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• W2009195662 abstract "RGS3 belongs to a family of the regulators of G protein signaling (RGS). We previously demonstrated that cytosolic RGS3 translocates to the membrane to inhibit Gq/11signaling (Dulin, N. O., Sorokin, A., Reed, E., Elliott, S., Kehrl, J., and Dunn, M. J. (1999) Mol. Cell. Biol. 19, 714–723). This study examines the properties of a recently identified truncated variant termed RGS3T. Both RGS3 and RGS3T bound to endogenous Gαq/11 and inhibited endothelin-1-stimulated calcium mobilization and mitogen-activated protein kinase activity to a similar extent. However, unlike cytosolically localized RGS3, RGS3T was found predominantly in the nucleus and partially in the plasma membrane. Furthermore, RGS3T, but not RGS3, caused cell rounding and membrane blebbing. Finally, 44% of RGS3T-transfected cells underwent apoptosis after serum withdrawal, which was significantly higher than that of RGS3-transfected cells (7%). Peptide sequence analysis revealed two potential nuclear localization signal (NLS) sequences in RGS3T. Further truncation of the RGS3T N terminus containing putative NLSs resulted in a significant reduction of nuclear versus cytoplasmic staining of the protein. Moreover, this truncated RGS3T no longer induced apoptosis. In summary, RGS3 and its truncated variant RGS3T are similar in their ability to inhibit Gq/11 signaling but are different in their intracellular distribution. These data suggest that, in addition to being a GTPase-activating protein, RGS3T has other distinct functions in the nucleus of the cell. RGS3 belongs to a family of the regulators of G protein signaling (RGS). We previously demonstrated that cytosolic RGS3 translocates to the membrane to inhibit Gq/11signaling (Dulin, N. O., Sorokin, A., Reed, E., Elliott, S., Kehrl, J., and Dunn, M. J. (1999) Mol. Cell. Biol. 19, 714–723). This study examines the properties of a recently identified truncated variant termed RGS3T. Both RGS3 and RGS3T bound to endogenous Gαq/11 and inhibited endothelin-1-stimulated calcium mobilization and mitogen-activated protein kinase activity to a similar extent. However, unlike cytosolically localized RGS3, RGS3T was found predominantly in the nucleus and partially in the plasma membrane. Furthermore, RGS3T, but not RGS3, caused cell rounding and membrane blebbing. Finally, 44% of RGS3T-transfected cells underwent apoptosis after serum withdrawal, which was significantly higher than that of RGS3-transfected cells (7%). Peptide sequence analysis revealed two potential nuclear localization signal (NLS) sequences in RGS3T. Further truncation of the RGS3T N terminus containing putative NLSs resulted in a significant reduction of nuclear versus cytoplasmic staining of the protein. Moreover, this truncated RGS3T no longer induced apoptosis. In summary, RGS3 and its truncated variant RGS3T are similar in their ability to inhibit Gq/11 signaling but are different in their intracellular distribution. These data suggest that, in addition to being a GTPase-activating protein, RGS3T has other distinct functions in the nucleus of the cell. regulator of G protein signaling GTPase-activating protein Chinese hamster ovary endothelin-1 endothelin-1 receptor type A Gα interacting protein inositol trisphosphate mitogen-activated protein kinase nuclear localization signal phosphate-buffered saline fetal bovine serum polymerase chain reaction RGS3 belongs to the family of the regulators of G protein signaling (RGS)1 which are united by a sequence homology of a core RGS domain and by function as GTPase-activating proteins (GAP) for the α subunit of heterotrimeric G proteins (2.Koelle M.R. Horvitz H.R. Cell. 1996; 84: 115-125Abstract Full Text Full Text PDF PubMed Scopus (475) Google Scholar, 3.Druey K.M. Blumer K.J. Kang V.H. Kehrl J.H. Nature. 1996; 379: 742-746Crossref PubMed Scopus (403) Google Scholar, 4.Hunt T.W. Fields T.A. Casey P.J. Peralta E.G. Nature. 1996; 383: 175-177Crossref PubMed Scopus (306) Google Scholar, 5.Watson N. Linder M.E. Druey K.M. Kehrl J.H. Blumer K.J. Nature. 1996; 383: 172-175Crossref PubMed Scopus (472) Google Scholar, 6.Berman D.M. Kozasa T. Gilman A.G. J. Biol. Chem. 1996; 271: 27209-27212Abstract Full Text Full Text PDF PubMed Scopus (298) Google Scholar). We and others have previously shown that RGS3 is a potent inhibitor of Gq/11-mediated signaling, such as gonadotropin-releasing hormone-induced production of inositol trisphosphate (IP3) (7.Neill J.D. Duck L.W. Sellers J.C. Musgrove L.C. Scheschonka A. Druey K.M. Kehrl J.H. Endocrinology. 1997; 138: 843-846Crossref PubMed Scopus (86) Google Scholar), and endothelin-1-induced calcium mobilization and activation of mitogen-activated protein (MAP) kinase (1.Dulin N.O. Sorokin A. Reed E. Elliott S. Kehrl J. Dunn M.J. Mol. Cell. Biol. 1999; 19: 714-723Crossref PubMed Google Scholar). One of the unique features of RGS3 is that it is normally a cytosolic protein, which translocates to the membrane and binds to Gαq/11 upon agonist stimulation. Translocation of RGS3 involves the recruitment of its N-terminal non-RGS domain, whereas C-terminal RGS domain provides interaction with G proteins (1.Dulin N.O. Sorokin A. Reed E. Elliott S. Kehrl J. Dunn M.J. Mol. Cell. Biol. 1999; 19: 714-723Crossref PubMed Google Scholar).Recently, a truncated isoform of RGS3, termed RGS3T, has been identified by polymerase chain reaction (PCR) analysis (8.Chatterjee 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). RGS3T is a much smaller molecule, which lacks a large portion of the N-terminal domain of RGS3 but retains a core RGS domain and a smaller (about 70 amino acids) N-terminal tail. RGS3T is expressed ubiquitously in various tissues, whereas full length RGS3 is more selectively expressed in heart, lung, testis, skeletal, and smooth muscle (3.Druey K.M. Blumer K.J. Kang V.H. Kehrl J.H. Nature. 1996; 379: 742-746Crossref PubMed Scopus (403) Google Scholar, 8.Chatterjee 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), suggesting the possibility of distinct function(s) of these proteins. Because RGS3T lacks a domain which is implicated in cytosolic localization of RGS3 and its agonist-induced recruitment to the membrane (1.Dulin N.O. Sorokin A. Reed E. Elliott S. Kehrl J. Dunn M.J. Mol. Cell. Biol. 1999; 19: 714-723Crossref PubMed Google Scholar), we sought to determine whether this deletion affects intracellular localization of RGS3T as well as its ability to bind Gαq/11 and to inhibit Gq/11-mediated signaling.Here we demonstrate that RGS3 and RGS3T are similar in their ability to bind Gαq/11 and inhibit Gq/11-mediated signaling but are different in their intracellular distribution, wherein full length RGS3 is localized in the cytoplasm, whereas RGS3T is localized predominantly in the nucleus and partially in the plasma membrane. Moreover, nuclear localization parallels with the unique ability of RGS3T to induce apoptosis, suggesting that RGS3T is not just a regulator of G protein signaling, but has other function(s) in the nucleus.DISCUSSIONIt is now established that the main function of RGS proteins is to stimulate GTPase activity of heterotrimeric G proteins, and therefore, to inhibit G protein signaling. RGS3 is a potent inhibitor of Gq/11-mediated signaling such as gonadotropin-releasing hormone-induced IP3 production (7.Neill J.D. Duck L.W. Sellers J.C. Musgrove L.C. Scheschonka A. Druey K.M. Kehrl J.H. Endocrinology. 1997; 138: 843-846Crossref PubMed Scopus (86) Google Scholar) and endothelin-1-induced calcium mobilization and MAP kinase activation (1.Dulin N.O. Sorokin A. Reed E. Elliott S. Kehrl J. Dunn M.J. Mol. Cell. Biol. 1999; 19: 714-723Crossref PubMed Google Scholar). Discovery of an endogenously expressed truncated isoform of RGS3, namely RGS3T (8.Chatterjee 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), raised several important questions about RGS3 function, two of which are addressed in the present study. First, does the N-terminal region regulate the ability of RGS3 to inhibit G protein signaling? Second, what is the difference between these two products of the same RGS3 gene (15.Chatterjee T.K. Eapen A. Kanis A.B. Fisher R.A. Genomics. 1997; 45: 429-433Crossref PubMed Scopus (14) Google Scholar), besides their differential tissue distribution (8.Chatterjee 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)?The N-terminal Domain of RGS3 Is Not Required for G Protein Inhibitory FunctionOur coimmunoprecipitation experiments (Fig.1) and signaling studies (Figs. 2 and 3) convincingly demonstrate that there is no difference between RGS3 and RGS3T in the binding to Gαq/11 and the ability to inhibit Gq/11-coupled endothelin signaling, suggesting that the N-terminal domain of RGS3 does not regulate its GAP function toward Gαq/11. This is different from previously published data (8.Chatterjee 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) demonstrating that RGS3T, but not full length RGS3, inhibited platelet-activating factor-induced IP3 production, which was presumably mediated by Gq/11. This discrepancy, however, may be explained by the recently published observation of receptor selectivity of RGS proteins, whereas RGS1, RGS4, and RGS16 were more potent to inhibit carbachol-dependent signaling than cholecystokinin, even though both agonists were coupled to Gq (16.Xu 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).Regarding the importance of the regions outside the RGS domain, several reports document their requirement for the GAP function of RGS proteins. Thus, deletion of the non-RGS N-terminal region of RGS4 significantly reduced its potency in inhibition of carbachol-induced signaling (17.Zeng 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). Furthermore, although the core RGS domain of RGS16 by itself retained G protein binding and GAP activity in vitro, it was not functional in vivo without its N-terminal region, indicating the essential role of the N terminus in the function of RGS16 (18.Chen C. Lin S.C. FEBS Lett. 1998; 422: 359-362Crossref PubMed Scopus (36) Google Scholar). Finally, RGS9-2, a splice variant of retinal RGS9-1, being about 200 amino acids longer than its retinal isoform, dampened the Gi/o-coupled μu-opioid receptor signaling, whereas RGS9-1 did not (19.Rahman Z. Gold S.J. Potenza M.N. Cowan C.W. Ni Y.G. He W. Wensel T.G. Nestler E.J. J. Neurosci. 1999; 19: 2016-2026Crossref PubMed Google Scholar). These examples demonstrate the importance of the regions outside of RGS domain in the GAP function of RGS proteins. In this respect, RGS3 seems to be different from RGS4 (17.Zeng 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), RGS16 (18.Chen C. Lin S.C. FEBS Lett. 1998; 422: 359-362Crossref PubMed Scopus (36) Google Scholar), and RGS9 (19.Rahman Z. Gold S.J. Potenza M.N. Cowan C.W. Ni Y.G. He W. Wensel T.G. Nestler E.J. J. Neurosci. 1999; 19: 2016-2026Crossref PubMed Google Scholar), because its N-terminal region does not regulate the ability of RGS3 to bind G proteins (Fig. 1) and to inhibit G protein signaling (Figs. 2 and 3).RGS3T is Localized to the Nucleus and Induces ApoptosisThe most striking difference between RGS3 and RGS3T revealed in the present work is in their intracellular localization (Figs. 4, 5 B, and 6). Consistent with the previous study (1.Dulin N.O. Sorokin A. Reed E. Elliott S. Kehrl J. Dunn M.J. Mol. Cell. Biol. 1999; 19: 714-723Crossref PubMed Google Scholar), full length RGS3 was found in the cytoplasm of the intact cells. By contrast, RGS3T was localized predominantly in the nucleus and partially in the membrane.The membrane localization of RGS3T may provide a mechanism by which RGS3T regulates G protein-mediated signaling from seven transmembrane receptors in the plasma membrane. Compared with full length RGS3, which translocates to the membrane from the cytosol upon agonist stimulation (1.Dulin N.O. Sorokin A. Reed E. Elliott S. Kehrl J. Dunn M.J. Mol. Cell. Biol. 1999; 19: 714-723Crossref PubMed Google Scholar), RGS3T seems to associate with the membrane under basal conditions. This would suggest a higher potency of RGS3T in the regulation of G protein signaling. However, this hypothesis did not appear to be true, as evidenced from the present study (Figs. 2 and 3). Unfortunately, because of the high variability in the shape and size of transiently transfected cells, we were not able to assess the possibility of agonist-induced translocation of RGS3T as we previously described for full length RGS3 in stably transfected cells (1.Dulin N.O. Sorokin A. Reed E. Elliott S. Kehrl J. Dunn M.J. Mol. Cell. Biol. 1999; 19: 714-723Crossref PubMed Google Scholar). Stable expression of RGS3T appeared to be unattainable probably due to the ability of RGS3T to induce apoptosis (Fig. 8). Therefore, this issue will be addressed in future studies using inducible expression systems.The question also remains regarding the mechanism by which RGS3T is targeted to the membrane. For some RGS proteins, such as GAIP (20.De Vries L. Elenko E. Hubler L. Jones T.L.Z. Farquhar M.G. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 15203-15208Crossref PubMed Scopus (156) Google Scholar), RGSZ1 (21.Wang 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), RGS4, and RGS10 (22.Tu Y. Popov S. Slaughter C. Ross E.M. J. Biol. Chem. 1999; 247: 38260-38267Abstract Full Text Full Text PDF Scopus (87) Google Scholar), the membrane binding is proposed to be mediated by palmitoylation. Sequence analysis of RGS3T did not reveal the presence of putative sites for palmitoylation and myristoylation at its N terminus. However, RGS3T contains conserved cystein (Cys427 of full length RGS3) inside its RGS domain. In RGS4 and RGS10, analogous cysteins have been recently shown to be palmitoylated, which provided the membrane binding of these proteins (22.Tu Y. Popov S. Slaughter C. Ross E.M. J. Biol. Chem. 1999; 247: 38260-38267Abstract Full Text Full Text PDF Scopus (87) Google Scholar). Thus, the possibility of RGS3T palmitoylation still exists. Interestingly, palmitoylation of RGS16 is not necessary for its membrane localization and GAP activity in vitro but is somehow required for its function in vivo (23.Druey K.M. Ugur O. Caron J.M. Chen C.K. Backlund P.S. Jones T.L. J. Biol. Chem. 1999; 274: 18836-18842Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar). The membrane binding of RGS16 is provided by the non-RGS, N-terminal “membrane association domain,” which is also present in RGS4 and RGS5, and possibly contributes to the membrane localization of these proteins as well (24.Chen C. Seow K.T. Guo K. Yaw L.P. Lin S.C. J. Biol. Chem. 1999; 274: 19799-19806Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar). Based on our fractionation experiments, the N-terminal 65-amino acid region of RGS3T, outside the RGS domain, is responsible for membrane targeting of RGS3T, because its truncation eliminates membrane binding (Fig. 5 A). The active component of this region will be studied in the future.The nuclear localization of RGS3T is a new finding. Importantly, the cells expressing low levels of RGS3T, close to the limit of detection, still retained nuclear staining and revealed a punctate pattern of RGS3T localization within the nucleus (Fig. 6). This strongly suggests the nuclear localization of endogenous RGS3T. Moreover, the present study also provides a possible mechanism of nuclear localization of RGS3T by recruitment of its putative nuclear localization signal (NLS) sequences, truncation of which decreases nuclear distribution of RGS3T (Figs. 3 and 4). What is unclear is why full length RGS3, which obviously contains the same NLSs, is not targeted to the nucleus. One possibility is that the N-terminal tail of RGS3 somehow prevents nuclear localization, for example, by masking NLSs as a result of the three-dimensional structure. Alternatively, the N-terminal domain, which by itself contains structurally distinct regions (3.Druey K.M. Blumer K.J. Kang V.H. Kehrl J.H. Nature. 1996; 379: 742-746Crossref PubMed Scopus (403) Google Scholar), may keep RGS3 in the cytoplasm and antagonize the influence of NLSs. In support of this, RGS3-(1–379), which lacks the RGS domain but still retains putative NLSs at its C terminus, behaves similar to the full length RGS3 in terms of localization in the cytoplasm (1.Dulin N.O. Sorokin A. Reed E. Elliott S. Kehrl J. Dunn M.J. Mol. Cell. Biol. 1999; 19: 714-723Crossref PubMed Google Scholar).Another important question is the function of RGS3T in the nucleus. Some data supports the idea that an RGS3T nuclear function could be related to heterotrimeric G proteins, which have been also found to localize to the nucleus. Thus, an ADP-ribosylated 40-kDa protein recognized by the antibodies against Gαi1 and Gαi2 has been found in the nuclei prepared from the rat liver (25.Takei Y. Kurosu H. Takahashi K. Katada T. J. Biol. Chem. 1992; 267: 5085-5089Abstract Full Text PDF PubMed Google Scholar). In 3T3 fibroblasts, Gαi has been shown to translocate to the nucleus upon stimulation with epidermal growth factor, insulin, or thrombin, where it may regulate mitosis (26.Crouch M.F. Simson L. FASEB J. 1997; 11: 189-198Crossref PubMed Scopus (54) Google Scholar). Finally, Gαz has been reported to translocate to the nucleus of neuronal cells after being activated at the nerve terminal (27.Hendry I.A. Johanson S.O. Heydon K. Brain Res. 1995; 700: 157-163Crossref PubMed Scopus (20) Google Scholar). There are also examples of nuclear localization of G protein-coupled receptors. Functional prostaglandin E2receptors, EP3 and EP4, have been demonstrated in the nuclear envelope, where they affect intranuclear calcium transients and transcription of genes such as inducible nitric-oxide synthase (28.Bhattacharya M. Peri K. Ribeiro-da-Silva A. Almazan G. Shichi H. Hou X. Varma D.R. Chemtob S. J. Biol. Chem. 1999; 274: 15719-15724Abstract Full Text Full Text PDF PubMed Scopus (204) Google Scholar). Furthermore, type 1 angiotensin II receptors, generally coupled to Gq/11, have been shown to translocate to the nucleus upon stimulation of neuronal cells with angiotensin II (29.Lu D. Yang H. Shaw G. Raizada M.K. Endocrinology. 1998; 139: 365-375Crossref PubMed Google Scholar). Although Gαq and Gα11 have not been previously described in the nucleus, our fractionation experiments revealed the presence of a small amount of Gαq/11 in the nuclei of CHO cells (Fig.4 C). However, the significance of this is not clear.Alternatively, the nuclear function of RGS3T may be unrelated to G proteins. There is an increasing number of examples where RGS proteins recruiting their non-RGS domains are shown to bind targets distinct from heterotrimeric G proteins. Thus, protein kinase A-anchoring protein D-AKAP2 binds to the regulatory subunit of protein kinase A (30.Huang L.J. Durick K. Weiner J.A. Chun J. Taylor S.S. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 11184-11189Crossref PubMed Scopus (200) Google Scholar). A number of RGS proteins, such as RGS6, RGS7, RGS9, RGS11, and EGL-10, directly bind the β5 subunit of heterotrimeric G proteins (31.Cabrera J.L. de Freitas F. Satpaev D.K. Slepak V.Z. Biochem. Biophys. Res. Commun. 1998; 249: 898-902Crossref PubMed Scopus (113) Google Scholar, 32.Snow B.E. Krumins A.M. Brothers G.M. Lee S.F. Wall M.A. Chung S. Mangion J. Arya S. Gilman A.G. Siderovski D.P. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 13307-13312Crossref PubMed Scopus (228) Google Scholar). GAIP interacts with the PDZ domain of GIPC (GAIP-Interacting Protein C terminus) (33.De 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). Finally, but not lastly, a guanine nucleotide exchange factor for a small GTPase RhoA, p115RhoGEF, is also an RGS which binds to Gα12 and Gα13(34.Kozasa T. Jiang X. Hart M.J. Sternweis P.M. Singer W.D. Gilman A.G. Bollag G. Sternweis P.C. Science. 1998; 280: 2109-2111Crossref PubMed Scopus (735) Google Scholar) as well as a transducer of G13 signaling on RhoA (35.Hart M.J. Jiang X. Kozasa T. Roscoe W. Singer W.D. Gilman A.G. Sternweis P.C. Bollag G. Science. 1998; 280: 2112-2114Crossref PubMed Scopus (671) Google Scholar). Although alternative targets of RGS3T have not been described, the possibility exists and will be examined in future.In the present work, the nuclear localization of RGS3T was functionally linked to its ability to induce apoptosis (Fig. 6). This was based on our observations that RGS3T-transfected cells underwent cell rounding, membrane blebbing, and significant reduction in cell number and was confirmed by digoxigenin nucleotide labeling of DNA fragments in RGS3T-overexpressing cells. Importantly, this effect was specific for RGS3T, because RGS3 and RGS3C failed to induce apoptosis, although they were expressed to a similar extent. Inability of RGS3C to induce apoptosis, even though it was present in the nucleus in significant amounts, suggests the importance of the N terminus of RGS3T in this effect. The fact that RGS3T failed to induce apoptosis at low levels of expression suggests that it serves other function(s) in the nucleus, whereas apoptosis may reflect pathological conditions or certain developmental stages where RGS3T expression is up-regulated. This hypothesis will be examined in the future when detection of RGS3T with specific antibodies becomes possible. At present, the reported RGS3 antibodies have been generated against the N-terminal peptide of RGS3 (1.Dulin N.O. Sorokin A. Reed E. Elliott S. Kehrl J. Dunn M.J. Mol. Cell. Biol. 1999; 19: 714-723Crossref PubMed Google Scholar) or against purified RGS3 fusion proteins (7.Neill J.D. Duck L.W. Sellers J.C. Musgrove L.C. Scheschonka A. Druey K.M. Kehrl J.H. Endocrinology. 1997; 138: 843-846Crossref PubMed Scopus (86) Google Scholar, 36.Gruning W. Arnould T. Jochimsen F. Sellin L. Ananth S. Kim E. Walz G. Am. J. Physiol. 1999; 276: F535-F543PubMed Google Scholar), and their specificity to RGS3T was not examined. The significance of the present study is that it demonstrates and provides a mechanism for the nuclear localization of RGS3T and links it to apoptosis as one of its nuclear effects. RGS3 belongs to the family of the regulators of G protein signaling (RGS)1 which are united by a sequence homology of a core RGS domain and by function as GTPase-activating proteins (GAP) for the α subunit of heterotrimeric G proteins (2.Koelle M.R. Horvitz H.R. Cell. 1996; 84: 115-125Abstract Full Text Full Text PDF PubMed Scopus (475) Google Scholar, 3.Druey K.M. Blumer K.J. Kang V.H. Kehrl J.H. Nature. 1996; 379: 742-746Crossref PubMed Scopus (403) Google Scholar, 4.Hunt T.W. Fields T.A. Casey P.J. Peralta E.G. Nature. 1996; 383: 175-177Crossref PubMed Scopus (306) Google Scholar, 5.Watson N. Linder M.E. Druey K.M. Kehrl J.H. Blumer K.J. Nature. 1996; 383: 172-175Crossref PubMed Scopus (472) Google Scholar, 6.Berman D.M. Kozasa T. Gilman A.G. J. Biol. Chem. 1996; 271: 27209-27212Abstract Full Text Full Text PDF PubMed Scopus (298) Google Scholar). We and others have previously shown that RGS3 is a potent inhibitor of Gq/11-mediated signaling, such as gonadotropin-releasing hormone-induced production of inositol trisphosphate (IP3) (7.Neill J.D. Duck L.W. Sellers J.C. Musgrove L.C. Scheschonka A. Druey K.M. Kehrl J.H. Endocrinology. 1997; 138: 843-846Crossref PubMed Scopus (86) Google Scholar), and endothelin-1-induced calcium mobilization and activation of mitogen-activated protein (MAP) kinase (1.Dulin N.O. Sorokin A. Reed E. Elliott S. Kehrl J. Dunn M.J. Mol. Cell. Biol. 1999; 19: 714-723Crossref PubMed Google Scholar). One of the unique features of RGS3 is that it is normally a cytosolic protein, which translocates to the membrane and binds to Gαq/11 upon agonist stimulation. Translocation of RGS3 involves the recruitment of its N-terminal non-RGS domain, whereas C-terminal RGS domain provides interaction with G proteins (1.Dulin N.O. Sorokin A. Reed E. Elliott S. Kehrl J. Dunn M.J. Mol. Cell. Biol. 1999; 19: 714-723Crossref PubMed Google Scholar). Recently, a truncated isoform of RGS3, termed RGS3T, has been identified by polymerase chain reaction (PCR) analysis (8.Chatterjee 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). RGS3T is a much smaller molecule, which lacks a large portion of the N-terminal domain of RGS3 but retains a core RGS domain and a smaller (about 70 amino acids) N-terminal tail. RGS3T is expressed ubiquitously in various tissues, whereas full length RGS3 is more selectively expressed in heart, lung, testis, skeletal, and smooth muscle (3.Druey K.M. Blumer K.J. Kang V.H. Kehrl J.H. Nature. 1996; 379: 742-746Crossref PubMed Scopus (403) Google Scholar, 8.Chatterjee 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), suggesting the possibility of distinct function(s) of these proteins. Because RGS3T lacks a domain which is implicated in cytosolic localization of RGS3 and its agonist-induced recruitment to the membrane (1.Dulin N.O. Sorokin A. Reed E. Elliott S. Kehrl J. Dunn M.J. Mol. Cell. Biol. 1999; 19: 714-723Crossref PubMed Google Scholar), we sought to determine whether this deletion affects intracellular localization of RGS3T as well as its ability to bind Gαq/11 and to inhibit Gq/11-mediated signaling. Here we demonstrate that RGS3 and RGS3T are similar in their ability to bind Gαq/11 and inhibit Gq/11-mediated signaling but are different in their intracellular distribution, wherein full length RGS3 is localized in the cytoplasm, whereas RGS3T is localized predominantly in the nucleus and partially in the plasma membrane. Moreover, nuclear localization parallels with the unique ability of RGS3T to induce apoptosis, suggesting that RGS3T is not just a regulator of G protein signaling, but has other function(s) in the nucleus. DISCUSSIONIt is now established that the main function of RGS proteins is to stimulate GTPase activity of heterotrimeric G proteins, and therefore, to inhibit G protein signaling. RGS3 is a potent inhibitor of Gq/11-mediated signaling such as gonadotropin-releasing hormone-induced IP3 production (7.Neill J.D. Duck L.W. Sellers J.C. Musgrove L.C. Scheschonka A. Druey K.M. Kehrl J.H. Endocrinology. 1997; 138: 843-846Crossref PubMed Scopus (86) Google Scholar) and endothelin-1-induced calcium mobilization and MAP kinase activation (1.Dulin N.O. Sorokin A. Reed E. Elliott S. Kehrl J. Dunn M.J. Mol. Cell. Biol. 1999; 19: 714-723Crossref PubMed Google Scholar). Discovery of an endogenously expressed truncated isoform of RGS3, namely RGS3T (8.Chatterjee 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), raised several important questions about RGS3 function, two of which are addressed in the present study. First, does the N-terminal region regulate the ability of RGS3 to inhibit G protein signaling? Second, what is the difference between these two products of the same RGS3 gene (15.Chatterjee T.K. Eapen A. Kanis A.B. Fisher R.A. Genomics. 1997; 45: 429-433Crossref PubMed Scopus (14) Google Scholar), besides their differential tissue distribution (8.Chatterjee 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)?The N-terminal Domain of RGS3 Is Not Required for G Protein Inhibitory FunctionOur coimmunoprecipitation experiments (Fig.1) and signaling studies (Figs. 2 and 3) convincingly demonstrate that there is no difference between RGS3 and RGS3T in the binding to Gαq/11 and the ability to inhibit Gq/11-coupled endothelin signaling, suggesting that the N-terminal domain of RGS3 does not regulate its GAP function toward Gαq/11. This is different from previously published data (8.Chatterjee 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) demonstrating that RGS3T, but not full length RGS3, inhibited platelet-activating factor-induced IP3 production, which was presumably mediated by Gq/11. This discrepancy, however, may be explained by the recently published observation of receptor selectivity of RGS proteins, whereas RGS1, RGS4, and RGS16 were more potent to inhibit carbachol-dependent signaling than cholecystokinin, even though both agonists were coupled to Gq (16.Xu 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).Regarding the importance of the regions outside the RGS domain, several reports document their requirement for the GAP function of RGS proteins. Thus, deletion of the non-RGS N-terminal region of RGS4 significantly reduced its potency in i" @default.
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