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• W1987606367 abstract "RGS (regulator of Gprotein signaling) proteins are GTPase-activating proteins that attenuate signaling by heterotrimeric G proteins. Whether the biological functions of RGS proteins are governed by quantitative differences in GTPase-activating protein activity toward various classes of Gα subunits and how G protein selectivity is achieved by differences in RGS protein structure are largely unknown. Here we provide evidence indicating that the function of RGS2 is determined in part by differences in potency toward Gq versus Gi family members. RGS2 was 5-fold more potent than RGS4 as an inhibitor of Gq-stimulated phosphoinositide hydrolysis in vivo. In contrast, RGS4 was 8-fold more potent than RGS2 as an inhibitor of Gi-mediated signaling. RGS2 mutants were identified that display increased potency toward Gi family members without affecting potency toward Gq. These mutations and the structure of RGS4-Giα1 complexes suggest that RGS2-Giα interaction is unfavorable in part because of the geometry of the switch I binding pocket of RGS2 and a potential interaction between the α8-α9 loop of RGS2 and αA of Gi class α subunits. The results suggest that the function of RGS2 relative to other RGS family members is governed in part by quantitative differences in activity toward different classes of Gα subunits. RGS (regulator of Gprotein signaling) proteins are GTPase-activating proteins that attenuate signaling by heterotrimeric G proteins. Whether the biological functions of RGS proteins are governed by quantitative differences in GTPase-activating protein activity toward various classes of Gα subunits and how G protein selectivity is achieved by differences in RGS protein structure are largely unknown. Here we provide evidence indicating that the function of RGS2 is determined in part by differences in potency toward Gq versus Gi family members. RGS2 was 5-fold more potent than RGS4 as an inhibitor of Gq-stimulated phosphoinositide hydrolysis in vivo. In contrast, RGS4 was 8-fold more potent than RGS2 as an inhibitor of Gi-mediated signaling. RGS2 mutants were identified that display increased potency toward Gi family members without affecting potency toward Gq. These mutations and the structure of RGS4-Giα1 complexes suggest that RGS2-Giα interaction is unfavorable in part because of the geometry of the switch I binding pocket of RGS2 and a potential interaction between the α8-α9 loop of RGS2 and αA of Gi class α subunits. The results suggest that the function of RGS2 relative to other RGS family members is governed in part by quantitative differences in activity toward different classes of Gα subunits. guanine nucleotide-binding regulatory protein GTPase-activating protein green fluorescent protein inositol 1,4,5-trisphosphate phospholipase C guanosine 5′-3-O-(thio)triphosphate Many hormones, neurotransmitters, and sensory stimuli exert their effect on target tissues by activating receptors that are coupled to heterotrimeric G proteins1(1Bourne H.R. Sanders D.A. McCormick F. Nature. 1990; 348: 125-132Crossref PubMed Scopus (1824) Google Scholar, 2Hepler J.R. G i lman A.G. Trends Biochem. Sci. 1992; 17: 383-387Abstract Full Text PDF PubMed Scopus (919) Google Scholar). Receptor activation results in exchange of GTP for GDP on Gα subunits, dissociation of GTP-bound Gα subunits from the Gβγ heterodimers, and activation of downstream effector pathways. Signals are terminated following Gα-catalyzed hydrolysis of GTP and reformation of G protein heterotrimers. Thus, G proteins are molecular switches that coordinate physiological responses elicited by a variety of stimuli.RGS (regulator of G proteinsignaling) proteins are a family of more than 20 members that regulate G protein signaling in part by acting as GTPase-activating proteins (GAPs) for several classes of G protein α subunits (3Berman D.M. Wilkie T.M. Gilman A.G. Cell. 1996; 86: 445-452Abstract Full Text Full Text PDF PubMed Scopus (648) Google Scholar, 4Watson N. Linder M.E. Druey K.M. Kehrl J.H. Blumer K.J. Nature. 1996; 383: 172-175Crossref PubMed Scopus (472) Google Scholar, 5Ingi T. Krumins A.M. Chidiac P. Brothers G.M. Chung S. Snow B.E. Barnes C.A. Lanahan A.A. Siderovski D.P. Ross E.M. G i lman A.G. Worley P.F. J. Neurosci. 1998; 18: 7178-7188Crossref PubMed Google Scholar, 6Kozasa T. Jiang X. Hart M.J. Sternweis P.M. Singer W.D. G i lman A.G. Bollag G. Sternweis P.C. Science. 1998; 280: 2109-2111Crossref PubMed Scopus (736) Google Scholar). The GAP activity of RGS proteins decreases the lifetime of active, GTP-bound Gα subunits, thereby attenuating responses or accelerating the kinetics of signal termination (7Saitoh O. Kubo Y. Miyatani Y. Asano T. Nakata H. Nature. 1997; 390: 525-529Crossref PubMed Scopus (191) Google Scholar, 8Doupnik C.A. Davidson N. Lester H.A. Kofuji P. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 10461-10466Crossref PubMed Scopus (296) Google Scholar). Binding of RGS proteins to active Gα subunits can also antagonize effector activation, thereby blocking signal production (9Hepler J.R. Berman D.M. G i lman A.G. Kozasa T. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 428-432Crossref PubMed Scopus (335) Google Scholar). These activities are mediated by the conserved RGS domain of ∼120 amino acids that is characteristic of this protein family.Higher eukaryotes express several types of RGS proteins, potentially to provide selective regulation of distinct types of G protein signaling pathways. Consistent with this hypothesis, RGS proteins are structurally diverse, distinguished by various domains that are likely to confer specific functions. For example, the N terminus of RGS4 confers receptor-selective regulation of Gq-coupled responses (10Xu X. Zeng W. Popov S. Berman D.M. Davignon I. Yu K. Yowe D. Offermanns S. Muallem S. Wilkie T.M. J. Biol. Chem. 1999; 274: 3549-3556Abstract Full Text Full Text PDF PubMed Scopus (232) Google Scholar, 11Zeng W. Xu X. Popov S. Mukhopadhyay S. Chidiac P. Swistok J. Danho W. Yagaloff K.A. Fisher S.L. Ross E.M. Muallem S. Wilkie T.M. J. Biol. Chem. 1998; 273: 34687-34690Abstract Full Text Full Text PDF PubMed Scopus (220) Google Scholar), the PDZ domain of RGS12 binds peptides from the C termini of certain G protein coupled receptors (12Snow B.E. Hall R.A. Krumins A.M. Brothers G.M. Bouchard D. Brothers C.A. Chung S. Mangion J. G i lman A.G. Lefkowitz R.J. Siderovski D.P. J. Biol. Chem. 1998; 273: 17749-17755Abstract Full Text Full Text PDF PubMed Scopus (182) Google Scholar), and the GGL domain of RGS7 selectively binds Gβ5 (13Cabrera J.L. de Freitas F. Satpaev D.K. Slepak V.Z. Biochem. Biophys. Res. Commun. 1998; 249: 898-902Crossref PubMed Scopus (113) Google Scholar, 14Snow B.E. Krumins A.M. Brothers G.M. Lee S.F. Wall M.A. Chung S. Mangion J. Arya S. G i lman A.G. Siderovski D.P. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 13307-13312Crossref PubMed Scopus (228) Google Scholar). Differences in expression pattern (15Hong J.X. Wilson G.L. Fox C.H. Kehrl J.H. J. Immunol. 1993; 150: 3895-3904PubMed Google Scholar, 16Druey K.M. Blumer K.J. Kang V.H. Kehrl J.H. Nature. 1996; 379: 742-746Crossref PubMed Scopus (404) Google Scholar), subcellular localization (17De Vries L. Elenko E. McCaffery J.M. Fischer T. Hubler L. McQuistan T. Watson N. Farquhar M.G. Mol. Biol. Cell. 1998; 9: 1123-1134Crossref PubMed Scopus (88) Google Scholar, 18De Vries L. Lou X. Zhao G. Zheng B. Farquhar M.G. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 12340-12345Crossref PubMed Scopus (186) Google Scholar, 19Wylie F. Heimann K. Le T.L. Brown D. Rabnott G. Stow J.L. Am. J. Physiol. 1999; 276: C497-C506Crossref PubMed Google Scholar), and interaction with other signaling or regulatory proteins are also likely to give RGS family members distinct biological functions.It is less clear to what extent differences in Gα subunit selectivity govern the biological function of various RGS proteins. Whereas a few RGS proteins, such as RGSZ1 (20Wang J. Ducret A. Tu Y. Kozasa T. Aebersold R. Ross E.M. J. Biol. Chem. 1998; 273: 26014-26025Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar) and the RGS domain of p115Rho-GEF (6Kozasa T. Jiang X. Hart M.J. Sternweis P.M. Singer W.D. G i lman A.G. Bollag G. Sternweis P.C. Science. 1998; 280: 2109-2111Crossref PubMed Scopus (736) Google Scholar), are highly specific for certain Gα subunits, many RGS proteins are promiscuous in vitro. For example, RGS1, RGS2, RGS4, and RGS-GAIP stimulate the GTPase activity of Giα family members and Gqα in vitro. Less selective members of the RGS family do display quantitative differences in GAP activity toward various classes of Gα subunits in vitro(21Heximer S.P. Watson N. Linder M.E. Blumer K.J. Hepler J.R. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 14389-14393Crossref PubMed Scopus (311) Google Scholar, 22Lan K.L. Sarvazyan N.A. Taussig R. Mackenzie R.G. DiBello P.R. Dohlman H.G. Neubig R.R. J. Biol. Chem. 1998; 273: 12794-12797Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar), but the significance of this in vivo is unknown. Transfection studies have shown that RGS proteins can be more selective for certain types of signaling pathways in vivo than they are as GAPs in vitro (23Zhang Y. Neo S.Y. Han J. Yaw L.P. Lin S.C. J. Biol. Chem. 1999; 274: 2851-2857Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar, 24Bowman E.P. Campbell J.J. Druey K.M. Scheschonka A. Kehrl J.H. Butcher E.C. J. Biol. Chem. 1998; 273: 28040-28048Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar, 25Mao J. Yuan H. Xie W. Simon M.I. Wu D. J. Biol. Chem. 1998; 273: 27118-27123Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar). Whether the regulatory specificity of RGS proteins observed in intact cells is achieved by differences in G protein selectivity or other mechanisms remains unknown. Accordingly, the mechanisms that govern the intrinsic Gα subunit selectivity of RGS proteins remain poorly understood.To examine the importance of Gα selectivity as a determinant of the biological functions of RGS proteins, we have focused on RGS2 and RGS4. RGS2 or RGS4 can act in vivo and in vitro on Gq or Gi class α subunits, although RGS2 is much less potent than RGS4 as a GAP for Giα subunitsin vitro. Here we have examined whether RGS2 and RGS4 differ in potency as inhibitors of Gq versusGi signaling in intact cells and used mutagenesis to determine whether differences in intrinsic G protein selectivity govern the function of RGS2. The results suggest that RGS2 preferentially regulates Gq, an effect that is mediated by unique structural features of its G protein-binding interface. They further implicate G protein selectivity as an important determinant of RGS2 function.DISCUSSIONIn this study we have provided three lines of evidence indicating that RGS2 inhibits signaling by Gq in a more potent manner than it inhibits Gi class G proteins. RGS2 is 5-fold more potent than RGS4 as an inhibitor of Gq function in vivo and in vitro. In contrast, RGS2 is 8-fold less potent than RGS4 as an inhibitor of yeast mating pheromone signaling, which is mediated by a Gi class α subunit. Finally, RGS2 mutants have been identified that are more potent toward Giclass α subunits without affecting potency toward Gq. Thus, RGS2 and RGS4 differ considerably in terms of their relative intrinsic potencies as inhibitors of Gq versusGi class G proteins.Mechanism of G Protein Discrimination by RGS2A mechanism whereby RGS2 discriminates between classes of Gα subunits is suggested by our studies of RGS2 mutants and by the crystal structure of the RGS4-Giα1 transition state complex (33Tesmer J.J.G. Berman D.M. G i lman A.G. Sprang S.R. Cell. 1997; 89: 251-261Abstract Full Text Full Text PDF PubMed Scopus (680) Google Scholar). This information indicates why RGS2 interacts inefficiently with Gi class α subunits. It also suggests why RGS2 retains affinity for Gqα, but this aspect of the model remains to be tested experimentally. Two features of RGS2 are proposed to attenuate interaction with Gi class α subunits while maintaining affinity for Gqα (Fig.7). First, the geometry of the pocket of RGS2 that binds a conserved threonine residue in switch I of Gα subunits appears to decrease affinity for Gi class α subunits. We suggest this because increasing the potency of RGS2 toward Gi class α subunits requires substituting residues predicted to form part of the floor and lip of the pocket (Cys106 and Asn184, respectively) with their RGS4 counterparts (Ser85 and Asp163; Fig. 7). Indeed, the pocket of RGS2 is predicted to be smaller than that of RGS4, based on modeling studies using RGS4 as a template.2For example, the closest approach between the side chains of Cys106 and Asn184 in the pocket of RGS2 is predicted to be about 1 Å shorter than that of the corresponding residues of RGS4.We suggest two ways that the pocket of RGS2 could favor binding of Gq over Gi class α subunits. In the first model, the observed selectivity is due somewhat to different conformations of switch I in Gq and Gi class α subunits, as suggested by sequence differences (Lys180and Val185 of Giα1 are proline and isoleucine, respectively, in Gqα; Fig. 7). This allows the invariant threonine residue of switch I of Gqα to dock with the shallower or smaller pocket of RGS2 with higher affinity than the homologous threonine residue of Giα. RGS4 binds Gq and Gi class α subunits because its threonine binding pocket is bigger and docks either substrate relatively well. In the second model, switch I regions of Gi and Gq class α subunits are assumed to have approximately the same conformation. Sequence differences in and around the threonine binding pockets of RGS2 and RGS4 affect the structure of adjacent regions of their Gα binding surfaces. These adjacent regions in turn dictate selectivity for Gα subunits. A combination of these models could also occur.The second structural feature of RGS2 that appears to attenuate activity toward Gi class α subunits is at the edge of the interaction footprint defined by the RGS4-Giα1 crystal structure. This interfering interaction is proposed to involve the putative α8-α9 loop of RGS2 and αA of Gi class α subunits, because we have found that a E191K substitution in the α8-α9 loop of RGS2 is required to increase activity toward Gi class subunits. One interpretation is that charge repulsion occurs between Glu191 of RGS2 and a conserved glutamic acid residue (Glu65) in αA of Giα1. However, the closest approach between the equivalent residue in the α8-α9 loop of RGS4 (Lys170) and Glu65 of αA in Giα1 is 6.5 Å, a distance over which charge repulsion may be negligible. Nevertheless, this distance could be shorter earlier in the reaction mechanism, disfavoring a high affinity interaction. Alternatively, the structure of RGS2 may differ from that of RGS4, placing the α8-α9 loop closer to αA of the Gα subunit. Gqα may bind tightly to wild type RGS2 because the equivalent of E65 in αA (D71) is positioned farther away from Glu191 in the α8-α9 loop, either because of its shorter side chain or conformational differences of Gq- relative to Gi class α subunits. Consistent with these hypotheses, recent studies suggest that sequences in the helical domain (which includes αA) of transducin α subunits mediate preferential interaction with the RGS9 core domain (34Skiba N.P. Yang C.-S. Huang T. Bae H. Hamm H.E. J. Biol. Chem. 1999; 274: 8770-8778Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar). Indeed, it would be interesting to determine whether such interactions generally affect the selectivity of RGS-Gα interaction.Two findings suggest that the relative inability of RGS2 to interact with Gi class α subunits could also involve other sequence or structural features. None of the RGS2 mutants we have analyzed is as active toward yeast or mammalian Gi class α subunits as RGS4. Furthermore, the RGS4-triple mutant, which is identical to RGS2 at the three residues proposed to attenuate Gi interaction, is 2-fold more potent than RGS2 as an inhibitor of yeast mating pheromone response. Potentially, sequences flanking the RGS domain could influence the Gα selectivity of RGS2.G protein selection by other RGS family members may use somewhat different mechanisms. We suggest this because the RGS domain of p115Rho-GEF, which is specific for G12/13, is highly diverged from other RGS family members (6Kozasa T. Jiang X. Hart M.J. Sternweis P.M. Singer W.D. G i lman A.G. Bollag G. Sternweis P.C. Science. 1998; 280: 2109-2111Crossref PubMed Scopus (736) Google Scholar), suggesting that its Gα interaction surface is quite different. Furthermore, RGSZ1 (which acts on Gzα but not other Gi family members or Gq) is identical to RGS4 (which acts on all Gifamily members including Gzα) at the two of the three positions we have identified as G protein selectivity determinants in RGS2 (20Wang J. Ducret A. Tu Y. Kozasa T. Aebersold R. Ross E.M. J. Biol. Chem. 1998; 273: 26014-26025Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar, 35Glick J.L. Meigs T.E. Miron A. Casey P.J. J. Biol. Chem. 1998; 273: 26008-26013Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar). The selectivity of RGSZ1 may be mediated by other features of its RGS domain or by flanking sequences.Biological Implications of the G Protein Selectivity of RGS2G protein selectivity is likely to be one of several factors that govern the function of RGS2. Gq selectivity may be important because RGS2 does not yet appear to have preference for certain types of G protein coupled receptors (10Xu X. Zeng W. Popov S. Berman D.M. Davignon I. Yu K. Yowe D. Offermanns S. Muallem S. Wilkie T.M. J. Biol. Chem. 1999; 274: 3549-3556Abstract Full Text Full Text PDF PubMed Scopus (232) Google Scholar). This is in contrast to RGS1, RGS4, and RGS16, which display pronounced selectivity toward different types of Gq-coupled receptors (10Xu X. Zeng W. Popov S. Berman D.M. Davignon I. Yu K. Yowe D. Offermanns S. Muallem S. Wilkie T.M. J. Biol. Chem. 1999; 274: 3549-3556Abstract Full Text Full Text PDF PubMed Scopus (232) Google Scholar). Expression level is also likely to determine which types of G protein signaling pathways are regulated by RGS2. Lower level expression of RGS2 may be sufficient to attenuate Gq signaling without affecting Gi-mediated responses, whereas higher level expression of RGS2 could attenuate both types of signaling pathway. Thus, regulated expression of RGS2, as occurs in response to rises in cAMP or changes in neuronal activity (5Ingi T. Krumins A.M. Chidiac P. Brothers G.M. Chung S. Snow B.E. Barnes C.A. Lanahan A.A. Siderovski D.P. Ross E.M. G i lman A.G. Worley P.F. J. Neurosci. 1998; 18: 7178-7188Crossref PubMed Google Scholar, 10Xu X. Zeng W. Popov S. Berman D.M. Davignon I. Yu K. Yowe D. Offermanns S. Muallem S. Wilkie T.M. J. Biol. Chem. 1999; 274: 3549-3556Abstract Full Text Full Text PDF PubMed Scopus (232) Google Scholar, 36Pepperl D.J. Shah-Basu S. VanLeeuwen D. Granneman J.G. MacKenzie R.G. Biochem. Biophys. Res. Commun. 1998; 243: 52-55Crossref PubMed Scopus (72) Google Scholar), may provide a mechanism whereby various types of stimuli regulate Gq and Gisignaling pathways separately or in a coordinated fashion, depending on the expression level of RGS2 that is achieved. Other processes such as Gα modification (e.g. palmitoylation or phosphorylation) or association with other signaling or regulatory molecules could further augment the ability of RGS2 to effect various regulatory outcomes.In conclusion, it appears that quantitative differences in GAP activity toward Gi and Gq class α subunits is one factor that governs the biological function of RGS2 and presumably other RGS isoforms as well. Expression of mutants having altered G protein selectivity should provide a means of determining to what extent intrinsic selectivity for various classes of G proteins, receptors, or effectors governs the function of RGS2 mammalian cells. Many hormones, neurotransmitters, and sensory stimuli exert their effect on target tissues by activating receptors that are coupled to heterotrimeric G proteins1(1Bourne H.R. Sanders D.A. McCormick F. Nature. 1990; 348: 125-132Crossref PubMed Scopus (1824) Google Scholar, 2Hepler J.R. G i lman A.G. Trends Biochem. Sci. 1992; 17: 383-387Abstract Full Text PDF PubMed Scopus (919) Google Scholar). Receptor activation results in exchange of GTP for GDP on Gα subunits, dissociation of GTP-bound Gα subunits from the Gβγ heterodimers, and activation of downstream effector pathways. Signals are terminated following Gα-catalyzed hydrolysis of GTP and reformation of G protein heterotrimers. Thus, G proteins are molecular switches that coordinate physiological responses elicited by a variety of stimuli. RGS (regulator of G proteinsignaling) proteins are a family of more than 20 members that regulate G protein signaling in part by acting as GTPase-activating proteins (GAPs) for several classes of G protein α subunits (3Berman D.M. Wilkie T.M. Gilman A.G. Cell. 1996; 86: 445-452Abstract Full Text Full Text PDF PubMed Scopus (648) Google Scholar, 4Watson N. Linder M.E. Druey K.M. Kehrl J.H. Blumer K.J. Nature. 1996; 383: 172-175Crossref PubMed Scopus (472) Google Scholar, 5Ingi T. Krumins A.M. Chidiac P. Brothers G.M. Chung S. Snow B.E. Barnes C.A. Lanahan A.A. Siderovski D.P. Ross E.M. G i lman A.G. Worley P.F. J. Neurosci. 1998; 18: 7178-7188Crossref PubMed Google Scholar, 6Kozasa T. Jiang X. Hart M.J. Sternweis P.M. Singer W.D. G i lman A.G. Bollag G. Sternweis P.C. Science. 1998; 280: 2109-2111Crossref PubMed Scopus (736) Google Scholar). The GAP activity of RGS proteins decreases the lifetime of active, GTP-bound Gα subunits, thereby attenuating responses or accelerating the kinetics of signal termination (7Saitoh O. Kubo Y. Miyatani Y. Asano T. Nakata H. Nature. 1997; 390: 525-529Crossref PubMed Scopus (191) Google Scholar, 8Doupnik C.A. Davidson N. Lester H.A. Kofuji P. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 10461-10466Crossref PubMed Scopus (296) Google Scholar). Binding of RGS proteins to active Gα subunits can also antagonize effector activation, thereby blocking signal production (9Hepler J.R. Berman D.M. G i lman A.G. Kozasa T. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 428-432Crossref PubMed Scopus (335) Google Scholar). These activities are mediated by the conserved RGS domain of ∼120 amino acids that is characteristic of this protein family. Higher eukaryotes express several types of RGS proteins, potentially to provide selective regulation of distinct types of G protein signaling pathways. Consistent with this hypothesis, RGS proteins are structurally diverse, distinguished by various domains that are likely to confer specific functions. For example, the N terminus of RGS4 confers receptor-selective regulation of Gq-coupled responses (10Xu X. Zeng W. Popov S. Berman D.M. Davignon I. Yu K. Yowe D. Offermanns S. Muallem S. Wilkie T.M. J. Biol. Chem. 1999; 274: 3549-3556Abstract Full Text Full Text PDF PubMed Scopus (232) Google Scholar, 11Zeng W. Xu X. Popov S. Mukhopadhyay S. Chidiac P. Swistok J. Danho W. Yagaloff K.A. Fisher S.L. Ross E.M. Muallem S. Wilkie T.M. J. Biol. Chem. 1998; 273: 34687-34690Abstract Full Text Full Text PDF PubMed Scopus (220) Google Scholar), the PDZ domain of RGS12 binds peptides from the C termini of certain G protein coupled receptors (12Snow B.E. Hall R.A. Krumins A.M. Brothers G.M. Bouchard D. Brothers C.A. Chung S. Mangion J. G i lman A.G. Lefkowitz R.J. Siderovski D.P. J. Biol. Chem. 1998; 273: 17749-17755Abstract Full Text Full Text PDF PubMed Scopus (182) Google Scholar), and the GGL domain of RGS7 selectively binds Gβ5 (13Cabrera J.L. de Freitas F. Satpaev D.K. Slepak V.Z. Biochem. Biophys. Res. Commun. 1998; 249: 898-902Crossref PubMed Scopus (113) Google Scholar, 14Snow B.E. Krumins A.M. Brothers G.M. Lee S.F. Wall M.A. Chung S. Mangion J. Arya S. G i lman A.G. Siderovski D.P. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 13307-13312Crossref PubMed Scopus (228) Google Scholar). Differences in expression pattern (15Hong J.X. Wilson G.L. Fox C.H. Kehrl J.H. J. Immunol. 1993; 150: 3895-3904PubMed Google Scholar, 16Druey K.M. Blumer K.J. Kang V.H. Kehrl J.H. Nature. 1996; 379: 742-746Crossref PubMed Scopus (404) Google Scholar), subcellular localization (17De Vries L. Elenko E. McCaffery J.M. Fischer T. Hubler L. McQuistan T. Watson N. Farquhar M.G. Mol. Biol. Cell. 1998; 9: 1123-1134Crossref PubMed Scopus (88) Google Scholar, 18De Vries L. Lou X. Zhao G. Zheng B. Farquhar M.G. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 12340-12345Crossref PubMed Scopus (186) Google Scholar, 19Wylie F. Heimann K. Le T.L. Brown D. Rabnott G. Stow J.L. Am. J. Physiol. 1999; 276: C497-C506Crossref PubMed Google Scholar), and interaction with other signaling or regulatory proteins are also likely to give RGS family members distinct biological functions. It is less clear to what extent differences in Gα subunit selectivity govern the biological function of various RGS proteins. Whereas a few RGS proteins, such as RGSZ1 (20Wang J. Ducret A. Tu Y. Kozasa T. Aebersold R. Ross E.M. J. Biol. Chem. 1998; 273: 26014-26025Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar) and the RGS domain of p115Rho-GEF (6Kozasa T. Jiang X. Hart M.J. Sternweis P.M. Singer W.D. G i lman A.G. Bollag G. Sternweis P.C. Science. 1998; 280: 2109-2111Crossref PubMed Scopus (736) Google Scholar), are highly specific for certain Gα subunits, many RGS proteins are promiscuous in vitro. For example, RGS1, RGS2, RGS4, and RGS-GAIP stimulate the GTPase activity of Giα family members and Gqα in vitro. Less selective members of the RGS family do display quantitative differences in GAP activity toward various classes of Gα subunits in vitro(21Heximer S.P. Watson N. Linder M.E. Blumer K.J. Hepler J.R. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 14389-14393Crossref PubMed Scopus (311) Google Scholar, 22Lan K.L. Sarvazyan N.A. Taussig R. Mackenzie R.G. DiBello P.R. Dohlman H.G. Neubig R.R. J. Biol. Chem. 1998; 273: 12794-12797Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar), but the significance of this in vivo is unknown. Transfection studies have shown that RGS proteins can be more selective for certain types of signaling pathways in vivo than they are as GAPs in vitro (23Zhang Y. Neo S.Y. Han J. Yaw L.P. Lin S.C. J. Biol. Chem. 1999; 274: 2851-2857Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar, 24Bowman E.P. Campbell J.J. Druey K.M. Scheschonka A. Kehrl J.H. Butcher E.C. J. Biol. Chem. 1998; 273: 28040-28048Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar, 25Mao J. Yuan H. Xie W. Simon M.I. Wu D. J. Biol. Chem. 1998; 273: 27118-27123Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar). Whether the regulatory specificity of RGS proteins observed in intact cells is achieved by differences in G protein selectivity or other mechanisms remains unknown. Accordingly, the mechanisms that govern the intrinsic Gα subunit selectivity of RGS proteins remain poorly understood. To examine the importance of Gα selectivity as a determinant of the biological functions of RGS proteins, we have focused on RGS2 and RGS4. RGS2 or RGS4 can act in vivo and in vitro on Gq or Gi class α subunits, although RGS2 is much less potent than RGS4 as a GAP for Giα subunitsin vitro. Here we have examined whether RGS2 and RGS4 differ in potency as inhibitors of Gq versusGi signaling in intact cells and used mutagenesis to determine whether differences in intrinsic G protein selectivity govern the function of RGS2. The results suggest that RGS2 preferentially regulates Gq, an effect that is mediated by unique structural features of its G protein-binding interface. They further implicate G protein selectivity as an important determinant of RGS2 function. DISCUSSIONIn this study we have provided three lines of evidence indicating that RGS2 inhibits signaling by Gq in a more potent manner than it inhibits Gi class G proteins. RGS2 is 5-fold more potent than RGS4 as an inhibitor of Gq function in vivo and in vitro. In contrast, RGS2 is 8-fold less potent than RGS4 as an inhibitor of yeast mating pheromone signaling, which is mediated by a Gi class α subunit. Finally, RGS2 mutants have been identified that are more potent toward Giclass α subunits without affecting potency toward Gq. Thus, RGS2 and RGS4 differ considerably in terms of their relative intrinsic potencies as inhibitors of Gq versusGi class G proteins.Mechanism of G Protein Discrimination by RGS2A mechanism whereby RGS2 discriminates between classes of Gα subunits is suggested by our studies of RGS2 mutants and by the crystal structure of the RGS4-Giα1 transition state complex (33Tesmer J.J.G. Berman D.M. G i lman A.G. Sprang S.R. Cell. 1997; 89: 251-261Abstract Full Text Full Text PDF PubMed Scopus (680) Google Scholar). This information indicates why RGS2 interacts inefficiently with Gi class α subunits. It also suggests why RGS2 retains affinity for Gqα, but this aspect of the model remains to be tested experimentally. Two features of RGS2 are proposed to attenuate interaction with Gi class α subunits while maintaining affinity for Gqα (Fig.7). First, the geometry of the pocket of RGS2 that binds a conserved threonine residue in switch I of Gα subunits appears to decrease affinity for Gi class α subunits. We suggest this because increasing the potency of RGS2 toward Gi class α subunits requires substituting residues predicted to form part of the floor and lip of the pocket (Cys106 and Asn184, respectively) with their RGS4 counterparts (Ser85 and Asp163; Fig. 7). Indeed, the pocket of RGS2 is predicted to be smaller than that of RGS4, based on modeling studies using RGS4 as a template.2For example, the closest approach between the side chains of Cys106 and Asn184 in the pocket of RGS2 is predicted" @default.
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• W1987606367 title "G Protein Selectivity Is a Determinant of RGS2 Function" @default.
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