G proteins in cellular control

Allen M. Spiegel

National Institutes of Health, Bethesda, Maryland, USA

In the past year, cDNA cloning has revealed substantial diversity in G protein a, fi and y subunits. The number of cellular functions recognized to be controlled by G proteins is also increasing. Most G proteins are associated with the cytoplasmic surface of the plasma membrane, and molecular mechanisms for membrane association of certain C protein subunits have been defined recently. Mutations in C protein subunits, both artificially induced and naturally occurring, have provided important

insights into C protein structure and function.

Current Opinion in Cell Biology 1992, 4:203-211

Introduction

Heterotrimeric G proteins were discovered originally as plasma membrane-bound signal transducers that senre to couple receptors such as rhodopsin and the fi-adrenergic receptor to effecters such as cGMP phosphodiesterase and adenylyl cyclase, respectively. It is now clear that G protein a subunits are members of a large GTP-binding protein superfamily that share common structural and functional features [ 11. Alpha subunits bind guanine nu- cleotides, possess intrinsic GTPase activity, are substrates for ADP-ribosylation by bacterial toxins, and are thought to confer specificity in receptor-effecter coupling. Beta and y subunits form a tightly, but non-covaIentIy, bound heterodimer that dissociates reversibly from a subunits when the latter are activated.

As with all members of the superfamily, the state of a subunit activation is controlled by binding of GTP versus GDP (Fig. 1). Ligand-activated receptors act catalytically as guanine nucleotide exchange proteins. GTP-bound, activated a subunits have been shown to modulate di- rectly various effecters. Whether or not direct modula- tion of effector activity by & subunits is physiologically relevant is unclear. In the yeast mating factor pathway, genetic evidence strongly suggests that j3r rather than a subunits modulate effector activity [2]. Inhibition of adenylyl cyclase activity has been attributed to an indi- rect action of py via binding to G,-a. Recent studies with baculovirus-expressed adenylyl cyclase, however, show direct inhibition of the type 1 enzyme by Pr subunits [3**]. Alpha subunit GTPase hydrolyzes bound GTP to GDP, thereby inactivating the a subunit and permitting Pr reassociation. This then allows the high-affinity cou- pling of the a subunit to a receptor.

This review will focus on recent data that shows a sub- stantial diversity in the structure and function of G pro- tein subunits. Given the diversity of receptors, effecters and G proteins within any given cell, elucidation of the

specificity of receptor-effecter coupling by G proteins poses a major challenge. A variety of experimental ap- proaches attempting to address this problem are re- flected in many of the studies discussed here.

Low-molecular-weight GTP-binding proteins may be even more diverse than G protein a subunits. Despite the overall conservation in their GTP-binding domains [ 11, a subunits and low-molecular-weight GTP-binding proteins show some key differences. First, only a subunits are acti- vated by fluoride; this anion, complexed with aluminum, mimics the y phosphate, converting bound GDP into the equivalent of GTP (4-l. Second, whereas some low- molecular-weight GTP-binding proteins such as ras p21 are clearly involved in transmembrane signaIling, there is no evidence that they play a role in the direct coupling of receptors and effecters. A discussion of low-molecular- weight GTP-binding proteins is beyond the scope of this review.

G protein diversity

Recent polymerase chain reaction (PCR)-based cloning studies have brought the total of distinct genes encoding mammalian a subunits to at least 15 (Table 1). Further diversity is achieved through alternative splicing of G,-a and G,-a genes [5,6]. Mammalian a subunits can be grouped into four classes on the basis of amino acid identity and presumed evolutionary distance: G, and Golf, G,lt (3~7 Gil, Gi2, G,, Go and G,; Gq, G11, (314 and G15/16; and Gt2 and G,, [7*,8,9]. G15 is apparently the murine equivalent of human G16. Similarly, bovine liver a subunit cDNAs termed G,, and Grr [ 101 correspond to murine G,j and Gt,, respectively. Progress has been made in defining the distribution of these novel a subunit mRNAs as well as their encoded proteins but, for most, details bf receptor-effecter coupling remain undefined (Table 1).

Abbreviations EGF--epidermal growth factor; C protein--CTP-binding protein; PCR-polymerase chain reaction.

@ Current Biology Ltd ISSN 0955+674 203

204 Cell regulation

(4

(a) Extracellular

(b) %

CTP GDP

Fig. 1. The C protein CTPase cycle.

(a) The a subunits of C proteins in their basal (inactive) state contain tightly

bound GDP and are associated with the pu complex. (b) Interaction with an ac- tivated receptor (R’) catalyzes exchange

of bound GDP for CTP. Binding of CTP leads to dissociation of G protein from

the receptor and of the a subunit from py. (c) GTP-bound a (activated form; a*) interacts with and regulates the effec- tor (E; shown as a transmembrane pro-

tein, but may also be a peripheral mem- brane protein). Whether the by complex may also directly regulate certain effec-

tor activities is not clear. (d) The intrinsic GTPase activity of the a subunit leads

to hydrolysis of bound GTP to GDP; this ‘turns off’ the a subunit. The latter dis-

sociates from the effector and reasso- ciates with By to re-enter the GTPase

cycle. Bacterial toxins transfer ADP-ri- bose from NAD to G-protein a subunits. Pertussis toxin (PT) leads to uncoupling

of its G protein substrates from recep- tors. This blocks signal transduction by

preventing exchange of GDP for GTP. Cholera toxin (CT) acts on G, to reduce

its intrinsic rate of CTPase activity. This

1

causes more long-lived G protein (and thereby, effector) activation. L

Table 1. Receptor-effector coupling by G proteins.

a Subunit Receptor Effector Toxin Expression

G, (four forms) P-Adrenergic Adenylyl cyclase’ C Ubiquitous

Voltage-sensitive Ca channel

G elf Odorant Adenylyl cyclase C Olfactory

G,,, Gt2 (two genes) Opsins cG-PDE P/C Rods/cones

G2 a2-Adrenergic Adenylyl cyclase P Ubiquitous

WW~, Somatostatin Adenylyl cyclase’ P Mainly neuronal

Ion channels K&&J.

Phospholipase C Other (G,,)

GZ ? ? - Neuronal, platelets

$1 %I Thromboxane A, Phospholipase C-p - Ubiquitous

C14 ? Phospholipase C - Liver, lung, kidney

G15/16 ? Phospholipase C - Blood cells

C12, $3 ? ? - Ubiquitous

Selected examples of receptors given, rather than a comprehensive list. C, cholera toxin-sensitive; CC-PDE, cCMP phosphodiesterase;

P, pertussis toxin-sensitive; - , toxin-insensitive. ‘Stimulation by G, and inhibition by pertussis toxin-sensitive G proteins.

At least four distinct p subunit cDNAs and four distinct y subunit cDNAs have also been cloned [ 111. Additional

protein-derived peptide sequences suggest even further y subunit diversity [ 111, The functional significance of

C proteins in cellular control Spiegel 205

Py subunit diversity is as yet unclear. There could be specificity among p and y subunits in forming py het- erodimers, and in py subunit interaction with a sub- units, receptors [12] or even effecters, but further work is needed to define the degree of specificity, if any.

G proteins are highly conserved in evolution. Alpha, p and y subunit homologs function as transducers in the mating factor pathway of budding [2] and fission [ 131 yeast. Multicellular organisms such as Drosophilci and Cuenorhabdifis ekgans [ 141 possess a subunit homo- logs of mammalian G,-a and G,,-a, as well as unique a subunits. Mutations in a novel a subunit expressed in Drosophila embryos lead to defective gastrulation [ 15**]. This protein, termed concertina, shows limited amino acid homology to Glz and G,,, and may be involved in cel1Lcell communication and movement. Sensory trans- duction is mediated by specialized G proteins in mam- mals (Table 1). In Drosophila, an a subunit homolog of G,, is expressed only in photoreceptors (7.1, and a unique p subunit, expressed only in photoreceptors, has also been cloned [ 161.

G protein localization and membrane association

Covalent modifications with lipid are critical for mem- brane association of y and certain a subunits [ 171. Gamma subunits undergo three sequential modifications characteristic of proteins (including ras ~21) possessing a carboxy-terminal CAAX (C. cysteine; A, aliphatic; X, any) motif (Fig. 2 ). Isoprenylation of y subunits (farnesT when X is serine; geranylgeranyl when X is leucine) can be demonstrated in ilz z&-o translation systems [ 1%201, Mutation of the CAAX cysteine blocks isoprenylation and subsequent modifications [ 18,20,21**],

Pertussis toxin-sensitive a subunits end in an appar- ent CAAX motif but do not undergo isoprenylation [l&20,22]. Expression of By heterodimers in cos cells depends on cotransfection of both p and y subunit cDNks [ 21**]; isolated fi and y polypeptides appear to be unstable. Mutation of the CAAX cysteine blocks iso- prenylation and membrane association, but not assem- bly, of the Py heterodimer. Thus, modification of y is critical for membrane localization but not for interac- tion with p [21**], Mutations that block prenylation of the yeast y subunit homolog lead to a loss-of-function phenotype - sterility [2]. Prenylation and proteolysis of AAX residues appear to be irreversible modifications performed in the cytoplasm. In contrast, methylation oc- curs in a membrane-bound compartment and may be reversible as well as regulated 1231.

Mammalian Gi and G,, a subunits and the yeast mating factor pathway a subunit require cotranslational modif- cation at amino-terminal glycine for membrane binding [2,17]. Non-myristoylated Gi and G,, a subunits show reduced affinity for Py [ 17,24**]. G,-a (and probably sev- eral of the newly cloned a subunits such as G,,) are not myristoylated, but are membrane-associated nonetheless.

Amino-terminal-deletion mutants of G,-a retain the ability to bind to the plasma membrane, but are defective in binding to py [ 251. Carboxyl-terminal residues 367-376 have been suggested to be critical for G,-a membrane association, but no covalent modifications of this region have yet been found 1261. Protein kinase C phospho- rylates G,-a in platelets in response to agonist stimula- tion [27], and the yeast sterile-r p subunit homolog is phosphorylated in response to pheromone [28*]. The latter mediates an adaptation response, but the functional significance of G,-a phosphorylation is unknown.

Although solubilized G proteins behave as approx- imately 1OOkD proteins of composition a, plyI on gel filtration and sucrose density gradient centrifuga- tion, G proteins in native membranes may be part of more complex macromolecular assemblies. Studies in squid giant axons suggest that G proteins are assem- bled az part of receptor+Sector complexes, and de- livered to axon and synaptic terminal plasma mem- branes by fast axonal transport [29]. There is in r+tr*o evidence for association of a subunits with cy- toskeletal elements, in particular tubulin [30], and some studies suggest that agonist stimulation leads to dis- aggregation of a subunits from large-molecular-weight membrane-associated complexes [ 311. Alpha subunits may not be permanently membrane-associated. In m;Lst cells, agonist stimulation causes translocation of Giz-a from membrane to cytosol (321, and in T-cells, T-cell receptor activation causes loss of pertussis toxin-sensitive G proteins from the membrane [33].

III polarized epithelia, a subunits have been localized to apical membranes where they may regulate sodium [3-l] and chloride [35] channels. Gi,-a has been iden- tified in association with Golgi membrdnes [36**]. Low- molecular-weight GTP-binding proteins are known to be involved in vesicle transport in the endoplasmic reticu- lum and Golgi, but recent evidence suggests that het- erotrimeric G proteins such as Gi, may also play a role in Golgi t&Sing [36**], perhaps through assembly of P-COP (coat protein) onto the Golgi membrane [37*].

Specificity of receptor-effecter coupling by G proteins

The number of G protein-coupled receptors is in excess of several hundred, and there are probably as many as two dozen different G proteins and effecters. How spe- cifc then is receptor-elfector coupling by G proteins, and what accounts for this specificity? A restricted range for the expression of receptors, G proteins (Table 1) and ef- fectors is one mechanism for achieving specific coupling, but in many cells, multiple receptors, G proteins and ef- fectors are coexpressed. Available evidence suggests that G-protein-mediated signal transduction functions more like a complex network than a simple linear pathway. Multiple receptors may converge on a single G protein, but individual receptors may also ‘talk’ to more than one G protein. Likewise single G proteins may modulate more

206 Cell regulation

(i) Polypeptide synthesis (i) Polypeptide synthesis

~~~n~~~ ~(~~~ Myristoyl- o H (iii) By assembly

CoA Myristate 1

7 /VAp ky 0 a peptide cq

P Y

Oii ys-OH

n f-

+ lsoprenoid

(iii) Membrane association

(iv) Membrane association and

Intracellular

Extracellular

Fig. 2. Biosynthesis and membrane association of C protein subunits. (a) a subunits are presumably synthesized on free ribosomes in the cytoplasm. Alpha, and a,, subunits are known to undergo cotranslational myristoylation as depicted: cleavage of amino-terminal methionine, amidation of the next residue (must be a glycine) by myristate, followed by membrane association. Alpha, and probably other a subunits such as a do not undergo myristoylation, yet are also membrane-associated. (b) Beta and y polypeptides are also presumed to be synthesize a on free ribosomes in the cytoplasm. They y subunit undergoes ‘CAAX box’ processing, which consists of three sequential post-translational modifications: isoprenylation of the cysteine fourth from the carboxyl terminus (with farnesyl or geranylgeranyl moiety depending on residue X of the partrcular y subunit); proteolysis of AAX resrdues; and methylation of the resultant cysteine-free carboxyl group. It is proposed that the last modification probably occurs on the membrane rather than in the cytoplasm on the basis of the subcellular localization of the carboxymethyltransferase (particulate rather than soluble like the prenyltransferase; the protease has not yet been defined). Individual 0 and y polypeptides are unstable in the cell. Here, p and y polypeptides are shown assembling to form heterodimers after y subunit modification, but the order of events is unknown; py heterodimer formation may in fact precede y modification, as modification is critical for membrane association but not for py assembly.

than one effector, and several G proteins may also con- verge on a single effector. To the extent that receptoreffector coupling by G pro teins is specific, it depends on key structural differences within each signal transduction component. Extensive

studies (detailed discussion of which is beyond the scope of this review) indicate that certain portions of the in- tracellular loops (especially the third) of G-protein-cou- pled receptors are critical for detemlining specificity of G protein interaction [38-1. A wasp venom peptide, mastoparan, mimics such a portion of a G-protein-cou-

change on a subunits, Cross-finking studies show that mastoparan binds to the amino terminus of a subunits, indicating that not only the carboxyl but also the amino ternlinus is probably important for receptor coupling [39-l. A comparable peptide corresponding to residues 259-273 of the human P2-adrenergic receptor potently activates G,-a. Interestingly, protein kinase A-catalped phosphotylation of a serine within this peptide decreases its ability to activate G,-a and potentiates its otherwise weak activation of Gt-a [ 40**]. Thus, coupling specificity may not be a static property of receptors, but may rather

pled receptor, and promotes guanine nucleotide ex- be dynamically regulated.

G proteins in cellular control Spiegel 207

Reconstitution of a signal transduction pathway using purified components has been a powerful method for defining specificity. Purified a subunits of the G, family, for example, were shown to activate purified phospho- lipase C-p1 isozyme [41*,42*]. Reconstitutions of recep- tors and G proteins (using assays such as stimulation of GTPase or guanine nucleotide binding to monitor cou- pling) have been performed with purified native proteins 1431, as well as recombinant proteins expressed in Es- cberichiu coli [44,45] and baculovirus [46*]. M2 mus- carinic receptors, for example, coupled efficiently to Gis, G, and, interestingly, G,, whereas the Ml receptor did so poorly [ 46*]. The fi-adrenergic receptor preferentially activated G, [45]. The Al adenosine receptor (purified from bovine brain) was shown to couple preferentially to G, in one study [44] but, in another study, coupling to Gil, Giz and G,, was demonstrated by reconstitution and antibody identification of G proteins copurifying with the receptor [ 431. Similar techniques were used to show that Gil and Gi, couple to somatostatin receptors [47], and that G,-a interacts directly, not only with adenylyl cyclase, but also with skeletal muscle calcium channels [48].

Expression of muscarinic receptor mRNAs in Xenopus oocytes has shown that specific receptor subtypes are coupled to distinct G proteins; in addition, although both G proteins stimulate phosphoinositide breakdown, the subsequent subcellular pattern of calcium mobilization differs markedly [38**]. Two forms of G,, presumably arising via alternative splicing, were found to differ in subtle aspects of guanine nucleotide binding kinetics, but both promoted an increase in chloride current upon in- jection of purified protein into Xenopus oocytes [17]. Coupling of the calcitonin receptor to distinct G pro- teins was shown to be cell-cycle-dependent in a kidney tubule cell line [SO], but the possibility of calcitonin re- ceptor heterogeneity was not definitively excluded. Stud- ies with transfected, defined receptor cDNAs demonstrate clearly, however, that a single receptor can couple to more than one G protein [51,52]. DrosophiluG,-a homo- logs expressed in murine S47 cells were seen to stim- ulate mammalian adenylyl cyclase, but interact poorly with mammalian G,-coupled receptors [53*]. Alignment of Drosophila and mammalian forms of G,-a revealed a highly variable region near the carboxyl terminus that may account for the observed differences in receptor coupling.

Reconstitution and transfection studies help reveal pos- sible receptor-effecter coupling by G proteins, but other techniques permit identification of endogenous G pro- teins coupled to specific receptors and effecters in a given cell. Agonist-promoted GTP photoaffinity analog la- beling has been used to demonstrate coupling of opiate and a2-adrenergic receptors to multiple forms of Gi and G,, in neuroblastoma-glioma hybrid (NG-108) and rat insulinoma cells, respectively 154,551.

Immunoprecipitation of members of the G, family photoaI%nitylabeled in response to vasopressin in liver membranes provides evidence for Vl receptor coupling to the G, family [56*]. Antibodies against the carboxyl terminal decapeptide of a subunits disrupt receptor-(;

protein coupling in native membranes, and have been used extensively to define coupling specificity. For exam- ple, using this approach, the thromboxane A2 receptor in platelets [57*], and bradykinin, angiotensin and his- tamine receptors in various cell types [58*] were shown to couple to the G in insulinoma to 8.

family. Coupling of galanin receptors ,2 was also observed [57]. Injection

of antisense oligonucleotides into mouse pituitary tumor (growth hormone-secreting) cells was used to decrease G protein expression, and demonstrated highly specific coupling between muscarinic and somatostatin receptors to the G,,l and Go2 subtypes, respectively [60**].

G proteins may not couple solely to receptors predicted to have seven membrane-spanning domains. A peptide corresponding to residues 241&2423 of the cytoplasmic portion of the insulin-like growth factor II receptor (sin- gle membrane-spanning) was shown to activate Giz in a manner analogous to mastoparan [61]. It is possible that Gil might mediate certain of the actions of insulin- like growth factor 11. Similarly, the epidermal growth fac- tor (EGF) receptor (tyrosine kinase family) was found to activate phosphoinositide breakdown in hepatocytes, but not in other cell types such as A431, via a pertussis toxin-sensitive G protein [62]. Coprecipitation of a Gi-a subunit with tyrosine-phosphorylated phospholipase C-y was demonstrated in hepatocytes treated with EGF [63**], but the precise role of a Gi-a in EGF stimula- tion of phospholipase C remains to be delined. Recently, the T-cell-antigen receptor has been shown to be as- sociated with a novel 32 kD GTP-binding protein [64]. The relationship of this protein to G protein a subunits, and its role in T-cell-signal transduction require further study. There is also evidence that G proteins may be involved in cell-cell communication via neural cell adhe- sion molecules. Pertussis toxin blocks neurite outgrowth in PC12 cells in response to stimulation by neural cell adhesion molecules [65]. GAP43, an intracellular neu- ronal growth cone-associated protein, has been shown to promote guanine nucleotide exchange on G,,, the ma- jor non-cytoskeletal protein in growth cone membranes [ 661. The mechanism of GAP-43 action, and its possible relation to neural cell adhesion molecule effects, deserve further study.

G protein mutations as probes of structure and function

Site-directed mutagenesis of G protein a subunits has been used to identify residues in the carboxyl-terminal third of the molecule critical for interaction of G,-a with adenylyl cyclase [ 67.1. Mutation of key residues known to be involved in guanine nucleotide binding [ 11 may either reduce intrinsic GTPase activity or prevent activation by GTP [68,67]. Cells expressing GTPase-deficient mutants are constitutively activated for the pathway subserved by the relevant G protein. Such mutations of the yeast mating factor pathway a subunit cause growth and cell morphol- ogy defects [ 701. Similar mutants of Giz-a constitutively inhibit CAMP accumulation [69,71,72], and cause com-

208 Cell regulation

plex changes in phospholipase A2 activity [72]. Trans- fection of fibroblasts with GTPase-deficient Giz-a mutant cDNAs causes increased cell proliferation 169,731, whereas a mutant that blocks activation by GTP inhibits cell proliferation [69]. These data are consistent with the suggestion that GTPase-deficient a subunit mutants ma) function as oncogenes in certain cell types. InterestingI!., however, knockout of Gil-U. expression by lw~~wlogous

recombination in embryonic stem cells cawed no ap-

parent defect in growth or dift‘erentiation [7+], perhaps reflecting redundanq of Gi subtypes.

G protein mutations have also been linked to hum:In

diseases. An inherited form of generalized hornlone re- sistance is associated with a 50% delicienq. of G,-a in all tissues. Mutations in the G,-a gene leading to re- duced mRNA formation an&or stability have been found in affected subjects [ 751. GTPase-deficient mutants (at the arginine modified by chol&a tosin or the glutamine equivalent to position 61 of ras p21 ) of G,-a and GiL-a have been found in certain human tumors [ 11. A mo-

saic distribution of constitutivehr acti\ated G,-a mutant- containing cells has been iden&ed in subjects nith an acquired disease characterized by autonomous h)per- function of multiple endocrine and other tissues [?6*]. The constitutively activated G,-a mutant behaves like ;I

dominant ‘oncogene’, promoting receptor-independent CAMP stimulation and resultant hyperplasia and h!q>er- function. In some cell gpes, such as pituita? lactotrophs ‘and p cells in the pancreas, pertussis toxin-sensiti\re G proteins exert tonic negative control over cellular gronTh and function. An intriguing theoretical possibilin. is that the genes encoding these G proteins could represent ‘tumor suppressor’ genes; loss-of-function mutations in such genes could represent another mechanism for dys- regulated growth and function.

Conclusion

An increasing number of G proteins are now recognized to control an ever more diverse array of cellular func- tions. Distinct lipid modifications halve been identified that are critical for association of certain G protein suh-

units with the inner surface of the plasma membrane, but additional mechanisms responsible for G protein tar- geting to the plasma membrane and perhaps other sub- cellular compartments remain to be elucidated. Kecon- stitution with puritied native and recombinant subunits, as well as transfection strategies using defned cDNAs, antibodies or antisense RNAs, have all been uscvi to de- line specificity of receptor-effecter coupling by G pro teins. In general, such studies have re\fealed specilicir), that is relative rather than absoLte. Mutational analysis has helped define regions critical for membrane asso- ciation and important protein-protein interactions of G protein subunits, as well as residues critical for a subunit activation by GTP and for GTPase activity. Both gain-

of-function (constitutive activation caused by reduced GTPase) and loss-of-function (null) mutants, primaril) of G,-a, have been linked with certain human diseases;

further efforts to identib mutations of other a subunits in various disorders are clearly warranted.

References and recommended reading

Papers of l’anicular interest. published nithin the annu:~l peri(~I of rc- \ie\v. h:tve been highlighted as: . of special intercht . . of outstanding interest

I I~OI’KSI; I1R. S~NINW DA. ~~CC~KMI:K F: The GTPase Super- family Consewed Structure and Molecular Mechanism. ,Vlc /n)‘e 1991. 349:l l--12’.

1 I\~.t’hw~ yI. TIIOKSI:R I, Receptor-G Protein Signaling in Yeast. :I?I,,// KU /‘+Yo/ 1991, 5337-57.

3 TASG W:l. KHI’IWXI j. GII.\L.IS AG: Expression and Character- . . izltion of Calmodulin-activated (Type I) Adenylylcyclase. .I

/M ~hwt I99 I, 266:tW-H603.

4. I IIG~WII~I~I:\ T. GR~WIANO MP. SIG.! II, ~G\INOSIK~ hl. GII.\I:\u . . AG: 19F and 31P NIMR Spectroscopy of G Protein Alpha

Subunits. .I IGo! Char 199 I, 266:3j9G3+0 I. NhlH slwctroscopj~ n’:t.s usecl to show that an :tluminum tlu~~riclc~ mngn~sium c~~mplcs binds to and acti\atrs GDP-b~~md G protcm ‘1 subunits.

5. 13wrk.w) I’. S~NIOUJ J. KI~~M~I~II I’. COI~~A J. I~IKNI~AI~~:K I.. At Least Three Alternatively Spliced mRh%s Encoding Two ‘1 Subunits of the Go GTP-binding Protein can be Expressed in a Single Tissue. ./ Rio/ c/w~~ 1990. 265.1X5% IX%+).

6. T~~KA.w~~ T. To~..L\L~ R. Iwt I I I. K;ozw T. 1l~vrw )K.\ Il. K.WHO \I’. Structure of the Human Gene and Two Rat cDNAs Encoding the ‘x Chain of GTP-binding Regulator) Protein G,,: Two Different mRNAs are Generated by Altcr- native Splicing. /%K- ,Vtr// &trc/ Scr 1 ‘S ,.I 1991, WL\)‘+LU’X

S’rw’rII.w9S XI. SI.W>N Ml G Protein Diversiw a Distinct . Class of z Subunits is Present in Vertebrates and Invertc-

hntes. /‘UK ,Vc:;rll Aitrtl Sci I’ .S A 1990. 8731 13-91 1’. PCH bxs uwcl to ;tmpli~ novel cDNA5 encocling G protein 5 whunits of tk G,, fkiiily. (‘sing a similar approach. this group ha.. grc:ttly cslxinckd the list of lincw,~~ G protein genes (SLY also II-W.1 1 ] 1.

x.

9.

IO.

I I.

IL.

13.

Ii.

S.rltti-IIwss Ml’. S~stoh’ hll: Gal2 and Gal 3 Subunits Define a Fourth Class of G Protein a Subunits. /Jr~x’ ,‘Gt/ .&trt/ Set 1’ .S :I I99i. t38:i5K-ii~6.

AWTHI IM 1-r III. Sn:iiui DA. Si&xw \%. S~xiox XII, GzlO. a G Protein a Subunit SpeciIically Expressed in Hematopoictic Cells. /kc ,\‘tr// Aitrtl Sir 1’ 5 .-I 1‘991, FHiiX--ii9l,

N.4li.wtw I:. Oc..crr\ K. SHI~~~I K. ~~.UI~~Y~LUA li. 0‘1 IAK.\ ti, II:\<;;\ 1‘. NI’K.u>.A T: Identification of Two Novel GTP-hind- ing Protein Y-Subunits that Lack Apparent ADP-rihosylation Sites for Pertussis Toxin. ./ Hiol ~~KWI 1991, 266: I .!6’h I.ZU-4 I

SIVOS All. SI~K,U~IAL%U Ml’. GAI ‘I.:LV N, Diversity of G Pro- teins in Signal Tranduction. .S~~WKL~ 1991, 252:XOL-X0X.

I:hW%I b’& F:\Y DS. blI’KI’tIY 1%. ~fhhllH ll. l<KIX)\ JI, bk)K~IIll’l’ jK: Rhodopsin and the Retinal <;-protein Distinguish Among G-protein Beta Gamma Subunit Forms. ./ Hid Chat 1991, 266: I2 19+ 12200.

OI~AKA T. NAMH xl Al, \‘?\?&\.wl’O M. KUIKO Y: Isolation and Characterization of a Gene Encoding a G-protein Alpha Suhunit from Schizosaccharomyces pomhe: Involvement in Mating and Sporulation Pathways. /-‘)7x ,Vcct/ &rtd .Sci I .S .-I 1991, 88:=JX”7-5XH1.

lIX:IlKII: MA. hlt?w~ii. Jl:. S’rI:KSIII;Kc; IW’. Siw)r All: Homolo- gous and Unique G Protein Alpha Subunits in the Nema- tode Caenorhahditis elegrtns. Cdl Kept/ 1991, 2: 135-I i-1.

C proteins in cellular control Spiegel 209

15. PARKS S, WIESCHN~ E: The Drosophila Castrolation Gene . . concerfinu Encodes a Ga-like Protein. Cell 1991, 64+14745X. A mulam Drmop/h Rene causing defective gzzstrulation w&s ckmed and shown to encode a G protein a subunit homolog (tith a large amino-terminal extension). A possible role for G proteins in cell<ell communication was su~ggeslrd.

16.

17.

1X

19.

20.

21. . .

Ywrlz S, NIUII GA. MCCONNIXL Jl., Frrct~ Cl., HIIKII\’ JI% A G- beta Protein in rhe Drosophila Compound Eye is Different Tom that in the Brain. Netrro?t 1991. 7:-129-138.

SI~IKXI. AM. Ba~Kl.l’NI) PSJR. BlVO’NXI JE, JONES TIZ. Sl~tONl)5 WF: The G Protein Connection: Molecular Basis of Mem- brane Association. ‘li-e~rds Biocknr Sci 1991, 16:33%-j+ 1.

b~AI.TlX WA, R0I~Istl,\\~ JB: Isoprenylation of C-terminal Cys- teine in a G-protein Gamma Subunit. .I Rid Chwr 1990. 265:18071-18074.

SCH,MIIX CJ. NEER EJ: In Vifro Synthesis of G Protein oy Dimers. ,/ Hid C!wr~ 1991, 266:-153X--f%+.

Sllhl~otw J. COIIINA J. BIRNt3,v:hlliR 11 y-subunits of G Pro- teins. but Not their a- or B-Subunits, are Polyisoprenylated. Studies on Post-translational Modifications Using In Vim Translation with Rabbit Reticulocyte Lysates. .I Hid Char 199 1, 266:9570-9579.

SIXIONIX WF. Bl“IH,‘N5KI JE. G~I”l~h~l N. I’USON CG, SI’IE(;lil. AM: G.protein Beta/Gamma Dimers: Membrane Targeting Requires Subunit Coexpression and Intact Gamma CAAX Domain. .I Hid C~~I 1’991, 266:5363%5.%X1

G protein py subunits were expressed successft~lly in cos cells onl! afrrr corransfection of both subunit cDNAs. Mutation of the carho\? rrrminal c)sreinc blocked prenylation and memhranr associarion. hut permitted assembly of the hetcrtdimer,

22 JON13 Tl%. SPIKXI. AM: Isoprenylation of an Inhibitory G Prorein a Subunit Occurs Only Llpon Mutagenesis of the Carboxyl Terminus. .I Rio1 Chw 1990. 265:1938%19397

23. ~%RI:%-SAIA D. Tk\: EW. Ch~Ai)r\ I-1. lL\six~ RR Methylation and Demcthylation Reactions of Guanine Nucleotide-bind- ing Proteins of Retinal Rod Outer Segments. /‘).oL‘ N&l .4io(l .Sci 1’ S d 1991, B&3043-30-16.

14. IISIKH Ml?. i’hN<; 1 11, DI’ROSIO RJ. GOlUX,S Ji. STliK~V’lil5 PC. . . GII.U~LU AG: Lipid Modifications of G Protein Subunits. Myris-

toylation of G,,a Increases its Atfinity for py. .I Niol C/xwr 1991, 266:-l6it-1659.

G pro’cin a subunits expressed in f:‘ coli xc n8)t mynstoylattul :mcl 4lolv lower :tliIni~ for py subunits rhan the crmesponcling native ‘1 subunits. C~~~prc4on of the myrisrovl rransfcrzse v.ith the ? suhunitb in fz’ c.o/iIed to cspression of mynsrc,yl&d r huhunits nith higher atfn ity for py suhunirs.

25.

26.

27.

2x. .

3x. Lt%:tIiJirrl;H J, GI~QHI~ S. Cll\rati;l\! 1). Ptm~.‘r~ E: Subcellular . . Patterns of Calcium Release Determined by G Protein-

specific Residues of Muscarinic Receptors. ,V&rre 1991, 350:505-50x.

Jwtwo’r l., P:w-r,uoxl C, Btx.tbui~~ J. A’i)iwx \I’: Deletion Within the Amino-terminal Region of G,‘1 Impairs its Ahility to Intcrdct with py Subunits and to Activate Adenylate Cyclase. ./ Rid Chwl 199 1. 266:900+90 Ii.

JoitHNo’r I, l~,~Nrhi.osi C, 1’01’1. hl A. MUXXUI. H, Boc~tw J. AI’I)I~;II:R \I’: Amino Acids 367-376 of the Gs-alpha Sub- unit Induce Membrdne Association when Fused to Soluble Amino-terminal Deleted Gi 1 -alpha Subunit. /‘IX ,Vtrll ,-lc‘rrrl Sci 1 ’ .Y A 199 1, 88: 1005-1~ 1005X.

ML and Xl3 muscannic. recelX”r mRNA.. v.‘cre injected into Sr?rop!rs c)c,c~~cs. and thr etf>cts of agonist stimulation on intracellular calcium mc,bili;l;nion \vere monitored with confocal micrc)scopy. Nor only are rhe hvc> rtrcp’or suhtvpr5 c~~ul~led to distinct G proreins. hut the pat- arms of calcium mc,h&ticm resulting from their respective acthation ditler holh spatialh and tcmpomlly.

39 I il(;~wl.wi -r. Kc)\\ EM: Mapping of the Mastoparan- . binding Site on G Proteins. Cross-linking of [lr51-

TyrA.Cysll]Mastoparan to G,,. ./ Niol Cl~cwr 1991, 266:17655- 12661.

~~I~N~I3I‘RY KM. C.kw~ PJ. BRh\\ 1.F. b1;\NSINC; DR. Phos- phorylation of Gz in Human Platelets. .I Rid Char 1991.

Careful protein chemisl? srudy sho\vs that a mastoparan-like peptide

266:2205 l-22056. hinds to Cys3 of G,, 2. The implicarions for sires of G protein-recepror imeraction are discussed.

Cou: GM. RIXI) Sl: Pheromone-induced Phosphorylation of a G Protein p Subunit in S. cercvisiae is Associated with an Adaptive Response to Mating Pheromone. Cell 1991. 64:703--16.

-10. OK,\.\i(xro .r. ,\IUL~~,\.u\ I.. tL\YrX\lll Y. lN,\(ihlil M, 0CAT.S . . I:. Ki\iii>io’ro 1. identification of a Gs Activator Region

of the Beta2-adrencrgic Receptor that is Autoregulated Via Protein Kinase A-dependent Phosphorylation. Cell 1991, 67:~23m'30.

The aurhors clemons~ratc phosphor?l:lric,n of the yeasr mating factor path\Xly p suhunir in a region 1101 li)uncl in mammalian homologs.

29. V~UX 55. CIIIS GJ. Scu’,wr/. 11 1. Klili’rli ‘1‘5: Pertussis Toxin- sensitive G Proteins are Trdnsported Toward Synaptic Ter- minals by Fast Axonal Transport. f’rw ~Vtrrl ~lsctd Sci I’ 5 .-I 1991. 88:1=5-l’??.

30.

31.

32.

33.

3-I.

35.

36. . .

WANG N. RAS~NICK IMM: Tubulin-G Protein Interactions In- volve Microtubule Polymerization Domains. Eiochemstry 1991, 30:10957-10965.

NNG\hIlIIu S, Ror)atx. M: Glucagon Induces Disaggregation of Polymer-like Structures of the a Subunit of the Stimulatory G Protein in Liver Membranes. Proc N4 As& Sci I/ S A 1991. 88:715&715-i.

T,\tbut,wll 5. NEC;IS~II M. IctitLw~ A: Cytosol Promotes the Guanine Nucleotide-induced Release of the a Subunit of Gi, from the Membrane of Mouse Mastocytoma P-815 Cells. J Hid Cl~ewr 1991( 266:5367-5370.

GI:RARIX.SCI wt IN R. MrrrRt:ctw I IW, Sctii UZE U. F~IIKHEK 1% Selective Loss of Pertussis Toxin-sensitive G-proteins from the Plasma IMembrane after Antibody-induced Inter- nalization of T-cell Surface Molecules. J Rio/ Chew 1991. 266:69-1249+7.

C,\SlXll.O 1 IF. P;VI-I:N,\I’lX CR, COIXNA J. BIRNHAIlXlEH L, Al’wxo DAI G Alpha i-3 Regulates Epithelial Na+ Chan- nels by Activation of Phospholipase A2 and Lipoxygenase Pathways. J Hid Chetu 1990. 265:2 162+21628.

TII.I.S BC. KAC\N\I!S hl. V:!s G,%tiiIn,Nlc PG. VA.V DEN BER(;HI: S. GAi,IMt<i) II. lSi]him J. IX Jozlc;t: IlR: G-proteins Mediate Intestinal Chloride Channel Acth’ation. J Hio/ U~tv?r 1991, 266:203&70-10.

S-K\\ JI, Dii AI.~;II~A JB. NARI’IA N. t lol:rxwN EJ, ERCOIUI 1.. AI’SIIUO DA: A Heterotrimeric G Protein, Gai.+ on Golgi Membranes Regulates the Secretion of a Heparan Sulfate Proteoglycan in LLC-PK, Epithelial Cells. ./ Cc4 Hio/ 1991, 1141113~llL.i.

lmmunolocalization by light and electron microscopy identilitul G,,-z in rhe Golgi of cc’IIs rransfecttul wiirh rhc corresponding cDNA Func- tional assays of Golgi rratfcking suggested a role for this protein in regulation of rhis process.

37 DONALIWN JG. KNIN KA. L~t’I~i~co’rr Sct1\Y’AH’r/. J. KL~~‘~NEH . RI): Binding of ARF and Beta-COP to Golgi Membranes:

Possible Regulation by a Trimeric G Protein. Scietfce 1991, 254:l I’)‘-1 199.

In an i,r /‘//ro assa!’ of coat protein binding to Golgi mrmhranes, hc)lh non hydroI!zthle GTP :m:tl~,g and fluoride tvt’rc stimulate?. and excjge nous By suhunit.s n’cre inhihiroF. The d:ua suggest that hrterotrimrric G prorcins may he m\~olvcul in regulating protein traficking.

mc authors tested rhe ahiliv of several peptides corresponding to in. tracellular k~)ps of the F2 adrcnergi; rtyeptor to aclivdte G,-a. Nor only did rhey tind one that acri\;ued the purititul protein potently. but thr) were also able IO show acti\;ltion in wildnpr hut not G,-a mutant-con- taining S-49 ccl1 memhmnes.

210 Cell regulation

41. SMRCKA AV. HEPIIR IR. BROWN KO. STERNU’I:IS PC: Regulation . of PoIyphosphoino%ide-specitic Phospholipase C Activity

by Purified Gq. Sciencc~ 1991, 251:8Ot807. First demonstration that a member of the novel G protein family. Gq, stimulates phospholipase C.

42. TAYLOR SJ, CHAE HZ, RIIK~: SG, EXTON JH: Activation of the . pl Isozyme of Phospholipase C by a Subunits of the Gq

Class of G Proteins. iVu0trr 1991, 350:51&51X. A member of the novel G protein class, G,. is shonn to activate a spe- cific phospholipase C suhc)pe.

43. MIINSHI R. PANG I H, STERNWEIS PC, UNDI:N J: Al Adcnosine Receptors of Bovine Brain Couple to Guanine Nucleotide- binding Proteins Gil, Gi2, and Go. 1 Hiol W~et?r 1991. 266;22285-22289,

44. FRErnhlrrni M. Sctirw” W. LIxrx~ ME: Interactions of the Bovine Brain A,-adenosine Receptor with Recombinant G Protein a-Subunits. Selectivity for rGia-3. J f%ol Uwnr 1991. 266: 1777% I 7783.

45. RLIBENSTEIN RC, LINDER ME, Ross EM: Selectivity of the Beta. adrenergic Receptor Among Gs, Gi’s. and Go: Assay Using Recombinant Alpha Subunits in Reconstituted Phospholipid Vesicles. Ric~J~erni.rlq~ 1991, 30:10769-l 0 7’7.

r6. PARKER EM, KA&~EYAA~~ K. HIG~~HIJI~W T. Ross EM: Recon- . stitutively Active G Protein-coupled Receptors Purified

from Baculovirus-infected Insect Cells. J Rio/ &,!?r 1991. 2665 13-527.

Several G.proteincoupled receptors are expressed successfully using haculo\irus. These are then used in receptor-G protein reconstitution experiments to define G protein coupling specificin.

47.

-ia.

49.

50.

51.

52.

53 .

IAU~ SF, MANNING D, VISING T: Identification of the Subunits of GTP-binding Proteins Coupled to Somatostatin Recep- tOTS. .I Bid dJCt?l lw)1, 26(,:1x-?%-17897.

MILTON SL CODINA J, 11~~~s IMJ. YAT~I A SA~,.U,A T. STIUCKIAND FM, FROEI~NEK SC, SI’IEGEI. AM, TOKO L, STEF,!NI E. BIR~AIIMER L. BRO%N AM: Evidence for Direct Interaction of &-alpha with the Calcium Channel of Skeletal Muscle. J Biol Uxm 1991, 266:19528-19535.

PAI)wLI. E. CARED’ DJ. MORLAR~ TM, I~IL~XBIUNIIT JD. k4I)AI EM, hFNG.4R R: Two Forms of the Bovine Brain G,, that Stimulate the Inositol Trisphosphate-mediated Cl- Currents in Xenoprrs Oocytes. Distinct Guanine Nucleotide Binding Properties. J Rio! UJ~I 1991. 266:9771-9777.

CHAMAHORIY M. CHAII%RJEE D. KULOK~~~!PI. S. R~%hll’wi~ H. BARON R: Cell Cycle-dependent Coupling of the Cal- citonin Receptor to Different G Proteins. Scic~ncc~ 1991. 251:107%1082.

RAYMOND JR, AI&& FJ. MIDD~TON JP. LE~~(owrrz RJ. C,ARO?; MG, OREID LM, DENNIS VW: 5-HTlA and Histamine HI Re- ceptors in HeLa Cells Stimulate Phosphoinositide Hydroly- sis and Phosphate Uptake Via Distinct G Protein Pools. ./ Biol Cbem 1991, 266372-379.

MUCAN G, CARK C, G~~II.II GW, Muuxwy 1. ,AvAS BE: Agonist-dependent, Cholera Toxin-catalyzed ADP-rlbosy- lation of Pertussis Toxin-sensitive G-proteins Following Transfection of the Human a,-Cl0 Adrenergic Receptor into Rat 1 Fibroblasts. Evidence for the Direct Interaction of a Single Receptor with Two Pertussis Toxin-sensitive G-proteins, Gi2 and Gi3. J Rio1 Uxm 1991. 266:6+-17&55.

QUAN F. THOhtAs L, FORTE M: Drosophila Stimulatory G Protein a Subunit Activates Mammalian Adenylyl Cyclase but Interacts Poorly with Mammalian Receptors: Implica- tions for Receptor-G Protein Interaction. Ptw Nd Acad Sci u s A 1991, 88:189~1902.

Vaccinia virus infection wz used IO express insect and mammalian a subunits in cyc- S49 mutine cells lacking G,-a, hut containing recep- tors and adenylyl cyclase.

54. OFFER~~ANS S. SCHLIL’II G. ROSE- W: Evidence for Opioid Receptor-mediated Activation of the G-proteins, Go and

Gi2, in Membranes of Neuroblastoma x Glioma (NC-108) Hybrid Cells. .I Rio1 UJCW 1991, 2(4:336%3368.

55. SCH~!IDT A. HESCHEUR J, OI;FER~~ANNS S, SIJICHER K. HINSCH K-D, KUNZ FJ. CODINA J. BiIuwALlhw 1 GAL~SE~OHL Ii. FKGVK R. SCHI’LTZ G. R~SEKIY+L W: Involvement of Pertus- sis Toxin-sensitive G-proteins in the Hormonal Inhibition of Dihydropyridine-sensitive CaZ+ Currents in an Insulin- secreting Cell Line (RINmSF). ./ Rio/ Chew I(99l. 26618025-18033.

56. WANLX RI.. ShlRcM AV. S?www~s PC, EXTON JH: Phototinit) . Labeling of Two Rat Liver Plasma Membrane Proteins with

[jzP]gamma-azidoanilido GTP in Response to Vasopressin. Immunologic Identification as a Subunits of the Gq Class of G Proteins. ./ Hiol UJWI 1991, 2661140~11412.

A comhinarion of agonist-stimulated photoaffinity labeling ‘and im- munoprrtcipitation R-.,s used to detine receptor4 protein coupling speciliciv.

5’. SHI:XUR A. Gou%hlinl P, LINSON CC, SI~I~XXL AM: The G Pro- . tein Coupled to the Thromboxane A2 Receptor in Human

Platelets is a Member of,the Novel G, Family. J Rio/ Uwnr 1991, 266:930$&9313.

Anntihcxlies to the carhox$terminal decapeptides of several G protein a subunits found in platelets were tested for ability to inhibit stimulalion of GTPase by various agonists.

58. Gtz~oas~ S, Shmcta A, Nowti L, Wu D, SIMON M. S’nxw~is . PC: Antibodies to the aq Subfamily of Guanine Nucleotide-

binding Regulatory Protein a Subunits Attenuate Activation of Phosphatidylinositol 4.5.bisphosphate Hydrolysis by Hor- mones. .I Hid Chm 1991, 266:2051?2052+.

Antibodies similar to those described in 157.1 were able (o inhibit ag(onist-stimulatevl phosphoinositide breakdown in membranes from \ari011s cell npes.

i9. CORx~lOi\T M, U MAKCHAN~~-BRLls7FL Y, V/Iv Okil3l3GHl<N E. SPIEGEL AM, SHARP GWG: Identification of G Protein a-Sub- units in RINm5F Cells and their Selective Interaction with Galanin Receptor. Dinhetes 1991, 40:1170-1176.

60. KI.I<~:% C. H~sctltilli~ J. E\Y’EL C. ROSENTHAL W. Sc~l~l.li! G. . . WITTIC; B: Assignment of G-protein Subtypes to Specific

Receptors Inducing Inhibition of Calcium Currents. Nrrflrr~ 1991) 353:-13-+x.

Antisense oligonucleotides selective for various a subunits were in- jected into the nuclei of GH3 cells. Those against the two subtypes of G,, nere shonn 10 reduce protein expression (monitored hy immuno- c)~ochcmist~). and were able to block selectively the calcium channel inhibition in response to either muscarinic or somatostatin agonists.

61. NISHlhlOTO 1. OGAI’A E. OKA&!OTO T: Guanine Nucleotide- binding Protein Interacting but Unstimulating Sequence Lo- cated in Insulin-like Growth Factor 11 Receptor. Its Autoin- hibitory Characteristics and Structural Determinants. J Rio/ Uwtn 1991, 26612747-12751.

62. IJAW M. G,wu>o~ JC: The Epidermal Growth Factor Re- ceptor is Coupled to a Pertussis Toxin-sensitive Guanine Nucleotide Regulatory Protein in Rat Hepatocytes. ./ Rio/ Uwn 1991, 266: 13342-13349.

63. YAW I.. BAI;I~ G, RHEE SG. MANNING D, tH,wsEN CA, . . WII.ILL\I~~S JR: Pertussis Toxin-sensitive Gi Protein Involve-

ment in Epidermal Growth Factor-induced Activation of Phospholipase C-gamma in Rat Hepatocytes .I Rio/ U~etn 1991. 266:224ilb7~45H. --

EGF trealment of hrpartx>qes was shown 10 lead not only to tyrosine phosphonlation of phospholipase C-y, hul also to coprecipitation of the latter with a G,-? subunit. In cell types in which pertussis toxin &es not inhibit EGF action. no coprecipitation was derected.

6i. TIUXR JC. Rlwl) CE: A 32.kD GTP-binding Protein Associ- ated with the CDI-p5@ and CD%p5&k T CeU Receptor Complexes. Scietze 1991, 254:439+4 1.

65. Dotiwm P. AsilTON SV. IMOORF: SE, WAISH FS: Morphoregula- tory Activities of NCAM and N-cadherin can be Accounted for by G Protein-dependent Activation of L- and N-type Neuronal Calcium Channels. Cell 1991. 672-33.

C proteins in cellular control Spiegel 211

66. STHlTt’MA7TER SM. VAU;.N~~IIU D, sun0 Y. LENDER ME. FlStthL4N 73. PACE AM, WONG YH, BOURNE HR: A Mutant a Subunit of MC: An Intracellular Guanine Nucleotide Release Protein GQ Induces Neoplastic Transformation of Rat-1 Cells. Proc for Go. .I Hid UJCW 1991, 266:22i165-22471. Nad Acad Sci U S A 1991, 88:7031-7035.

67. I’t’oti H, GIIWAN AG: Expression and Analysis of Gs Alpha . Mutants with Decreased Ability to Activate Adenylylcyclase.

J Hid GT3L'??I 1991, 2661622616231. Three residues critical for interaction with adenylyl ~ycl;lse were iden- tified by site-directcxl mutagenesis of G,-a. These appear to he lo- cated in a putatively surface-cxposcxl loop that may he involved in protein-protein contxw

74. MORTKNSEN RM, ZtIHIAtIa M, NEER EJ. SEIDMAN JC: Embryonic Stem Cells Lacking a Functional Inhibitory G-protein Sub- unit (Alpha i2) Produced by Gene Targeting of Both Alleles. Pmx N&l Acad Sci 11 S A 1991, 88:70367040.

75, WEINSTEIN IS. GEJ~~AN PV. FRIED~&N E. KAoow~~l T, COU~NS KM, GE~HON ES, SPIKXL AM: Mutations of the G,-a-subunit Gene in Albright Hereditary Osteodystrophy Detected by Denaturing Gradient Gel Electrophoresis. Proc Nafl Acud Sci U 5 A 1990. 87:8287-8290.

6x.

69.

70.

71.

72.

OSAWA S, JOHNSON GL: A Dominant Negative G-alpha-s Mu- tant is Rescued by Secondary Mutation of the Alpha Chain Amino Terminus. J Hiol Chat! 1991, 266-1673-4676.

I lt~two~~r S. MIXENI)INO JJ JR. G~:TKINI) JS, S~wctx AM: Acti- 76. W~INST;~~IY LS. S~I~NKKR A. GEJhIAN PV, MERINO MJ. FRIEDMAN

vating and inactivating Mutations of the Alpha Subunit of . E. SPII~GEI. AM: Activating Mutations of Stimulatory G Pro- Gi2 Protein have Opposite Effects on Proliferation of NIH tein in the McCune-Albright Syndrome. N Engl J Med 1991, 3T3 Cells. /‘IX IV& /lcctr/ Sci I! S A 1991, 88:10455-10-159. 325:16+X?-1695.

KIIRJAS J, IIIHSCII Jl’. DII:-TLEI. C: Mutations in the Guanine Nucleotide-binding Domains of a Yeast Ga Protein Con- fer a Constitutive or Uninducible State to the Pheromone Response Pathway. &w~.x /Irrl 1991. 5:-17%1X3.

WON<; 111. Flitxww A, l’hcli AM, LACI+\HY 1. E\‘,\N\ T. I’O~YW~GI~H J. f~ot~vti IIK: Mutant Alpha Subunits of Gi2 Inhibit Cyclic AMP Accumulation. N~I/II)P 1991, 351:63X15.

Mutations at the cholera toxin-sensitive arginine of G,.a leading to con- stitutive actkation were found in hyperplastic tissues from subjects with a discqsc characterized hy autonomous endocrine hyperfunction. These findings, and prexious studies showing similar mutations in sporadic en- docrine tumors. sugest that G protein mutations can cause receptor. independent stimulation of signal transduction pathways.

Imv~tm JM. G~WTA SK. OSAWA 5. Jott~‘\o~ GL: GTPase- deficient GQ Oncogene ,@2 Inhibits Adenylylcyclase and Attenuates Receptor-stimulated Phospholipase AZ Activity. J Rid OJlWl 199 1, 266: 1 -t 193-1-f 197.

A&l Spiegel. Molecular Pathophysiology I~ranch, National lnstitute of Diabetes and Digestive and Kidney Diseases, National Institutes of IHealth, Bethesda. Maryland 20892. LISA.