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• W2068432279 abstract "RGS proteins comprise a family of proteins named for their ability to negatively regulate heterotrimeric G protein signaling. Biochemical studies suggest that members of this protein family act as GTPase-activating proteins for certain Gα subunits, thereby accelerating the turn-off mechanism of Gα and terminating signaling by both Gα and Gβγ subunits. In the present study, we used confocal microscopy to examine the intracellular distribution of several RGS proteins in COS-7 cells expressing RGS-green fluorescent protein (GFP) fusion proteins and in cells expressing RGS proteins endogenously. RGS2 and RGS10 accumulated in the nucleus of COS-7 cells transfected with GFP constructs of these proteins. In contrast, RGS4 and RGS16 accumulated in the cytoplasm of COS-7 transfectants. As observed in COS-7 cells, RGS4 exhibited cytoplasmic localization in mouse neuroblastoma cells, and RGS10 exhibited nuclear localization in human glioma cells. Deletion or alanine substitution of an N-terminal leucine repeat motif present in both RGS4 and RGS16, a domain identified as a nuclear export sequence in HIV Rev and other proteins, promoted nuclear localization of these proteins in COS-7 cells. In agreement with this observation, treatment of mouse neuroblastoma cells with leptomycin B to inhibit nuclear protein export by exportin1 resulted in accumulation of RGS4 in the nucleus of these cells. GFP fusions of RGS domains of RGS proteins localized in the nucleus, suggesting that nuclear localization of RGS proteins results from nuclear targeting via RGS domain sequences. RGSZ, which shares with RGS-GAIP a cysteine-rich string in its N-terminal region, localized to the Golgi complex in COS-7 cells. Deletion of the N-terminal domain of RGSZ that includes the cysteine motif promoted nuclear localization of RGSZ. None of the RGS proteins examined were localized at the plasma membrane. These results demonstrate that RGS proteins localize in the nucleus, the cytoplasm, or shuttle between the nucleus and cytoplasm as nucleo-cytoplasmic shuttle proteins. RGS proteins localize differentially within cells as a result of structural differences among these proteins that do not appear to be important determinants for their G protein-regulating activities. These findings suggest involvement of RGS proteins in more complex cellular functions than currently envisioned. RGS proteins comprise a family of proteins named for their ability to negatively regulate heterotrimeric G protein signaling. Biochemical studies suggest that members of this protein family act as GTPase-activating proteins for certain Gα subunits, thereby accelerating the turn-off mechanism of Gα and terminating signaling by both Gα and Gβγ subunits. In the present study, we used confocal microscopy to examine the intracellular distribution of several RGS proteins in COS-7 cells expressing RGS-green fluorescent protein (GFP) fusion proteins and in cells expressing RGS proteins endogenously. RGS2 and RGS10 accumulated in the nucleus of COS-7 cells transfected with GFP constructs of these proteins. In contrast, RGS4 and RGS16 accumulated in the cytoplasm of COS-7 transfectants. As observed in COS-7 cells, RGS4 exhibited cytoplasmic localization in mouse neuroblastoma cells, and RGS10 exhibited nuclear localization in human glioma cells. Deletion or alanine substitution of an N-terminal leucine repeat motif present in both RGS4 and RGS16, a domain identified as a nuclear export sequence in HIV Rev and other proteins, promoted nuclear localization of these proteins in COS-7 cells. In agreement with this observation, treatment of mouse neuroblastoma cells with leptomycin B to inhibit nuclear protein export by exportin1 resulted in accumulation of RGS4 in the nucleus of these cells. GFP fusions of RGS domains of RGS proteins localized in the nucleus, suggesting that nuclear localization of RGS proteins results from nuclear targeting via RGS domain sequences. RGSZ, which shares with RGS-GAIP a cysteine-rich string in its N-terminal region, localized to the Golgi complex in COS-7 cells. Deletion of the N-terminal domain of RGSZ that includes the cysteine motif promoted nuclear localization of RGSZ. None of the RGS proteins examined were localized at the plasma membrane. These results demonstrate that RGS proteins localize in the nucleus, the cytoplasm, or shuttle between the nucleus and cytoplasm as nucleo-cytoplasmic shuttle proteins. RGS proteins localize differentially within cells as a result of structural differences among these proteins that do not appear to be important determinants for their G protein-regulating activities. These findings suggest involvement of RGS proteins in more complex cellular functions than currently envisioned. guanine nucleotide-binding protein Dulbecco's phosphate buffered saline enhanced GFP green fluorescent protein human immunodeficiency virus inhibitor κ B α nuclear export sequence phosphoinositide phospholipase C guanosine 5′-3-O-(thio)triphosphate polymerase chain reaction fluorescein isothiocyanate RGS proteins comprise a family of more than 20 known members that have been implicated in the negative regulation of heterotrimeric G protein1 signaling (1Dohlman H.G. Thorner J. J. Biol. Chem. 1997; 272: 3871-3874Abstract Full Text Full Text PDF PubMed Scopus (447) Google Scholar, 2Berman D.M. Gilman A.G. J. Biol. Chem. 1998; 273: 1269-1272Abstract Full Text Full Text PDF PubMed Scopus (444) Google Scholar). RGS proteins were discovered as a pheromone desensitization factor (Sst2p, supersensitive phenotype protein) by genetic studies in yeast and shown to negatively regulate signaling by the yeast homolog of Goα, Gpa1 (3Dohlman H.G. Apaniesk D. Chen Y. Song J. Nusskern D. Mol. Cell. Biol. 1995; 15: 3635-3643Crossref PubMed Scopus (166) Google Scholar). Genetic studies in Caenorhabditis elegansidentified a homolog of Sst2p involved in negative regulation of signaling by the Goα homolog GOA-1 (4Koelle M.R. Horvitz H.R. Cell. 1996; 84: 115-125Abstract Full Text Full Text PDF PubMed Scopus (475) Google Scholar). Subsequent studies have documented the existence of transcripts encoding proteins with RGS domains, a semi-conserved sequence of approximately 120 amino acids found in all RGS proteins, in species ranging from fungus to man (1Dohlman H.G. Thorner J. J. Biol. Chem. 1997; 272: 3871-3874Abstract Full Text Full Text PDF PubMed Scopus (447) Google Scholar, 2Berman D.M. Gilman A.G. J. Biol. Chem. 1998; 273: 1269-1272Abstract Full Text Full Text PDF PubMed Scopus (444) Google Scholar,5Lee B.N. Adams T.H. Mol. Microbiol. 1994; 14: 323-334Crossref PubMed Scopus (169) Google Scholar).Biochemical studies have supported the genetic evidence that the locus of RGS protein action is at the level of G protein α subunits. RGS proteins bind to Gα subunits in the Gi and Gqfamilies in vitro and dramatically enhance their intrinsic GTPase activity (6Berman D.M. Wilkie T.M. Gilman A.G. Cell. 1996; 86: 445-452Abstract Full Text Full Text PDF PubMed Scopus (648) Google Scholar, 7Berman D.M. Kozasa T. Gilman A.G. J. Biol. Chem. 1996; 271: 27209-27212Abstract Full Text Full Text PDF PubMed Scopus (298) Google Scholar). Thus, RGS proteins function as GTPase-activating proteins for certain heterotrimeric Gα subunits. Stimulation of GTP hydrolysis of Gα subunits leads to their conversion from the active Gα-GTP form to the inactive Gα-GDP form and their recombination with Gβγ subunits, effectively terminating signaling by both Gα and Gβγ subunits. The deduced crystal structure of RGS4 bound to Giα1 showed interaction of RGS domain residues with the G protein switch regions, suggesting that the mechanism of GTPase acceleration by RGS proteins may be due primarily to stabilization of the transition state of Gα residues directly involved in catalysis (8Tesmer J.J.G. Berman D.M. Gilman A.G. Sprang S.R. Cell. 1997; 89: 251-261Abstract Full Text Full Text PDF PubMed Scopus (680) Google Scholar). Additional studies have raised the possibility that RGS proteins may also interact with effectors or receptors to attenuate G protein signaling. These studies reported that recombinant RGS proteins can block PI PLC activation by Gqα-GTPγS in vitro (9Hepler J.R. Berman D.M. Gilman A.G. Kozasa T. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 428-432Crossref PubMed Scopus (335) Google Scholar) and produce receptor-selective attenuation of Gq signaling when added to permeabilized cells (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).Implicit in the proposed regulatory actions of RGS proteins in cells is their localization at or near the plasma membrane where G proteins, as well as receptors and effectors, are located. Here we examined the cellular localization of RGS proteins expressed as GFP fusion proteins in COS-7 cells or endogenously in other cells. Our results reveal a surprising diversity in subcellular localization and trafficking of RGS proteins. RGS proteins localize in the nucleus, the cytoplasm or shuttle between the nucleus and cytoplasm as nucleo-cytoplasmic shuttle proteins. None of the RGS proteins studied showed localization at the plasma membrane. We identified the molecular determinant for nuclear localization of RGS proteins to the conserved RGS domain present in all RGS proteins. We also identified sequence elements outside of the RGS domain that result in either nuclear-cytoplasmic transport or cytoplasmic retention of RGS proteins. These results show that RGS proteins localize differentially within cells as a result of structural differences among these proteins that do not appear to be important determinants for their G protein-regulating activities. This study is the first to investigate sequences responsible for and mechanisms underlying localization of RGS proteins within mammalian cells. Our findings suggest involvement of RGS proteins in more complex cellular functions than currently envisioned.DISCUSSIONThese results have elucidated an unexpected diversity in subcellular localization and trafficking of RGS proteins in mammalian cells. Our findings show that RGS proteins localize in the nucleus, in the cytoplasm, or shuttle between the nucleus and cytoplasm as nucleo-cytoplasmic shuttle proteins. We identified the molecular determinant for nuclear localization of RGS proteins to the conserved RGS domain present in all RGS proteins. We also identified sequence elements outside of the RGS domain that result in either nuclear-cytoplasmic transport or cytoplasmic retention of RGS proteins. A Rev-like sequence in the N termini of RGS4 and RGS16 was identified and shown to mediate nuclear-cytoplasmic export of these proteins by interactions with the exportin1-RanGTP complex. Our results suggest that cytoplasmic retention of some RGS proteins may be mediated by other types of sequences located in their N-terminal domains. Cytoplasmic retention and localization of RGSZ within the trans-Golgi network is mediated by sequences in its N-terminal domain, which shares a cysteine-rich motif found in the N terminus of RGS-GAIP, another trans-Golgi RGS protein. None of the RGS proteins we studied showed appreciable accumulation at the plasma membrane.RGS proteins are named for their ability to negatively regulate signaling by G proteins, located at the plasma membrane and, for some G proteins, in the Golgi complex (23Druey K.M. Blumer K.J. Kang V.H. Kehrl J.H. Nature. 1996; 379: 742-746Crossref PubMed Scopus (404) Google Scholar, 24Yan Y. Chi P.P. Bourne H.R. J. Biol. Chem. 1997; 272: 11924-11927Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar, 25Buckbinder L. Velasco-Miguel S. Chen Y. Xu N. Talbott R. Gelbert L. Gao J. Seizinger B.R. Gutkind J.S. Kley N. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 7868-7872Crossref PubMed Scopus (84) Google Scholar, 26Mao 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, 27Huang C. Hepler J.R. Gilman A.G. Mumby S.M. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 6159-6163Crossref PubMed Scopus (150) Google Scholar, 28Tseng C.-C. Zhang X.-Y. Endocrinology. 1998; 139: 4470-4475Crossref PubMed Scopus (62) Google Scholar). RGS proteins are thought to produce their regulatory effects by physically interacting with Gα subunits, particularly those in the Gi and Gqfamilies, and enhancing their intrinsic GTPase activity by stabilizing the transition state conformation of these proteins for this reaction (7Berman D.M. Kozasa T. Gilman A.G. J. Biol. Chem. 1996; 271: 27209-27212Abstract Full Text Full Text PDF PubMed Scopus (298) Google Scholar). Numerous studies have provided evidence for interaction of RGS proteins with Gα subunits. Watson et al. (29Watson N. Linder M.E. Druey K.M. Kehrl J.H. Blumer K.J. Nature. 1996; 383: 172-175Crossref PubMed Scopus (472) Google Scholar) demonstrated binding of recombinant RGS1, RGS2, and RGS4 to GDP·AlF4−-treated Gi/oα in bovine brain membranes, and Berman et al. (7Berman D.M. Kozasa T. Gilman A.G. J. Biol. Chem. 1996; 271: 27209-27212Abstract Full Text Full Text PDF PubMed Scopus (298) Google Scholar) showed that RGS4 forms a high affinity complex with recombinant GDP·AlF4−·Giα1. Co-precipitation of GTPase-deficient forms of Giα3 and Gzα with RGS10 (30Hunt T.W. Fields T.A. Casey P.J. Peralta E.G. Nature. 1996; 383: 175-177Crossref PubMed Scopus (306) Google Scholar), of endogenous G11α with RGS3 (31Dulin N.O. Sorokin A. Reed E. Elliott S. Kehrl J.H. Dunn M.J. Mol. Cell. Biol. 1999; 19: 714-723Crossref PubMed Google Scholar), and of receptor-activated Gsα with RGS2 (28Tseng C.-C. Zhang X.-Y. Endocrinology. 1998; 139: 4470-4475Crossref PubMed Scopus (62) Google Scholar) has been documented in lysates derived from cells over-expressing these RGS proteins. Hepler et al. (9Hepler J.R. Berman D.M. Gilman A.G. Kozasa T. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 428-432Crossref PubMed Scopus (335) Google Scholar) showed that addition of RGS4 and RGS-GAIP to membrane preparations inhibits receptor-mediated Giα signaling, and Heximer et al. (32Heximer 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) reported that recombinant RGS2 specifically inhibits Gq signaling in membrane preparations. Recombinant RGS proteins stimulate the GTPase activity of membrane-bound and recombinant Giα (6Berman D.M. Wilkie T.M. Gilman A.G. Cell. 1996; 86: 445-452Abstract Full Text Full Text PDF PubMed Scopus (648) Google Scholar, 7Berman D.M. Kozasa T. Gilman A.G. J. Biol. Chem. 1996; 271: 27209-27212Abstract Full Text Full Text PDF PubMed Scopus (298) Google Scholar, 9Hepler J.R. Berman D.M. Gilman A.G. Kozasa T. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 428-432Crossref PubMed Scopus (335) Google Scholar, 29Watson N. Linder M.E. Druey K.M. Kehrl J.H. Blumer K.J. Nature. 1996; 383: 172-175Crossref PubMed Scopus (472) Google Scholar,32Heximer 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), and mutant RGS proteins defective in binding Gα subunits are inactive in augmenting GTP hydrolysis by Giα (33Srinivasa S.P. Watson N. Overton M.C. Blumer K.J. J. Biol. Chem. 1998; 273: 1529-1533Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar).It also has been proposed that RGS proteins may interact directly with receptors or effectors. Xu et al. (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) showed that recombinant RGS1, RGS4, and RGS16, but not RGS2, produced receptor-selective inhibition of Gq signaling when added to permeabilized rat pancreatic acinar cells. The differential inhibitory effects of the same or different RGS protein on receptor-mediated Gq signaling were not due to differences in the type of Gq activated or the extent of Gq activation. These observations suggested that RGS proteins interact directly with receptors to modulate G protein-coupled receptor signaling. Zenget al. (34Zeng 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) reported that the N terminus of RGS4 was responsible for receptor-selective inhibition of Gq in pancreatic acinar cells dialyzed with recombinant RGS4 or portions thereof. Interestingly, the N-terminal 33 amino acids of RGS4 produced inhibitory effects on receptor-mediated Gq signaling that were synergistic with that of the RGS domain of RGS4 and that were not reversed by GTPγS. Thus, the N-terminal domain of RGS4 was suggested to interact with receptors to position the protein between Gq and the effector PI PLCβ. In such a position, RGS4 could impart receptor-selective inhibition of Gqactivation, act as a Gq GTPase-activating protein, and act as an effector antagonist toward PI PLCβ. Evidence for this latter concept was provided first by the observation that recombinant RGS4 and RGS-GAIP blocked activation of PI PLC by GTPγS or GTPγS plus bradykinin in NG-108 membranes (9Hepler J.R. Berman D.M. Gilman A.G. Kozasa T. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 428-432Crossref PubMed Scopus (335) Google Scholar). These workers also showed that PI PLCβ inhibited binding of Gqα to RGS4, suggesting that RGS4 may compete with Gq for binding to PI PLC to mediate effector antagonism. RGS4 had no effects on receptor-mediated nucleotide exchange.Implicit in the proposed regulatory actions of RGS proteins on cell surface receptors, Gα subunits or the effector PI PLC is their localization at or near the plasma membrane. However, our finding that certain RGS proteins (RGS2 and RGS10) are localized predominantly in the nucleus suggest that their interaction with plasma membrane-bound receptors, G proteins, or effectors is unlikely in intact resting cells. This, of course, does not preclude their interaction with such signaling proteins when added as recombinant proteins in vitro or to permeabilized or dialyzed cells as well as under certain cellular situations that are not as yet defined. Our results show that other RGS proteins (RGS4 and RGS16) are found predominantly in the cytoplasm where it seems more likely that they could interact with signaling proteins at the plasma membrane. We did not observe recruitment of RGS4 or RGS16 to the plasma membrane of COS-7 cells following stimulation of endogenous Gi-coupled lysophosphatidic acid receptors in these cells. Druey et al.(35Druey K.M. Sullivan B.M. Brown D. Fischer E.R. Watson N. Blumer K.J. Gerfen C.R. Scheschonka A. Kehrl J.H. J. Biol. Chem. 1998; 273: 18405-18410Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar) showed translocation of cytoplasmic RGS4 to the plasma membrane of HEK cells following expression of a GTPase deficient Giα2 (Q207L), although the specificity and physiological relevance of this response is unclear. The mechanism of this recruitment does not involve interaction of RGS4 with mutant Giα because similar recruitment was observed with an RGS4 mutant that does not interact with Giα, and previous studies have shown that RGS4 does not complex with GTPase-deficient Giα1 (7Berman D.M. Kozasa T. Gilman A.G. J. Biol. Chem. 1996; 271: 27209-27212Abstract Full Text Full Text PDF PubMed Scopus (298) Google Scholar). Dulin et al. (31Dulin N.O. Sorokin A. Reed E. Elliott S. Kehrl J.H. Dunn M.J. Mol. Cell. Biol. 1999; 19: 714-723Crossref PubMed Google Scholar) reported the presence of RGS3 immunoreactivity in endothelin- or A23187-induced membrane ruffles in HMG/RGS3 cells, although most RGS3 remained in the cytoplasm. Whether RGS3 localization in membrane ruffles is unique to these mesangial cell transfectants and/or important in the regulatory effects of RGS3 in these cells is unknown. However, it is known that expression of RGS4 in several mammalian cells including COS-7 cells attenuates receptor-mediated activation of mitogen-activated protein kinase via Gi and Gq(13Chatterjee T.K. Eapen A.K. Fisher R.A. J. Biol. Chem. 1997; 272: 15481-15487Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar, 23Druey K.M. Blumer K.J. Kang V.H. Kehrl J.H. Nature. 1996; 379: 742-746Crossref PubMed Scopus (404) Google Scholar, 24Yan Y. Chi P.P. Bourne H.R. J. Biol. Chem. 1997; 272: 11924-11927Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar). A key question that remains unanswered is whether these actions of RGS4 and other cytoplasmic RGS proteins requires their translocation and physical association with the plasma membrane.Two recent papers support our evidence that some members of the RGS protein family are localized in the Golgi complex and nucleus. De Vrieset al. (20De Vries L. Elenko E. McCaffery M. Fischer T. Hubler L. McQuistan T. Watson N. Farquhar M.G. Mol. Biol. Cell. 1998; 9: 1123-1134Crossref PubMed Scopus (88) Google Scholar) showed that RGS-GAIP is located on Golgi membranes and not the plasma membrane. Our results show that RGSZ is localized exclusively in the trans-Golgi complex and that this localization is dependent upon sequences in its N-terminal domain, a region that shares with RGS-GAIP 8 cysteines in an 11-amino acid stretch. Because RGSZ shares no homology with RGS-GAIP in this region apart from this cysteine string, we favor the idea that this motif is important in the cytoplasmic retention and targeting of these two RGS proteins to the Golgi complex. In addition, Bowman et al.(36Bowman 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) showed confocal images of L1/2 lymphoid cells transfected with GFP fusions of RGS1, RGS2, and RGS3. Although nuclear staining was not shown, the authors suggested that RGS2 was present in a region identified as the nucleus as well as near the plasma membrane. RGS1 was found throughout the cell with equivalent intensity, similar to the pattern of GFP alone, whereas RGS3 seemed to be excluded from the presumed nuclear region. Although these findings are not entirely consistent with our documentation of nuclear localization of RGS2, they support our evidence that some RGS proteins are transported to and remain in the nucleus after synthesis by virtue of their lack of cytoplasmic retention or nuclear export sequences.The experiments described here do not address whether RGS proteins have functions apart from their ability to regulate membrane-delimited events involved in G protein signaling. It is possible that they have only G protein regulatory functions. In such a case, the separation in space of G proteins at the plasma membrane and RGS proteins at other cellular sites may be of regulatory importance, with these two families of proteins interacting only under certain cellular conditions. Alternatively, it is possible that RGS proteins have functions apart from, or in addition to, their ability to negatively regulate G protein signaling. In view of our evidence for nuclear localization and nucleo-cytoplasmic shuttling of some RGS proteins, it is interesting to speculate that these proteins may possess currently unrecognized functions in nuclear processes. We are unaware of such evidence, although Chuang et al. (37Chuang H-H., Yu, M. Jan Y.N. Jan L.Y. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 11727-11732Crossref PubMed Scopus (107) Google Scholar) suggested that RGS4 expression in oocytes caused a larger population of G proteins to be accessible for receptor-mediated GIRK channel activation. Although it has been shown that stimulation of G protein-coupled receptors can induce expression of RGS proteins (23Druey K.M. Blumer K.J. Kang V.H. Kehrl J.H. Nature. 1996; 379: 742-746Crossref PubMed Scopus (404) Google Scholar, 28Tseng C.-C. Zhang X.-Y. Endocrinology. 1998; 139: 4470-4475Crossref PubMed Scopus (62) Google Scholar), it is unclear whether RGS protein expression can similarly influence expression of G proteins or other proteins. Of interest is our finding that RGS domains of RGS proteins possess, in addition to determinants required for interaction with Gα proteins, sequences that target these proteins to the cell nucleus. Thus, it seems appropriate to speculate that both conserved and divergent sequences of RGS proteins may encode functions apart from the G protein-binding and -regulating activities of these proteins.The existence of a large family of RGS proteins immediately prompted questions regarding functional differences among members of this family. Among many possibilities was the notion that sequence differences among these proteins may be involved in determining their specificity(s) toward G proteins. However, most studies have shown that RGS proteins are GTPase-activating proteins for G proteins in the Gi and Gq family, despite considerable sequence diversity outside of their conserved RGS domains. Thus, the RGS domain may represent the primary determinant for RGS protein interactions with G proteins, a finding supported by the deduced crystal structure of RGS4-Giα1 and in vitro studies (8Tesmer J.J.G. Berman D.M. Gilman A.G. Sprang S.R. Cell. 1997; 89: 251-261Abstract Full Text Full Text PDF PubMed Scopus (680) Google Scholar,12Brott B.K. Pinsky B.A. Erikson R.L. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 963-968Crossref PubMed Scopus (113) Google Scholar, 33Srinivasa S.P. Watson N. Overton M.C. Blumer K.J. J. Biol. Chem. 1998; 273: 1529-1533Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar). Here, we provide evidence that diversity within this protein family has consequences in terms of the localization of RGS proteins within cells. The present study is the first to investigate the sequences responsible for and mechanisms underlying localization of these proteins within mammalian cells. Our results show that RGS proteins localize differentially within cells as a result of structural differences among these proteins that do not appear to be important determinants for their G protein-regulating activities. It seems quite likely that RGS proteins may have different functions within cells as a result of these different localization patterns. Hopefully, the present results will facilitate identification of new activities of the proteins that comprise the RGS protein family. RGS proteins comprise a family of more than 20 known members that have been implicated in the negative regulation of heterotrimeric G protein1 signaling (1Dohlman H.G. Thorner J. J. Biol. Chem. 1997; 272: 3871-3874Abstract Full Text Full Text PDF PubMed Scopus (447) Google Scholar, 2Berman D.M. Gilman A.G. J. Biol. Chem. 1998; 273: 1269-1272Abstract Full Text Full Text PDF PubMed Scopus (444) Google Scholar). RGS proteins were discovered as a pheromone desensitization factor (Sst2p, supersensitive phenotype protein) by genetic studies in yeast and shown to negatively regulate signaling by the yeast homolog of Goα, Gpa1 (3Dohlman H.G. Apaniesk D. Chen Y. Song J. Nusskern D. Mol. Cell. Biol. 1995; 15: 3635-3643Crossref PubMed Scopus (166) Google Scholar). Genetic studies in Caenorhabditis elegansidentified a homolog of Sst2p involved in negative regulation of signaling by the Goα homolog GOA-1 (4Koelle M.R. Horvitz H.R. Cell. 1996; 84: 115-125Abstract Full Text Full Text PDF PubMed Scopus (475) Google Scholar). Subsequent studies have documented the existence of transcripts encoding proteins with RGS domains, a semi-conserved sequence of approximately 120 amino acids found in all RGS proteins, in species ranging from fungus to man (1Dohlman H.G. Thorner J. J. Biol. Chem. 1997; 272: 3871-3874Abstract Full Text Full Text PDF PubMed Scopus (447) Google Scholar, 2Berman D.M. Gilman A.G. J. Biol. Chem. 1998; 273: 1269-1272Abstract Full Text Full Text PDF PubMed Scopus (444) Google Scholar,5Lee B.N. Adams T.H. Mol. Microbiol. 1994; 14: 323-334Crossref PubMed Scopus (169) Google Scholar). Biochemical studies have supported the genetic evidence that the locus of RGS protein action is at the level of G protein α subunits. RGS proteins bind to Gα subunits in the Gi and Gqfamilies in vitro and dramatically enhance their intrinsic GTPase activity (6Berman D.M. Wilkie T.M. Gilman A.G. Cell. 1996; 86: 445-452Abstract Full Text Full Text PDF PubMed Scopus (648) Google Scholar, 7Berman D.M. Kozasa T. Gilman A.G. J. Biol. Chem. 1996; 271: 27209-27212Abstract Full Text Full Text PDF PubMed Scopus (298) Google Scholar). Thus, RGS proteins function as GTPase-activating proteins for certain heterotrimeric Gα subunits. Stimulation of GTP hydrolysis of Gα subunits leads to their conversion from the active Gα-GTP form to the inactive Gα-GDP form and their recombination with Gβγ subunits, effectively terminating signaling by both Gα and Gβγ subunits. The deduced crystal structure of RGS4 bound to Giα1 showed interaction of RGS domain residues with the G protein switch regions, suggesting that the mechanism of GTPase acceleration by RGS proteins may be due primarily to stabilization of the transition state of Gα residues directly involved in catalysis (8Tesmer J.J.G. Berman D.M. Gilman A.G. Sprang S.R. Cell. 1997; 89: 251-261Abstract Full Text Full Text PDF PubMed Scopus (680) Google Scholar). Additional studies have raised the possibility that RGS proteins may also interact with effectors or receptors to attenuate G protein signaling. These studies reported that recombinant RGS proteins can block PI PLC activation by Gqα-GTPγS in vitro (9Hepler J.R. Berman D.M. Gilman A.G. Kozasa T. Proc. Natl." @default.
• W2068432279 created "2016-06-24" @default.
• W2068432279 creator A5048166676 @default.
• W2068432279 creator A5080153002 @default.
• W2068432279 date "2000-08-01" @default.
• W2068432279 modified "2023-10-18" @default.
• W2068432279 title "Cytoplasmic, Nuclear, and Golgi Localization of RGS Proteins" @default.
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