reports are being published on this group of proteins, it isbecoming clear that modulation of GAP activity might not be the only way totherapeutically target RGS proteins.
I. Introduction _____________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________During the past several decades, G protein-coupled receptor (GPCR)
signaling has been greatly explored and targeted for drug discovery. Numer-ous clinically used drugs have been developed that target these receptors, andthe vast research has led to a comprehensive understanding of their physio-logical and pathophysiological function.
As is well understood, GPCRs signal through heterotrimeric G proteinsconsisting of an a- and a bg-subunit. Upon receptor activation, GDP on the a-subunit is exchanged for GTP and the heterotrimer dissociates. The signal isturned off by GTP hydrolysis to GDP and reformation of the heterotrimericcomplex.
In vitro, the intrinsic rate of GTP hydrolysis on the activated a-subunit isfairly slow, in the range of minutes. This correlates poorly with in vivostudies where this process is fast and the GTP hydrolysis occurs within acouple of seconds. The missing component, regulator of G protein signaling(RGS) proteins, was identified in the early 1990s. RGS proteins are a familyof proteins with the main function of accelerating GTPase activity at active(GTP-bound) Ga subunits. This action causes reduced amplitude and dura-tion of GPCR-mediated signaling (Hollinger & Hepler, 2002; Ross &Wilkie, 2000). RGS proteins all share a common domain termedthe RGS or RH (RGS homology) domain, which is responsible for thecatalytic activity toward Ga proteins. There is increasing literature on theroles for RGS proteins in regulating GPCR signaling as well asnon-GAP mechanisms, and this altogether suggests a potential role for RGSproteins as novel drug targets. This chapter gives an update of thecurrent state of RGS proteins in drug discovery with an emphasis on nonca-nonical functions and the regulation of RGS protein by posttranslationalmodifications.
II. RGS Proteins Regulate Signaling via GPCRs _____________________________________________________________________________________________________________________________________________________________________________________________________________________________________________Since their discovery more than 15 years ago, RGS proteins have been
characterized in their ability to regulate GPCR signaling, structure, tissuedistribution, and physiological function. Although there is still much to bediscovered, it is clear that RGS proteins are emerging as novel drug targets inseveral pathophysiological states. To date, 20 mammalian RGS proteins havebeen characterized and an additional 10 proteins that contain an RHdomain. The structure, function, and importance in physiology for RGSproteins have been extensively reviewed previously (Hollinger & Hepler,2002; Neubig & Siderovski, 2002; Ross & Wilkie, 2000; Sjögren et al.,2010; Tesmer, 2009; Zhong & Neubig, 2001).
RGS Proteins as Drug Targets: Current and Future 317
A. RGS Protein Families
RGS proteins are divided into subfamilies according to domain- andsequence-homology as well as specificity toward Ga subunits. The size andcomplexity of these molecules range from the simplest case, the R4 family,which, apart from the RGS domain, only contains small N- and C-terminalextensions, to more complex proteins. These can contain several additionaldomains that can participate in membrane-targeting and signal transductionmechanisms. An overview of the classical RGS proteins is presented in Table Iand has been previously described in a number of reviews (see, e.g., Hollinger& Hepler, 2002; Neubig & Siderovski, 2002; Ross & Wilkie, 2000).
The R4 family of RGS proteins (RGS1–5, 8, 13, 16, 18, and 21) containonly small N- and C-termini apart from the RGS domain. The N-terminalamphipathic helix present in most R4 family members serves an importantfunction in membrane association and can directly bind phospholipids(Bernstein et al., 2000; Saitoh et al., 2001). Despite the noncomplex structureof these proteins, several R4 family RGS proteins have been shown to possessadditional functions apart from the ability to work as GAPs at activated Gasubunits (see Section IV).
The members of the R7 family of RGS proteins (RGS6, 7, 9, and 11) aremore complex structures than the R4 family and are closely related to theCaenorhabditis elegans homologues EGL-10 and EAT-16 that were identi-fied in the early stage of RGS protein research (Hajdu-Cronin et al., 1999;Koelle & Horvitz, 1996). Several additional domains are present in theseproteins, that is, the Gg-like (GGL) domain, a disheveled-EGL10-Pleckstrin(DEP) homology domain, and a DEP helical extension domain (Andersonet al., 2009). These additional domains mediate protein–protein interactions,subcellular localization, and protein stability (Anderson et al., 2009).
The third family of classical RGS proteins is the R12 family consisting ofRGS10, 12, and 14. RGS12 and 14 are large proteins with additionaldomains that can participate in protein–protein interactions and other func-tions. The Gai/o-Loco (GoLoco) motif has GDI (guanine nucleotide dissocia-tion inhibitor) activity toward Gai1, Gai2, and Gai3 (Kimple et al., 2001;Siderovski & Willard, 2005). Through this activity, RGS12 and RGS14 caninhibit G protein signaling both by accelerating GTP hydrolysis and bypreventing G protein activation.
The RZ family of RGS proteins is less well characterized than the othersand consists of RGS17, 19, and 20. All members of this family contain an N-terminal cysteine string motif (reviewed in Nunn et al., 2006) which is a siteof palmitoylation which could serve functions in membrane targeting, pro-tein stability, or aid protein–protein interactions (reviewed in Linder &Deschenes, 2007). However, the function in the case of RZ family RGSproteins is not yet fully understood.
TABLE I Overview of Classical RGS Proteins
Family RGS proteins Domains Modifications Interactions References
RZ/A (reviewed inNunn et al., 2006)
RGS17/RGS-Z2 Cysteine string Palmitoylation Ogier-Denis et al.(2000), De Vries et al.(1996)
RGS19/GAIP Phosphorylation,palmitoylation
RGS20/RGS-Z1 PalmitoylationRet-RGS1
R4/B (reviewed inBansal et al., 2007)
RGS1 Amphiphatic a-helixRGS2 Phosphorylation,
palmitoylationACI, II, V, VI, eIF2e Cunningham et al.
(2001), Salim et al.(2003), Ni et al.(2006), Roy et al.(2006), Nguyen et al.(2009)
RGS3 Palmitoylation Castro-Fernandez et al.(2002)
RGS4 Palmitoylation PIP3, calmodulin Srinivasa et al. (1998),Popov et al. (2000),Ishii et al. (2005a,2005b)
RGS5RGS8RGS13 Phosphorylation CREB Xie et al. (2008, 2010)RGS16 Phosphorylation,
palmitoylationGa13 Druey et al. (1999),
Chen et al. (2001),Derrien and Druey(2001), Derrien et al.(2003), Hiol et al.(2003), Johnson et al.(2003)
RGS18RGS21
R7/C, (reviewed inAnderson et al.,2009)
RGS6 DEP, GGL, DHEX R7BP, Gb5 Benzing et al. (2002),Rose et al. (2000),Sandiford and Slepak(2009), Hu et al.(2001), Sokal et al.(2003)
Snow et al. (1998), Schiffet al. (2000), Snowet al. (2002)
RGS14 GoLoco
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As mentioned previously, there are a number of proteins that contain anRH domain in addition to the 20 classical RGS proteins described above(reviewed in Tesmer, 2009). These noncanonical RH domain-containingproteins include G protein-coupled receptor kinases (GRKs), Rho guaninenucleotide exchange factors (RhoGEFs), Axin, A-kinase-anchoring protein(DAKAP-2), and a subset of sorting nexins (SNXs). Although many of theRH domain-containing proteins (including GRK2, GRK3, and the three RHdomain-containing RhoGEFs) have been shown to interact with Ga, it is notyet clear whether they actually have GAP activity toward activated G pro-teins. Indeed, as described in a recent review (Tesmer, 2009), the mode ofinteraction with the RH domain in both the GRK/Gaq and the RhoGEF/Ga13complexes is very different as compared to the published RGS4/Ga. Clearly,more studies need to be performed to ascertain the function of the RHdomain in these proteins.
B. GAP Activity—The Classic Mechanism of Action forRGS Proteins
The mechanism by which RGS proteins regulate GPCR-mediated signal-ing has been well characterized. RGS proteins serve as GAPs on activated Ga-subunits. This mode of action was first demonstrated by Berman et al.(1996b). They showed that RGS proteins bind to activated Gai1 and increasethe rate of GTP hydrolysis. The proposed mechanism in this publication(Berman et al., 1996b) explained previous observations in yeast, where theprotein Sst2p negatively modulates G protein (Gpa1p)-dependent signalingby the a factor pheromone (Dohlman et al., 1995, 1996). This was laterbiochemically confirmed in studies with Gpa1p and Sst2p (Apanovitch et al.,1998). The initial study by Berman et al. was accompanied by others con-firming this mechanism of action for RGS proteins (Berman et al., 1996a;De Vries et al., 1996; Hunt et al., 1996; Watson et al., 1996).
Further support for the mode of action of RGS proteins has come fromthe structure of RGS4 in complex with Gai1 (PDB ID 1AGR; Tesmer et al.,1997). This structure demonstrated that RGS4 interacts with the Ga proteinin a state that is representative of the GTP-bound catalytic transition state ofthe G protein. It clearly showed that RGS proteins interact with activated Gaproteins. Since the publication of this first structure, a number of others havedemonstrated structures of RGS proteins (Tesmer, 2009) and have greatlycontributed to the knowledge of the mechanism of action of this family ofproteins.
C. G Protein Specificity
RGS proteins act on activated Ga proteins of the Gai/o, Gaq, and Ga12/13families. The specificity of different RGS proteins toward certain subtypes
R4: 1, 3, 4, 5, 8, 10, 13, 16, 18RZ: 17, 19
R12: 12, 14
G12 Gi
Go
Gz
Gq
RZ: 20
R7: 6, 7 9, 11
2, GRK
RhoGEFp115, PDZ, LARG
FIGURE 1 G protein selectivity among RGS proteins. Several RGS proteins are fairlypromiscuous in their selectivity toward Ga subunits, while others are more selective. Thiscontributes greatly to the specificity toward the regulation of specific signaling pathways. Notethat most of the data on G protein specificity are based on in vitro studies and may not reflect thesituation in biological systems.
RGS Proteins as Drug Targets: Current and Future 321
can vary between or even within RGS protein families and contributes to thespecificity in regulating GPCR signaling (Fig. 1). Although several RGSproteins are fairly promiscuous, there are some exceptions that could explaindifferential regulation within tissues and even within a single cell. The mem-bers of the R4 family are mostly nonselective toward all members of the Gi/oand Gq family of G proteins. RGS2 is the exception with great selectivitytoward Gaq (Heximer et al., 1997). The R7 family of RGS proteins onlypresent GAP activity toward Ga subunits of the Gi/o family and, untilrecently, thought to be nonspecific among these proteins. However, in vitrodata later showed differential GAP activity within the R7 family of RGSproteins. All four members show similar efficacy toward Gao, however,RGS9 and RGS11 are more promiscuous with comparable GAP activity onGai1, Gai2, and Gai3 as on Gao. In contrast, RGS6 and RGS7 are moreselective with 5- to 10-fold higher activity for Gao than the Gai subtypes(Hooks et al., 2003; Lan et al., 2000). Interestingly, even though RGS7 ismore selective for Gao over Gai isoforms, it is still 10- to 20-fold less potenttoward Gao than RGS4, a member of the R4 family (Lan et al., 2000).
Further examples of selectivity are members of the R12 and RZ family ofRGS proteins, as well as the “nonclassical” RhoGEF proteins p115, PDZ,and LARG (Fig. 1). In the R12 family of RGS proteins, RGS10 is fairlynonselective, whereas RGS12 and 14 show selectivity for members of the Gifamily of Ga subunits. However, this selectivity might be explained by GDIactions of the GoLoco motif present in these two proteins and not directlyattributed to GAP activity mediated by the RGS domain (Kimple et al.,2001). This will be further discussed in Section IV. RGS20, a member of
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the RZ family, is the only RGS protein showing selectivity toward Gaz (Glicket al., 1998). While the other members of the RZ family, RGS17 and 19, aswell as the majority of R4 family members, have GAP activity towards Gaz,they also have activity on the other members in the Gi/o and Gq families ofGa proteins.
Although RGS protein selectivity toward different Ga protein subtypescontributes to the physiological effect seen in vivo and in cellular systems, thisismost likely not the onlymechanism bywhichRGSproteins reach specificity.Tissue and subcellular distribution, cell background, and noncanonical func-tions (see Sections IV and V) also play important roles in the actions of RGSproteins. Also, most studies on G protein selectivity have been performedin vitro and may not completely reflect the situation in biological systems.This was demonstrated in a study by Anger et al. They studiedmembers of theR4 family in a recombinant cell system coexpressed withM2 orM3muscarin-ic receptors. All proteins studied (RGS2-5) were able to inhibit Gai/o-mediatedactivation of Akt, contradicting the hypothesis that RGS2 is selective for Gaq.Even more surprising was that Gaq-mediated Akt phosphorylation was onlyblocked by RGS3 and not by RGS2. However, ERK activation mediatedthrough Gaq could be blocked by RGS2, 3, and 5, whereas RGS2 wasincapable to inhibit Gai/o-mediated ERK activation (Anger et al., 2007).These data suggest that selectivity toward certain Ga subunits does notaccount for all specificity for RGS proteins to regulate signal transduction.Other mechanisms are clearly involved andmay be explained by the emergingdata on noncanonical functions of RGS proteins.
III. Regulation of RGS Protein Function and Expression ____________________________________________________________________________________________________________There are several mechanisms by which RGS protein function can be
regulated. Emerging data suggests that regulatory mechanisms controllingmembrane targeting, subcellular localization, and protein stability are keyplayers in modulating function of RGS proteins. The following sectionsdescribe some of the described mechanisms whereby RGS protein functioncan be regulated. An overview of classical RGS proteins and their modulationis also presented in Table I.
A. Membrane-Targeting Mechanisms
In order to act as GAPs on activated Ga subunits, RGS proteins need tobe localized at or near the plasma membrane. Membrane targeting can beachieved through several different mechanisms such as protein–protein inter-actions or through posttranslational modifications. Several groups haveidentified the N-terminus as a key structure for targeting signals (e.g.,Chatterjee & Fisher, 2000; Chen et al., 1999; Heximer et al., 2001;
RGS Proteins as Drug Targets: Current and Future 323
Hiol et al., 2003; Srinivasa et al., 1998). One common mechanism of mem-brane targeting is through palmitoylation. This was first demonstrated forRGS19 which, like all members of the RZ family, contains an N-terminalcysteine string (De Vries et al., 1996). Other proteins with this motif havepreviously been shown to be heavily palmitoylated, and De Vries et al.showed that this was also the case for RGS19. Interestingly, only the mem-brane-associated portion of RGS19 was palmitoylated leading to the conclu-sion that this posttranslational modification is a membrane-targeting signal.Subsequently, several other RGS proteins were shown to be modified bypalmitoylation and that this aids in membrane targeting (Castro-Fernandezet al., 2002; De Vries et al., 1996; Druey et al., 1999; Rose et al., 2000;Srinivasa et al., 1998).
Most members of the R4 family of RGS proteins contain an N-terminalamphiphatic helix that can be palmitoylated, anchor the protein to theplasma membrane, and bring it close to the site of action. Thus far, threesites for palmitoylation have been identified on RGS4. Two of these arelocated at the N-terminus and are important for plasma membrane localiza-tion (Srinivasa et al., 1998).
Although palmitoylation often serves as a membrane-targeting signal,this posttranslational modification can also have other consequences. Palmi-toylation of three cysteines (106, 116, and 199) on RGS2 was shown tomodulate conformation of the protein, and removal of either of these palmi-toylation sites inhibits RGS2 GAP activity toward Gaq in vitro (Ni et al.,2006). Similar results have been obtained in studies of other R4 familymembers, that is, RGS4, RGS10, and RGS16 (Hiol et al., 2003; Osterhoutet al., 2003; Tu et al., 1999). Clearly, palmitoylation of RGS proteins servesimportant functions in subcellular targeting as well as in function and con-formation of the proteins.
Another mechanism by which RGS proteins can be targeted to theplasma membrane is through interaction with other proteins. The mostwell-characterized case is the R7 family of RGS proteins which throughtheir DEP domain can interact with one of two membrane-targeting proteins(reviewed in Jayaraman et al., 2009). In the retina, RGS9-1 interacts withR9AP (RGS9-anchoring protein). R9AP has a transmembrane domain whichattaches the complex to the plasma membrane and enables RGS9-1 to act asa GAP on transducin (Gat), to regulate signaling via rhodopsin. The brain-specific R7BP (R7-binding protein) recruits all R7 family RGS proteins to theplasma membrane. The mechanism of membrane association for R7BP isdifferent from that of R9AP. R7BP is palmitoylated in the C-terminal regionand is hence more loosely attached to the plasma membrane (Drenan et al.,2005). This membrane-targeting mechanism increases R7 family RGS pro-tein efficiency of action toward Ga proteins as was shown in a study byDrenan et al. (2006). They showed that palmitoylation of R7BP is necessaryfor the RGS7/Gb5/R7BP complex to be translocated to the plasmamembrane
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and enhances the function of the complex. Heterotrimers, where R7BP wasnot palmitoylated, were just as inefficient in inhibiting G protein signaling asthe RGS7/Gb5 heterodimer.
Taken together, membrane targeting of RGS proteins is an importantregulatory mechanism for function. As will become clear in the next sectionin the case of RGS9, it seems that the interaction with R7BP is also necessaryfor protein stability (Anderson et al., 2007).
B. Protein Stabilization Mechanisms
As in many areas of biology, RGS proteins depend on additional cofac-tors to be stably expressed in cells. The members of the R7 family (RGS6, 7, 9,and 11) depend on the interaction with the Gb5 subunit for stable expression(reviewed in Anderson et al., 2009). This interaction has been well character-ized, and the crystal structure of the RGS9–Gb5 complex was recently pub-lished (Cheever et al., 2008). It revealed that the majority of the interactionoccurredwith theGGLdomain of RGS9 but also showed points of interactionwith the DEP domain. The interaction is crucial for stability of both proteinsas is demonstrated by the Gb5 knockout mice which lack protein expressionof all four members of the R7 family of RGS proteins (Chen et al., 2003).
Apart from Gb5, the members of the R7 family of RGS proteins alsointeract through their DEP domain with R7BP in the brain and R9AP in theretina as discussed above. This interaction serves as a membrane-targetingmechanism to bring the RGS protein to the site of action and, at least in thecase of RGS7, enhances effects on GPCR signaling (Drenan et al., 2006).The RGS–R7BP/R9AP interaction does not seem to be necessary forstable expression of R7 family RGS proteins. The exception being RGS9where Anderson et al. showed that the interaction also serves to stabilizethe protein. In the absence of R7BP or R9AP, RGS9 is degraded by cysteineproteases (Anderson et al., 2007). This was not the case for the RGS7 inthe same study. However, it seems that the interaction serves as an importantmodulator of function of all four members of the R7 family of RGS proteins.
Protein synthesis and degradation are tightly regulated processes for phys-iological function, and RGS proteins are no exception. The expression ofseveral RGS proteins is induced (or reduced) in response to various stimuli,for example, oxidative stress and apoptosis. The importance of altered RGSprotein expression was recently demonstrated in invasive cancer cell lines aswell as in a mouse xenograft model (Xie et al., 2009). This study demonstratedthat overexpression of RGS4 resulted in reduced cancer cell migration andinvasion. They also found that even thoughmRNAlevelswere greatly increasedin human breast cancer, the protein levels of RGS4were severely diminished. Itwas found that increased RGS4 protein degradation was responsible for thisdownregulation, and in fact, breast cancer tumor invasiveness could be blockedby inhibiting RGS4 protein degradation (Xie et al., 2009).
RGS Proteins as Drug Targets: Current and Future 325
The pathways for protein degradation are receiving more and moreinterest not only in drug discovery concerning RGS proteins but also inmany other fields of research. Several drugs are on the market that targetproteasomal degradation in the treatment of various cancers where the deg-radationmachinery has been compromised (Adams, 2004; Yang et al., 2009).The mechanisms for proteasomal degradation have been well characterizedand are reviewed elsewhere (see, e.g., Hershko & Ciechanover, 1998).
SeveralRGSproteins are rapidlydegraded in cells, and someof themechan-isms have been characterized. RGS4, 5, and 16 are degraded through the so-called N-end rule pathway (Bodenstein et al., 2007; Davydov & Varshavsky,2000; Lee et al., 2005), first proposed by Alexander Varshavsky (reviewed inVarshavsky, 1996). Thismechanism is based on the N-terminal residue follow-ing the initial methionine being either stabilizing or destabilizing. By posttrans-lationalmodification, themethionine is removed and the destabilizing residue isexposed. RGS4, 5, and 16 all have a cysteine in this position which is adestabilizing residue. Following oxidation of the cysteine, the enzyme R-trans-ferase (encoded by ATE-1) couples an arginine to the N-terminus which isdirectly recognizable by E3 ligases in the degradation machinery.
Early in vitro data demonstrated RGS4 and RGS16 as targets for the N-end rule pathway (Davydov & Varshavsky, 2000). This study showed thatRGS4 and RGS16 are rapidly degraded in cells and mutating the cysteine inposition 2 to either alanine or glycine stabilizes protein expression. Laterstudies in embryonic stem cells from ATE-1 knockout mice (Lee et al., 2005)confirmed this for RGS4 and also identified RGS5 as another target. In wild-type mice, both RGS4 and RGS5 are rapidly degraded. The degradation wascompletely diminished in cells from ATE-1 knockout mice, suggesting thatR-transferase is absolutely necessary for degradation of RGS4 and RGS5.The follow-up studies in vivo have not been performed due to the fact thatATE-1 knockout mice are embryonic lethal due to deficiencies in cardiovas-cular development (Kwon et al., 2002). This is evidence of the importance ofthe N-end rule pathway and tightly regulated mechanisms of protein degra-dation during development. Recently, a conditional ATE-1 knockout mousewas developed (Brower & Varshavsky, 2009). This line is viable and mayprovide further insight to the importance of protein degradation for RGS4, 5,16, and potentially other RGS proteins. Nevertheless, it seems clear thatexpression levels of several RGS proteins are tightly regulated at the levelof protein degradation (Sjogren & Neubig, 2010).
C. Regulation of RGS Proteins by Phosphorylation
As is clear from the aforementioned studies, degradation of RGS proteins isa tightly regulated process and can be regulated through different processes.Phosphorylation is awidespread cellularphenomenon that plays important rolesin the regulation of protein activity, expression, and subcellular localization.
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RGS proteins are not an exception in this context, and a lot of progress has beenmade in recent years in understanding the roles andmechanisms of RGS proteinphosphorylation.One example came froma recent study (Xie et al., 2010)whichshowed that RGS13 is protected from degradation by phosphorylation at Thr41
by protein kinaseA (PKA) via a cAMP-dependentmechanism. In the presence ofcAMP, the half-life of wild-type RGS13 was increased almost threefold.However, this effect was not present in the T41A mutant (Xie et al., 2010).This shows that although RGS proteins do not act directly on Gas, their expres-sion can be regulated by Gas-mediated signaling.
RGS16 is stabilized by phosphorylation mediated by Src kinase (Derrienet al., 2003). It was first demonstrated that RGS16 is phosphorylated at twoconserved Tyrosine residues (Tyr168 and Tyr177) and that these sites serveimportant regulatory functions (Derrien & Druey, 2001). The first studydemonstrated that phosphorylation at Tyr177, although not affecting GAPactivity in vitro, increases the ability of RGS16 to modulate Gi-mediatedcAMP inhibition (Derrien & Druey, 2001). Later, the same group showedthat RGS16 protein degradation is slowed down by phosphorylation atTyr168, the only other tyrosine residue in the protein (Derrien et al., 2003).This phosphorylation also has effects on GAP activity in that it increasesGAP activity in a single turnover GTPase assay. Another study came fromChen et al. showed that phosphorylation of RGS16 at two serine residues(Ser53 and Ser194) impairs GAP activity of RGS16 and its ability to regulatedownstream G protein signal transduction (Chen et al., 2001). Together,these studies show that phosphorylation of RGS16 plays an importantregulatory role and that depending on the residues phosphorylated, theactivity and protein expression can be differentially modulated.
RGS2 is phosphorylated at a serine residue in the RGS domain byprotein kinase C (PKC), and this attenuates GAP activity toward Gaq(Cunningham et al., 2001). In contrast, phosphorylation of RGS19 by Erkkinase actually increases RGS19 GAP activity toward Gai3 in vitro (Ogier-Denis et al., 2000). Further, phosphorylation of RGS10 at Ser168 by PKAinduces translocation of the protein to the nucleus (Burgon et al., 2001). Thisreduces effects on Ga by removing RGS10 from the site of action (i.e., theplasma membrane) without affecting GAP activity in vitro. This mechanismfurther strengthens the importance of RGS protein membrane targeting asdiscussed above.
Members of the R7 family of RGS proteins are also regulated by phos-phorylation events. The retina-specific isoform of RGS9 (RGS9-1) migratesto lipid rafts upon activation where it is phosphorylated at Ser475 by a kinase(Hu et al., 2001), later determined to belong to the PKC family (Sokal et al.,2003). This phosphorylation significantly reduces the affinity of RGS9-1 forits membrane anchor R9AP, suggesting a negative functional regulation.RGS7 interacts with 14-3-3 b through a mechanism that is dependent on
RGS Proteins as Drug Targets: Current and Future 327
phosphorylation at Ser434 in RGS7. This interaction inhibits the ability forRGS7 to interact with, and have GAP activity toward, Gao (Benzinget al., 2002).
As should be evident from the examples discussed above, phosphoryla-tion of RGS proteins serves an important function in modulating expressionand GAP activity (both directly and indirectly) toward Ga protein subunits.
IV. Noncanonical Functions of RGS Proteins _____________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________Apart from the canonical function as GAPs, many RGS proteins also
have other properties which have been a research area of great interest inrecent years. As discussed earlier, several RGS proteins contain additionaldomains to their RGS domain that can play a role in these functions.However, as will be outlined in this section, the presence of additionalfunctional domains is not always necessary for an RGS protein to havefunctions that are not directly related to the GAP activity. The followingsection brings up examples of noncanonical functions of RGS proteins.Many more have been characterized and are summarized in detail in a recentreview (Sethakorn et al., 2010).
The ability to participate in protein–protein interactions has been welldescribed for members of the R7 family of RGS proteins. Through the DEPdomain, these proteins can interact with and affect signaling through recep-tors and other proteins. Shuey et al. discovered one such phenomenonbetween RGS7 and the serotonin 2C receptor (5-HT2C). They found thatRGS7 inhibits Ca2þ signaling through this Gaq-coupled receptor (Shueyet al., 1998) and this was later discovered to be the result of a direct interac-tion between RGS7 and Gaq (Fig. 2, IV) as demonstrated by fluorescenceresonance energy transfer (FRET) (Witherow et al., 2003). Althoughthe R7 family members are selective GAPs for the Gi/o family of G proteins,this shows that RGS proteins can affect G protein signaling throughother mechanisms than through GAP activity. It has not yet been determined,however, whether RGS7 can have GAP activity toward Gaq. It is possible thatthis is actually an example of RGS7 acting on Gaq in a canonical fashion.
RGS7 has also been shown to regulate Gaq-mediated signaling via M3
muscarinic receptors through a direct interaction between the third intracel-lular loop of the receptor and the DEP domain of RGS7 (Fig. 2, III; Sandiford& Slepak, 2009). Clearly, the DEP domain is an important mediator ofprotein–protein interactions that enables R7 family RGS proteins to affectsignal transduction through noncanonical mechanisms.
A well-studied example of noncanonical RGS protein functions is theGai/o-Loco (GoLoco) motif present in the R12 family members, RGS12 andRGS14. Similar to the RGS domain, it binds to Ga but has GDI activitytoward Gai1, Gai2, and Gai3 (Fig. 2, VII; Kimple et al., 2001; Siderovski &
N-type calcium channel
Gai1–3
Gaq
DE
P
Gas
RGSPTBPDZRGS7
RGS13
CREB
RGS2RGS12
GoLoco
GTP
GDP
VIIVI
VIII
III
II
I V
AC
ATP
GDP
elF2 elF2
elF2Bε
GTP
40S ribosome
NUCLEUS
cAMP
IV
FIGURE 2 Overview of selected noncanonical functions of RGS proteins. The majority ofRGS proteins and RH domain-containing proteins possess functions apart from the GAPfunction toward Ga. Shown are some examples. (I) RGS2 suppression of Gas signaling throughinteraction with adenylate cyclase (AC). (II) RGS2 suppression of protein translation throughinteraction with eIF2Be. (III) Inhibition of GPCR signaling by RGS7 through interaction betweenthe third intracellular loop and the DEP domain of RGS7. (IV) Inhibition of Gq signaling byRGS7 through interaction between RGS7 and Gaq. (V) Interaction between the PDZ domain ofRGS12 and the C-terminus of GPCRs. (VI) Interaction between the PTB domain of RGS12 andthe pore-forming complex of N-type calcium channels. (VIII) Supression of transcription byRGS13 interaction with CREB.
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Willard, 2005). The interaction between the GoLoco motif and Ga inhibitsGTP exchange on the Ga subunit, thereby preventing G protein activation.Further, it blocks association of Ga with Gbg potentially leading toprolonged bg signaling. This demonstrates that these two RGS proteinscan inhibit G protein signaling through two distinct mechanisms. Inaddition, the actions through the GoLoco motif are not directly dependenton receptor activation. RGS proteins have the highest affinity for activated(GTP-bound) Ga, whereas the GoLoco motif binds to inactive (GDP-bound)Ga subunits.
An N-terminal PDZ domain in one splice variant of RGS12 can interactwith the C-terminus of several GPCRs (Fig. 2, V; Snow et al., 1998, 2002).Additional protein–protein interactions can be formed between a phospho-tyrosine binding (PTB) domain in RGS12 and the pore-forming unit of theN-type calcium channel in a phosphorylation-dependent manner (Fig. 2, VI;Schiff et al., 2000). Altogether, the presence of additional protein domains in
RGS Proteins as Drug Targets: Current and Future 329
RGS proteins plays important roles in regulating expression, subcellularlocalization, and noncanonical functions.
As mentioned above, additional protein domains are not always a neces-sary prerequisite for the ability of an RGS protein to serve noncanonicalfunctions. There are several examples of R4 family RGS proteins regulatingsignal transduction in a non-GAP manner, despite their small size. RGS16,but not RGS4, binds to Ga13 through its N-terminus, thereby inhibitingGa13-Rho signaling (Johnson et al., 2003). The functional significance ofthis interaction is not yet clear. However, it shows additional means ofreaching specificity among closely related RGS proteins.
Although there are no widely accepted publications on RGS proteinshaving GAP activity toward Gas, RGS2 has been shown to regulate Gassignaling through a direct interaction with certain adenylate cyclase subtypes(ACI, II, V, and VI; Fig. 2, I; Roy et al., 2006; Salim et al., 2003). Further,RGS2 has been shown to suppress protein translation by direct interactionwith eukaryotic initiation factor 2B e subunit (eIF2e; Fig. 2, II) as demon-strated by studies in transfected cells (Nguyen et al., 2009). Lastly, RGS2knockout mice were found to have increased total protein synthesis ascompared to wild-type mice.
Other examples of R4 family RGS proteins possessing noncanonicalfunction came from a recent study by Xie et al. where they showed thatRGS13 bound directly to the transcription factor CREB in B lymphocytes(Fig. 2, VIII), thereby acting as a nuclear transcription repressor (Xie et al.,2008). They also found that the nuclear accumulation of RGS13 was drivenby increased cAMP levels in response to activation of adrenergic b2 receptors.This shows a mechanism whereby RGS proteins can participate in regulationof gene expression in response to GPCR activation.
V. Biological Functions of RGS Proteins—Implications inDrug Discovery ___________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________
As more data is emerging on the function of RGS proteins, it is becomingincreasingly clear that these are promising targets for drug discovery. Resultsfrom biochemical, cellular, and in vivo studies all support this notion. Therehave been several reviews published in the past 10 years on the biologicalfunctions of RGS proteins (see Table II; e.g., Bansal et al., 2007; Blazer &Neubig, 2009; Gu et al., 2009; Hooks et al., 2008; Neubig & Siderovski,2002; Sjögren et al., 2010; Traynor & Neubig, 2005). The emerging data onnoncanonical functions of RGS proteins might help explain some of theresults obtained in vivo and open up new possibilities for drug discovery.For the purpose of this review, this section will focus on some selectedexamples where RGS proteins play a role and where modulation of theireffects could have therapeutic benefits.
TABLE II Summary of Some Clinical Implications for RGS Protein Modulators
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A. Inhibition of RGS Protein Function
Inhibition of RGS protein function would theoretically enhance signal-ing via certain GPCRs. Many RGS proteins have a very limited expression(Gold et al., 1997) and this could increase tissue specificity and enable lowerdosage of an endogenous or exogenous agonist. This notion has been previ-ously discussed (Blazer & Neubig, 2009) and the potential in drug discovery
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for RGS protein inhibitors has been demonstrated recently in mouse modelswith perturbed RGS protein function.
Early studies of RGS protein–Ga interactions found that mutation of onesingle residue in the switch I region of Ga (G184S) will disrupt the binding ofany RGS protein (Lan et al., 1998). This mutation has been used to developknock-in mice expressing RGS-insensitive Ga proteins. These models enablestudies of the overall effect of RGS proteins on a specific signaling pathway.Although the identity of specific RGS proteins involved in the genotype-specific differences will need to be determined, it is a good model to studythe effects of overall impaired RGS protein function.
Supporting data for the importance of RGS proteins in regulating Gao-mediated signaling came from a recent study that showed thatmice expressingRGS-insensitive Gao protein showed enhanced a2a adrenergic suppression ofhippocampal CA3 epileptiform bursts, suggesting a potential role for RGSprotein inhibitors in the treatment of epilepsy (Goldenstein et al., 2009). Thiseffect was specific forGao asmice expressingRGS-insensitive Gai2 protein didnot display this difference.
The genetic models using RGS-insensitive Ga proteins have also openedup a new understanding for the potential for RGS protein inhibitors in thetreatment of CNS disorders such as depression. Depression is commonlyassociated with low levels of serotonin (5-HT) and is commonly treatedwith SSRIs (selective serotonin reuptake inhibitors). However, better treat-ments are needed since these drugs are associated with problems such as lateonset of the effect as well as various side effects (Whittington et al., 2005).A recent study using mice expressing RGS-insensitive Gai2 showed that thesemutant mice had a spontaneously antidepressant phenotype as demonstratedby tail suspension test and the forced swim test (Talbot et al., 2010). Thiseffect was due to enhanced signaling through the 5-HT1A receptor as theantidepressant phenotype could be reversed by the selective 5-HT1A receptorantagonist, WAY-100635. It was also found that other 5-HT1A-mediatedresponses were unaffected, suggesting specificity for the antidepressant effectvia 5-HT1A receptors acting on Gai2. Identifying, and developing an inhibitorfor, the specific RGS protein involved in this pathway would enable increasedselectivity in SSRI treatment of depression.
Recent data suggest a potential benefit of RGS4 inhibitors in the treat-ment of diabetes. Ruiz de Azua et al. showed that glucose-stimulated insulinsecretion (GSIS) in a mouse b-cell line (MIN-6) was enhanced by activation ofthe muscarinic M3 receptor. SiRNA-mediated knockdown of RGS4 in thesecells further enhanced M3 receptor-mediated GSIS (Ruiz de Azua et al.,2010). Further, RGS4 knockout mice show increased insulin release fromb-cells and subsequent reduced plasma glucose levels in response to an M3
agonist compared to their wild-type littermate controls. This demonstrates apotential role for RGS4 inhibitors in the treatment of type 2 diabetes whereinsulin release is impaired.
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B. Enhancement of RGS Protein Function
Compounds that act by enhancing RGS protein function could be bene-ficial in the treatment of cardiovascular disease. Many forms of cardiovascu-lar disease (e.g., hypertension, heart failure, and arrhythmias) are associatedwith enhanced GPCR signaling through hormones and neurotransmitters,such as noradrenaline, angiotensin II, 5-HT, endothelin, and acetylcholine.There has been evidence from the genetic models with RGS-insensitive Gaproteins that RGS proteins play important roles for regulating cardiovascularfunction.
Our lab showed that embryonic stem cell-derived myocytes (Fu et al.,2006) as well as isolated hearts (Fu et al., 2007) from mice expressing RGS-insensitive Gai2 show an increased cholinergic response. Following in vivostudies confirmed that these mice are more sensitive to muscarinic-inducedbradycardia (Fu et al., 2006). In this context, an RGS enhancer could bebeneficial to reduce signaling via muscarinic receptors that couple to Gai2.
Similar to the previously mentioned studies, identifying the RGS protein(s) responsible for the phenotype will be crucial in developing novel mod-ulators of RGS protein function. RGS4 could be a candidate for the enhancedmuscarinic responses in the RGS-insensitive Gai2 expressing mice. RGS4knockout mice show increased responses to agonists at the M2 muscarinicreceptor as well as decreased GIRK channel desensitization (Bender et al.,2008) and altered kinetics of acetylcholine-activated Kþ currents (Cifelliet al., 2008) in the heart. This opens up for the potential for RGS4 enhance-ment in the regulation of cardiac automaticity.
Further evidence for potential beneficial roles of enhancers of RGSprotein function comes from the phenotype of the RGS2 knockout mice,which are hypertensive and prone to early heart failure (Heximer et al., 2003;Oliveira-Dos-Santos et al., 2000). This is related to increased signalingthrough several receptors known to mediate vasoconstriction such as PAR-1 receptors (Tang et al., 2003), noradrenaline, angiotensin II, vasopressin,and endothelin in vascular smooth muscle cells (VMSCs). It was recentlydemonstrated that the main mechanism by which RGS2 regulates bloodpressure may be through actions in the kidney. A recent study used kidneycross-transplantation to demonstrate that restoring RGS2 protein to thekidney alone was sufficient to restore normal blood pressure in RGS2 knock-out mice (Gurley et al., 2010).
RGS9 enhancers could be beneficial in the treatment of drug addictionand side effects of L-DOPA treatment in Parkinson’s disease (PD). RGS9 isinvolved in the regulation of motor activity through effects on dopaminergicsignaling in the striatum. Overexpression of RGS9 in the nucleus accumbensreduces locomotor activity in response to a dopamine D2 selective agonist(Rahman et al., 2003). In accordance with this, RGS9 knockout miceshow increased locomotor activity in response to morphine or cocaine
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(Kovoor et al., 2005). This suggests an important role for RGS9 in theregulation of both opioidergic and dopaminergic signaling. Additionally,RGS9 knockout mice have deficiencies in motor coordination and workingmemory (Blundell et al., 2008). Enhancers of RGS9 function could thereforehave benefits in improving motor coordination that has been impaired as aresponse to clinically used as well as abused drugs.
The beneficial role for enhanced RGS9 function in the treatment ofL-DOPA-induced dyskinesias is supported by findings in primate models ofPD. Local striatal overexpression of RGS9 in MPTP-treated nonhuman pri-mates reduces L-DOPA-induced dyskinesias (Gold et al., 2007). This, togetherwith previous findings in RGS9 knockout mice, suggests that enhancingRGS9 function could be a beneficial complementary treatment in PD.
VI. Advances in RGS Protein Drug Discovery ___________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________Given the clinical potential for drugs modulating RGS protein function,
several attempts have already been made to develop compounds with thismechanism of action. Most effort has been put on the development ofinhibitors, given that it is generally an easier approach to block functionthan to enhance it. As will be discussed below, these studies are focused oninhibiting the RGS–Ga interaction.
Inhibiting protein–protein interactions with small molecules is a chal-lenging task since these interaction surfaces are very flat and do not havetraditional “druggable”-binding pockets. In RGS proteins, this interactionsurface has been termed the “A-site” and many of the currently publishedinhibitors actually bind to an allosteric pocket, termed the “B-site”which is amore favorable drug interaction surface (Zhong & Neubig, 2001). In theRGS4–Gao structure, this site is located on the opposite side of the RGSdomain, far away from the interaction with Ga (Tesmer et al., 1997).
The first RGS inhibitor reported was from a yeast two-hybrid screen atWyeth Pharmaceuticals utilizing RGS4 as bait (Young et al., 2004). Howev-er, no follow-up studies have been published around these compound seriesand their structures were never made public. Our lab developed severalbiochemical assays to study the effects of small molecules and peptides onthe RGS–Ga interaction. This initially resulted in a series of papers identify-ing peptide inhibitors of the RGS4–Gai2 interaction. The initial peptidesmimic the switch I region of the G proteins that is crucial for the interactionand these were used to create a constrained peptide library (�2.5 millionpeptides) that identified several novel peptides with inhibitory actions againstseveral RGS proteins (Jin et al., 2004; Roof et al., 2006, 2008, 2009).Recently, the group at Wyeth Pharmaceuticals also identified peptide inhibi-tors for RGS4 using the same yeast two-hybrid approach as mentioned above(Wang et al., 2008).
RGS Proteins as Drug Targets: Current and Future 335
Our lab developed a novel high-throughput assay to identify inhibitorsof the RGS–Ga protein interaction, the flow cytometry protein interactionassay (FCPIA; Roman et al., 2007). This method has previously been de-scribed in detail (Blazer et al., 2010b; Roman et al., 2007, 2009). Briefly, theRGS protein of interest is immobilized on a polystyrene bead. This is mixedwith fluorescently labeled Ga, and the bead associated fluorescence can thenbe measured by flow cytometry. Using this assay, we identified the firstpublically disclosed small molecule inhibitor of the RGS4–Gao interaction,CCG-4986 (Fig. 3, left; Roman et al., 2007). Another approach we havetaken to identify RGS protein inhibitors is the use of time-resolved FRET(TR-FRET; Leifert et al., 2006). This is a biochemical approach utilizingpurified, labeled RGS4 (AlexaFluor 488) and Gao (LanthaScreen terbiumprobe). In screening over 200,000 small molecules, we identified a series ofcompounds that are the first example of reversible inhibitors of the RGS4–Gao interaction. CCG-63802 (Fig. 3, center) inhibits this interaction with anIC50 value of �10 mM and shows specificity for RGS4 over other RGSproteins (RGS4>19>16>8�7). Studies are ongoing to characterize thebinding site for this series of compounds, but the current data suggest thatthe mode of action is binding to the allosteric “B-site,” thereby altering theconformation of the RGS–Ga interface (Blazer et al., 2010a). This mecha-nism of action for small molecule RGS inhibitors seems to be common. Thepreviously identified CCG-4986 also seems to bind at this site (Roman et al.,2007, 2010).
A third series of small molecule inhibitors of the RGS4–Gao interactionwas recently published from our lab. CCG-50014 (Fig. 3, right) was identi-fied in a biochemical screen using FCPIA and is the first nanomolar potency
CCG-4986 CCG-63802 CCG-50014
O
S
S
S
Cl
O O
O–
N+
O
N
NN
N
N
CH3
H3C
H3CO
O O
OS
N
NF
CH3
FIGURE 3 Structures of small molecule RGS inhibitors. The three published RGS proteininhibitors CCG-4986 (Roman et al., 2007), CCG-63802 (Blazer et al., 2010a), and CCG-50014(Blazer et al., 2011) that were identified using biochemical high-throughput screening methods.CCG-4986 was the first published structure and inhibits the RGS4–Gao interaction with micro-molar potency in an irreversible manner. CCG-63802 was the first reversible RGS proteininhibitor and CCG-50014 is the first RGS inhibitor that shows activity in a cellular environment.
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RGS4 inhibitor to date (Blazer et al., 2011). It has selectivity toward RGS4over other RGS proteins tested (RGS4>RGS8>RGS16>RGS19; no bind-ing to RGS7). However, it also acts on RGS8 at least in a biochemical setting.Like the previously identified RGS inhibitors, CCG-50014 binds to theallosteric “B-site” of the RGS domain and is dependent on cysteine reactivity.The cysteinless RGS4 mutant (where all cysteines in the RGS domain havebeen mutated to alanine) did not bind CCG-50014 and mutation analysisof the binding of CCG-50014 to RGS8 showed that the two cysteines in the“B-site” are important for the potency of the compound (Blazer et al., 2011).
Importantly, in HEK-293 cells, CCG-50014 can inhibit RGS4 recruit-ment to the plasma membrane by Gao. This is the first example of an RGSinhibitor that has activity in a cellular environment (Blazer et al., 2011) andserves an important proof of concept in RGS protein drug discovery.
The allostericmodulation of RGS proteins by these smallmoleculesmightnot be too surprising given the challenging task of inhibiting a large protein–protein interaction surface with small molecules. In fact, the “B-site” in RGS4seems to be a site for general regulatory mechanisms. As discussed previously(Section III), RGS4 is palmitoylated at several sites. These can serve as mem-brane-targeting mechanisms (in the N-terminus), but the third site of palmi-toylation (Cys95) appears to have a role in regulating GAP activity, possiblyby inhibiting the interaction with Ga (Popov et al., 2000). Close to this site,RGS4 is regulated by interactionwith PIP3 and calmodulin (Ishii et al., 2005a,2005b; Popov et al., 2000). It seems that binding of PIP3 inhibits both Gainteraction and GAP activity of RGS4, and calmodulin binding can counter-act this effect (Luo et al., 2001; Muallem & Wilkie, 1999). Given the role ofthe “B-site” for endogenous allosteric modulation, it is not surprising that thesmall molecule RGS4 inhibitors identified thus far bind at or near this site.
VII. The Future of Targeting RGS Proteins inDrug Discovery ___________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________
The advances in RGS protein drug discovery have thus far been drivenby high- and medium-throughput screens for inhibitors of the RGS–Gainteraction as described above. The functional impact of these compoundshas in many cases been confirmed in follow-up studies, such as effects onGAP activity. However, as has been discussed here, there are several otherways by which RGS protein function could be altered and all the currentliterature only looks at one aspect (i.e., Ga interaction). Further, even thoughRGS inhibitors are clearly interesting both as pharmacological tools andpotential for clinical development, enhancers of RGS protein function areequally important to identify. This section summarizes some approaches thatcould be taken to approach these limitations. Some may already be in usethough reports have yet to be published.
RGS Proteins as Drug Targets: Current and Future 337
Biochemically, RGS protein function can be assessed by measuring GAPactivity. This method is already being used as a follow-up confirmation forhits identified in screens. However, until recently, it has not been feasible touse this approach in a high-throughput format. Bellbrook labs in collabora-tion with the laboratory of David Siderovski (Zielinski et al., 2009) reportedan adaption of this method making it amenable for high-throughput screen-ing. Important modifications to the protocol were the use of a mutant Gawith rapid GDP release in combination with a high-affinity GDP antibodyand a fluorescence polarization method to sensitively detect the releasedGDP. This improved protocol enables biochemical functional screening andis a big step forward in the development of RGS protein modulators.
As discussed above, one common issue in developing modulators of RGSproteins function is the transition from biochemical to cellular activity. Inmany cases, compounds loose potency in this transition due to availabilityissues (e.g., cell permeability), toxicity, and chemical modifications as a resultof the cellular environment. A way around this problem would be to directlyscreen for small molecule RGS protein modulators in a cell-based system.
Direct RGS–Ga interaction modulators could be identified in cells usingFRET or bioluminescence resonance energy transfer (BRET)-based methods(reviewed in, e.g., Bacart et al., 2008; Gales et al., 2005; Lohse et al., 2008;Pfleger & Eidne, 2006). These procedures utilize a fluorescent or biolumines-cent donor (e.g., CFP for FRET or Rluc for BRET) and acceptor (e.g., YFP,citrine or venus) proteins coupled to the two proteins involved in the studiedinteraction. This approach has already been used to study RGS–Gb5 inter-actions (Yost et al., 2007) and could easily be adapted to a more high-throughput format to study (and find modulators of) the interaction betweenRGS proteins and Ga.
Cellular assays are already in use to investigate the effects of RGSproteins on specific signaling pathways (e.g., effects on cAMP or Ca2þ levelsin response to receptor activation), and there are many commercially avail-able methods for this approach that are amenable for high throughput. Thisapproach would circumvent the transition from biochemical to biologicalfunction, although the mechanism of action for any identified compoundswould also have to be confirmed in a biochemical assay.
The abovementioned approaches, including published RGS inhibitors,all focus on the canonical action of RGS proteins. However, as has becomeevident in the previous sections, there are a number of additional functionsand regulatory mechanisms that could be targeted in RGS protein drugdiscovery. These could include inhibitors or enhancers for membrane target-ing, protein stability, or phosphorylation event as previously discussed(Section III). Also, specific inhibitors of protein–protein interactions otherthan RGS–Ga could be a feasible approach. For instance, inhibiting an RGSprotein interaction with a receptor could theoretically result in effects onactions through that receptor alone without affecting other functions of the
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RGS protein in question. It should be noted that in this sense, the field of RGSproteins is still in early stages of investigation and targeting a protein–proteininteraction, though a feasible approach, needs to be accompanied by im-mense testing for specificity. Indeed, there are most likely many interactionsyet to be discovered and the data on selectivity is not yet complete. Insummary, there are several avenues that could be taken in RGS proteindrug discovery, many of which have yet to be explored.
VIII. Conclusion ______________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________RGS proteins are novel drug targets in many areas of research. With
increasing knowledge of the canonical GAP function as well as noncanonicalfunctions and regulatory mechanisms, the understanding of these proteinsand their clinical importance has become apparent. Although the main focusin drug discovery has thus far been on developing modulators of the interac-tion with Ga proteins and GAP function of RGS proteins, it is expected thatmore effort will be put forth into identifying compounds that modulate otheraspects of RGS protein function. These could include modulators of proteinexpression or inhibitors of alternative protein–protein interactions. Moretools to study these mechanisms are rapidly becoming available and there isno doubt that the understanding of noncanonical functions and increasedknowledge of mechanisms controlling expression and posttranslational mod-ifications of RGS proteins will lead to exciting new discoveries in the nearfuture.
Conflict of Interest: The author has no conflict of interest to declare.
Abbreviations
BRET
bioluminescence resonance energy transfer DEP disheveled-EGL10-Pleckstrin FCPIA flow cytometry protein interaction assay FRET fluorescence resonance energy transfer GAP GTPase activating protein GDI guanine nucleotide dissociation inhibitor GDP guanine diphosphate GGL Gg-like GoLoco Gai/o-Loco GPCR G protein-coupled receptor GTP guanine triphosphate
RGS Proteins as Drug Targets: Current and Future 339
R7BP
R7 binding protein R9AP RGS9 associated protein RGS regulator of G protein signaling RH RGS homology
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