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• W2040296648 abstract "The signaling cascades evoked by G protein-coupled receptors are a predominant mechanism of cellular communication. The regulators of G protein signaling (RGS) comprise a family of proteins that attenuate G protein-mediated signal transduction. Here we report the characterization of RGS13, the smallest member of the RGS family, which has been cloned from human lung. RGS13 has been found most abundantly in human tonsil, followed by thymus, lung, lymph node, and spleen. RGS13 is a GTPase-activating protein for Gαi and Gαo but not Gαs. RGS13 binds Gαq in the presence of aluminum magnesium fluoride, suggesting that it bears GTPase-activating protein activity toward Gαq. RGS13 blocks MAPK activity induced by Gαi- or Gαq-coupled receptors. RGS13 also attenuates GTPase-deficient Gαq (GαqQL) mediated cAMP response element activation but not transcription evoked by constitutively active Gα12 or Gα13. Surprisingly, RGS13 inhibits cAMP generation elicited by stimulation of the β2-adrenergic receptor. These data suggest that RGS13 may regulate Gαi-, Gαq-, and Gαs-coupled signaling cascades. The signaling cascades evoked by G protein-coupled receptors are a predominant mechanism of cellular communication. The regulators of G protein signaling (RGS) comprise a family of proteins that attenuate G protein-mediated signal transduction. Here we report the characterization of RGS13, the smallest member of the RGS family, which has been cloned from human lung. RGS13 has been found most abundantly in human tonsil, followed by thymus, lung, lymph node, and spleen. RGS13 is a GTPase-activating protein for Gαi and Gαo but not Gαs. RGS13 binds Gαq in the presence of aluminum magnesium fluoride, suggesting that it bears GTPase-activating protein activity toward Gαq. RGS13 blocks MAPK activity induced by Gαi- or Gαq-coupled receptors. RGS13 also attenuates GTPase-deficient Gαq (GαqQL) mediated cAMP response element activation but not transcription evoked by constitutively active Gα12 or Gα13. Surprisingly, RGS13 inhibits cAMP generation elicited by stimulation of the β2-adrenergic receptor. These data suggest that RGS13 may regulate Gαi-, Gαq-, and Gαs-coupled signaling cascades. G protein signaling is an effective mechanism of cellular communication during both physiological and pathological conditions (1.Beadling C. Druey K.M. Richter G. Kehrl J.H. Smith K.A. J. Immunol. 1999; 162: 2677-2682PubMed Google Scholar, 2.Johnson, E. N., and Druey, K. M. (2002) J. Allergy Clin. Immunol., in pressGoogle Scholar, 3.Gilman A.G. Annu. Rev. Biochem. 1987; 56: 615-649Crossref PubMed Scopus (4682) Google Scholar). Regulators of G protein-signaling (RGS) 1The abbreviations used are: RGSregulator of G protein signalingGAPGTPase-activating proteinAMFaluminum magnesium fluorideMAPKmitogen-activated protein kinaseERKextracellular signal-regulated kinaseCREcAMP response elementSREserum response elementGFPgreen fluorescent proteinPCRpolymerase chain reactionGTPγSguanosine 5′-O-(thiotriphosphate)1The abbreviations used are: RGSregulator of G protein signalingGAPGTPase-activating proteinAMFaluminum magnesium fluorideMAPKmitogen-activated protein kinaseERKextracellular signal-regulated kinaseCREcAMP response elementSREserum response elementGFPgreen fluorescent proteinPCRpolymerase chain reactionGTPγSguanosine 5′-O-(thiotriphosphate) proteins are a relatively new family of proteins that attenuate G protein-mediated pathways by acting as GTPase-activating proteins (GAPs) for Gα subunits (4.Ross E.M. Wilkie T.M. Annu. Rev. Biochem. 2000; 69: 795-827Crossref PubMed Scopus (916) Google Scholar). RGS binding stabilizes a Gα conformation that favors hydrolysis of GTP to GDP, which hastens the termination of active Gα (5.Tesmer J.J. Berman D.M. Gilman A.G. Sprang S.R. Cell. 1997; 89: 251-261Abstract Full Text Full Text PDF PubMed Scopus (680) Google Scholar). The second mechanism of G protein inhibition by RGS proteins is effector antagonism in which the RGS protein binds GTP-bound Gαq and prevents Gαq/effector interaction (6.Hepler 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).RGS proteins share a common domain, the RGS box. This motif of 120 amino acids is highly conserved in all RGS family members and conveys the ability of RGSs to bind G proteins (7.De Vries L. Zheng B. Fischer T. Elenko E. Farquhar M.G. Annu. Rev. Pharmacol. Toxicol. 2000; 40: 235-271Crossref PubMed Scopus (504) Google Scholar). Although many RGS proteins exhibit GAP activity toward members of the Gαi (8.Berman D.M. Wilkie T.M. Gilman A.G. Cell. 1996; 86: 445-452Abstract Full Text Full Text PDF PubMed Scopus (648) Google Scholar, 9.Watson N. Linder M.E. Druey K.M. Kehrl J.H. Blumer K.J. Nature. 1996; 383: 172-175Crossref PubMed Scopus (472) Google Scholar) and Gαq (10.Mukhopadhyay S. Ross E.M. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 9539-9544Crossref PubMed Scopus (152) Google Scholar) families, only one RGS protein has been shown to possess GAP activity specifically toward Gαs (11.Zheng B. Ma Y.C. Ostrom R.S. Lavoie C. Gill G.N. Insel P.A. Huang X.Y. Farquhar M.G. Science. 2001; 294: 1939-1942Crossref PubMed Scopus (195) Google Scholar). In addition to the classical RGS family, an additional group of proteins exists termed the RhoGEF RGSs (rgRGS) (12.Chen Z. Wells C.D. Sternweis P.C. Sprang S.R. Nat. Struct. Biol. 2001; 8: 805-809Crossref PubMed Scopus (50) Google Scholar, 13.Longenecker K.L. Lewis M.E. Chikumi H. Gutkind J.S. Derewenda Z.S. Structure. 2001; 9: 559-569Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar). These proteins, which contain a highly diverged RGS homology domain, act as GAPs specifically for Gα12/13 and also stimulate GTP binding by Rho family members.Although all RGS proteins contain the conserved RGS box, they display significant variability in sequences outside of this region. For example, a cysteine string found in RGS-GAIP and RGS20 is a site of palmitoylation, which may assist in membrane anchorage (14.De Vries L. Elenko E. Hubler L. Jones T.L. Farquhar M.G. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 15203-15208Crossref PubMed Scopus (156) Google Scholar). The Gαs GAP RGS-PX1 also contains a Phox domain that may be involved in intracellular trafficking (11.Zheng B. Ma Y.C. Ostrom R.S. Lavoie C. Gill G.N. Insel P.A. Huang X.Y. Farquhar M.G. Science. 2001; 294: 1939-1942Crossref PubMed Scopus (195) Google Scholar).Unique motifs in certain RGSs may modify the G protein GTPase cycle or G protein effector stimulation in additional ways. RGS14, for example, contains a “GoLoco” motif that inhibits guanine nucleotide dissociation on Gαi (15.Siderovski D.P. Strockbine B. Behe C.I. Crit. Rev. Biochem. Mol. Biol. 1999; 34: 215-251Crossref PubMed Scopus (96) Google Scholar). Most RGS proteins do not exhibit GAP activity toward Gαs; however, a short form of RGS3 (RGS3T) blocks calcitonin gene-related peptide-induced cAMP generation (16.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). More recently, it was shown that RGS2 directly inhibits some isoforms of adenylyl cyclase, independently of Gαs (17.Sinnarajah S. Dessauer C.W. Srikumar D. Chen J. Yuen J. Yilma S. Dennis J.C. Morrison E.E. Vodyanoy V. Kehrl J.H. Nature. 2001; 409: 1051-1055Crossref PubMed Scopus (209) Google Scholar), suggesting that RGS proteins can affect signaling through interactions with proteins downstream of Gα.Here we report the characterization of RGS13, the smallest RGS found in mammalian tissues. Human RGS13 was cloned from lung cDNA and is expressed most prominently in immune tissues such as tonsil, thymus, lymph node, and spleen. As with other RGS proteins, recombinant RGS13 exhibits GAP activity toward Gαi family members and not Gαs. RGS13 binds Gαq in the presence of AMF, implying that it acts as a GAP for Gαq as well. Transfected RGS13 blunts MAPK activation evoked by either Gαq- or Gαi-coupled receptors. In addition, it blocks GαqQL-induced CRE-dependent transcription, consistent with a potential role for RGS13 as an effector antagonist of Gαq. RGS13 inhibits receptor-stimulated cAMP generation, suggesting that it regulates Gαs signaling despite its lack of Gαs GAP activity. Thus, RGS13 may regulate G protein-mediated processes in the lung and immune system.DISCUSSIONThis study represents the initial characterization of RGS13 expression and function. RGS13 is most abundant in human tonsil followed by thymus, lymph node, lung, and spleen with low levels or a lack of expression in various other tissues. Ectopically expressed GFP-RGS13 localizes in both membrane and nuclear fractions of 293T cells with a very small portion of RGS13 in the cytosol. Similar to other classical RGS proteins, RGS13 possesses GAP activity toward Gαi family members and inhibits signaling evoked by Gαi-coupled receptors. RGS13 binds to Gαqin the presence of AMF, suggesting that RGS13 is a GAP for Gαq. In addition to its GAP activity, RGS13 likely blocks Gαq signaling by effector antagonism as well because GTPase-deficient GαqQL-mediated CRE stimulation is attenuated by RGS13. RGS13 is not a GAP for Gαs, although it inhibits cAMP generation induced by stimulation of a Gαs-coupled receptor.The expression pattern of RGS family members is highly variable and may contribute to physiological specificity of RGS proteins that share similar biochemical properties. RGS16 is expressed predominantly in the retina, pituitary, and liver (22.Chen C. Zheng B. Han J. Lin S.C. J. Biol. Chem. 1997; 272: 8679-8685Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar, 23.Chen C.K. Wieland T. Simon M.I. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 12885-12889Crossref PubMed Scopus (124) Google Scholar, 24.Snow B.E. Antonio L. Suggs S. Siderovski D.P. Gene. 1998; 206: 247-253Crossref PubMed Scopus (32) Google Scholar). RGS18 is expressed in hematopoietic tissues and lung (25.Park I.K. Klug C.A. Li K. Jerabek L. Li L. Nanamori M. Neubig R.R. Hood L. Weissman I.L. Clarke M.F. J. Biol. Chem. 2001; 276: 915-923Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar, 26.Yowe D. Weich N. Prabhudas M. Poisson L. Errada P. Kapeller R. Yu K. Faron L. Shen M. Cleary J. Wilkie T.M. Gutierrez-Ramos C. Hodge M.R. Biochem. J. 2001; 359: 109-118Crossref PubMed Scopus (51) Google Scholar), whereas RGS2 and RGS3 are ubiquitously expressed (20.Druey K.M. Blumer K.J. Kang V.H. Kehrl J.H. Nature. 1996; 379: 742-746Crossref PubMed Scopus (404) Google Scholar, 27.Siderovski D.P. Heximer S.P. Forsdyke D.R. DNA Cell Biol. 1994; 13: 125-147Crossref PubMed Scopus (107) Google Scholar). RGS13 expression is highest in tonsil (about 6.8 × 106 times greater than CD4+ cells). There is 10-fold less RGS13 in thymus, lymph node, lung, and spleen, with much lower levels of RGS13 detected in other tissues such as brain, which is a reservoir for many of the RGS family members (28.Gold S.J. Ni Y.G. Dohlman H.G. Nestler E.J. J. Neurosci. 1997; 17: 8024-8037Crossref PubMed Google Scholar,29.Grafstein-Dunn E. Young K.H. Cockett M.I. Khawaja X.Z. Brain Res. Mol. Brain Res. 2001; 88: 113-123Crossref PubMed Scopus (74) Google Scholar). In contrast to earlier reports describing RGS13 expression in brain (29.Grafstein-Dunn E. Young K.H. Cockett M.I. Khawaja X.Z. Brain Res. Mol. Brain Res. 2001; 88: 113-123Crossref PubMed Scopus (74) Google Scholar, 30.Norlin E.M. Berghard A. Mol. Cell. Neurosci. 2001; 17: 872-882Crossref PubMed Scopus (28) Google Scholar), we failed to detect any RGS13 in brain cDNA. This discrepancy could be explained by localization of RGS proteins within the brain; that is, certain areas of the brain enriched with RGS13 may be underrepresented in total brain cDNA.Using the RGS13 peptide antibodies, we evaluated endogenous RGS13 expression in human lung, spleen, liver, pancreas, kidney, heart, and brain (data not shown). We concluded that although the peptide antibodies were able to detect recombinant RGS13 protein and not other closely related RGS proteins of similar size, the sensitivity of the peptide antibodies was insufficient to identify endogenous protein in tissue lysates by immunoblotting. To address this issue, we have initiated generation of knockout mice in which a LacZreporter gene is placed under control of the RGS13 promoter, which should allow us to better define the anatomical and developmental expression of RGS13.Transfected RGS13 localizes in the cell membrane and the nucleus. Several RGS proteins have been localized to the cell membrane (31.Heximer S.P. Lim H. Bernard J.L. Blumer K.J. J. Biol. Chem. 2001; 276: 14195-14203Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar) and have undergone post-translational modifications such as palmitoylation, which may assist in membrane anchorage (32.Peppelenbosch M.P. Tertoolen L.G. Vries-Smits A.M. Qiu R.G. M'Rabet L. Symons M.H. de Laat S.W. Bos J.L. J. Biol. Chem. 1996; 271: 7883-7886Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar, 33.Druey K.M. Ugur O. Caron J.M. Chen C.K. Backland P.S. Jones T.L. J. Biol. Chem. 1999; 274: 18836-18842Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar). RGS13 lacks several of the N-terminal residues that adopt an α-helical conformation in closely related RGSs such as RGS4, 5, and 16 and facilitate direct interaction with anionic membrane phospholipids (34.Tu Y. Woodson J. Ross E.M. J. Biol. Chem. 2001; 276: 20160-20166Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar, 35.Bernstein L.S. Grillo A.A. Loranger S.S. Linder M.E. J. Biol. Chem. 2000; 275: 18520-18526Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar). It will be interesting to determine whether palmitoylation of RGS13 is required for its membrane localization or whether another mechanism of membrane targeting is responsible. It has been reported that RGS2 and RGS10 are localized in the nucleus (36.Chatterjee T.K. Fisher R.A. J. Biol. Chem. 2000; 275: 24013-24021Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar). We show here that transfected RGS13 is also located in the nucleus, although the functional significance of nuclear RGS expression has yet to be determined.RGS13 functions similarly to many RGS proteins in that it bears GAP activity toward members of the Gαi family and likely has GAP activity toward Gαq as well. RGS13 may be an effector antagonist for Gαq as demonstrated by its ability to block GαqQL-mediated CRE activation. Although one could postulate that the capacity of RGS13 to block cAMP generation may have influenced the CRE-Luciferase reporter assay, the lack of significant decrease in either basal cAMP levels or basal CRE-Luciferase activity suggests that this mechanism would not account for the inhibition of GαqQL-stimulated CRE-Luciferase by RGS13.RGS13 blunted isoproterenol-evoked increases in cAMP but exhibited no GAP activity toward Gαs. There are several possible explanations for this result. An interaction between RGS9 and a phosphodiesterase has been reported (37.He W. Cowan C.W. Wensel T.G. Neuron. 1998; 20: 95-102Abstract Full Text Full Text PDF PubMed Scopus (305) Google Scholar). RGS13 may enhance cAMP metabolism by stimulating a phosphodiesterase, resulting in a net decrease in cAMP concentration. In that case one would expect to see decreased basal levels of cAMP, which we failed to detect. A truncated form of RGS3 (RGS3T) was shown to decrease receptor-mediated cAMP generation without decreasing basal cAMP levels (16.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 authors suggested that it was unlikely that RGS3T blocked adenylyl cyclase directly. Given the similarity in our results, RGS13 and RGS3T may be acting via the same uncharacterized mechanism. Another possibility is that like RGS2 (17.Sinnarajah S. Dessauer C.W. Srikumar D. Chen J. Yuen J. Yilma S. Dennis J.C. Morrison E.E. Vodyanoy V. Kehrl J.H. Nature. 2001; 409: 1051-1055Crossref PubMed Scopus (209) Google Scholar) RGS13 may block adenylyl cyclase directly. It has been demonstrated that RGS1, RGS2, and RGS3 (but not RGS4 or RGS5) block GTPγS/odorant-mediated cAMP generation in olfactory epithelium membranes. In addition, RGS2 was shown to block forskolin-evoked increases in cAMP (17.Sinnarajah S. Dessauer C.W. Srikumar D. Chen J. Yuen J. Yilma S. Dennis J.C. Morrison E.E. Vodyanoy V. Kehrl J.H. Nature. 2001; 409: 1051-1055Crossref PubMed Scopus (209) Google Scholar), indicating that some RGS proteins regulate certain isoforms of adenylyl cyclase directly. Interestingly, RGS2 failed to attenuate cAMP production induced by stimulation of endogenous adenylyl cyclase in HEK-293 cells (17.Sinnarajah S. Dessauer C.W. Srikumar D. Chen J. Yuen J. Yilma S. Dennis J.C. Morrison E.E. Vodyanoy V. Kehrl J.H. Nature. 2001; 409: 1051-1055Crossref PubMed Scopus (209) Google Scholar). In contrast we found that RGS13 inhibited cAMP generation in these cells, suggesting that RGS2 and RGS13 may regulate different isoforms of adenylyl cyclase. Given that the only identified domain in RGS13 is the RGS box, it will be of interest to examine whether the RGS box is the region that confers the ability of RGS proteins to inhibit cAMP production. If so, why then do RGS4 and RGS5 lack the ability to regulate this process?Functional analysis of RGS13 reported here was derived from cells transfected with GFP-RGS13. We utilized GFP-RGS13 only after several failed attempts to express RGS13 in untagged or His-tagged forms. Although we used the GFP vector or other GFP-tagged RGS proteins as controls, the difficulty in expressing untagged RGS13 is of interest. Future experiments are required to determine whether the difficulty in detecting untagged RGS13 is a result of such anomalies as unstable transcription or translation. This explanation seems unlikely because we could easily detect in vitro-translated untagged RGS13 in rabbit reticulocyte lysates (data not shown). Alternatively, RGS13 may require a binding partner or modification for stable expression in mammalian cells. An example of this type of protein instability is Gβ5, which requires expression of RGS9 in the brain (18.Chen C.K. Burns M.E. He W. Wensel T.G. Baylor D.A. Simon M.I. Nature. 2000; 403: 557-560Crossref PubMed Scopus (330) Google Scholar). These studies might also explain the difficulty in detecting endogenous levels of RGS13 by immunoblotting.Through the characterization of RGS13 we have added another piece to the puzzle of the RGS family. Unlike many RGS proteins RGS13 has no identified domain other than the RGS box, but because of its high expression in the immune system and lung and its ability to block Gαi, Gαq, and cAMP generation, the biological niche of RGS13 might be to regulate specific G protein-dependent signal transduction pathways in these regions. The physiological relevance of RGS13 and other RGS proteins is slowly being evaluated through the use of targeted gene disruption and expression of transgenes in mice. Through these lines of experimentation we hope to better understand the function of RGS13 and its relevance to health and disease. G protein signaling is an effective mechanism of cellular communication during both physiological and pathological conditions (1.Beadling C. Druey K.M. Richter G. Kehrl J.H. Smith K.A. J. Immunol. 1999; 162: 2677-2682PubMed Google Scholar, 2.Johnson, E. N., and Druey, K. M. (2002) J. Allergy Clin. Immunol., in pressGoogle Scholar, 3.Gilman A.G. Annu. Rev. Biochem. 1987; 56: 615-649Crossref PubMed Scopus (4682) Google Scholar). Regulators of G protein-signaling (RGS) 1The abbreviations used are: RGSregulator of G protein signalingGAPGTPase-activating proteinAMFaluminum magnesium fluorideMAPKmitogen-activated protein kinaseERKextracellular signal-regulated kinaseCREcAMP response elementSREserum response elementGFPgreen fluorescent proteinPCRpolymerase chain reactionGTPγSguanosine 5′-O-(thiotriphosphate)1The abbreviations used are: RGSregulator of G protein signalingGAPGTPase-activating proteinAMFaluminum magnesium fluorideMAPKmitogen-activated protein kinaseERKextracellular signal-regulated kinaseCREcAMP response elementSREserum response elementGFPgreen fluorescent proteinPCRpolymerase chain reactionGTPγSguanosine 5′-O-(thiotriphosphate) proteins are a relatively new family of proteins that attenuate G protein-mediated pathways by acting as GTPase-activating proteins (GAPs) for Gα subunits (4.Ross E.M. Wilkie T.M. Annu. Rev. Biochem. 2000; 69: 795-827Crossref PubMed Scopus (916) Google Scholar). RGS binding stabilizes a Gα conformation that favors hydrolysis of GTP to GDP, which hastens the termination of active Gα (5.Tesmer J.J. Berman D.M. Gilman A.G. Sprang S.R. Cell. 1997; 89: 251-261Abstract Full Text Full Text PDF PubMed Scopus (680) Google Scholar). The second mechanism of G protein inhibition by RGS proteins is effector antagonism in which the RGS protein binds GTP-bound Gαq and prevents Gαq/effector interaction (6.Hepler 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). regulator of G protein signaling GTPase-activating protein aluminum magnesium fluoride mitogen-activated protein kinase extracellular signal-regulated kinase cAMP response element serum response element green fluorescent protein polymerase chain reaction guanosine 5′-O-(thiotriphosphate) regulator of G protein signaling GTPase-activating protein aluminum magnesium fluoride mitogen-activated protein kinase extracellular signal-regulated kinase cAMP response element serum response element green fluorescent protein polymerase chain reaction guanosine 5′-O-(thiotriphosphate) RGS proteins share a common domain, the RGS box. This motif of 120 amino acids is highly conserved in all RGS family members and conveys the ability of RGSs to bind G proteins (7.De Vries L. Zheng B. Fischer T. Elenko E. Farquhar M.G. Annu. Rev. Pharmacol. Toxicol. 2000; 40: 235-271Crossref PubMed Scopus (504) Google Scholar). Although many RGS proteins exhibit GAP activity toward members of the Gαi (8.Berman D.M. Wilkie T.M. Gilman A.G. Cell. 1996; 86: 445-452Abstract Full Text Full Text PDF PubMed Scopus (648) Google Scholar, 9.Watson N. Linder M.E. Druey K.M. Kehrl J.H. Blumer K.J. Nature. 1996; 383: 172-175Crossref PubMed Scopus (472) Google Scholar) and Gαq (10.Mukhopadhyay S. Ross E.M. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 9539-9544Crossref PubMed Scopus (152) Google Scholar) families, only one RGS protein has been shown to possess GAP activity specifically toward Gαs (11.Zheng B. Ma Y.C. Ostrom R.S. Lavoie C. Gill G.N. Insel P.A. Huang X.Y. Farquhar M.G. Science. 2001; 294: 1939-1942Crossref PubMed Scopus (195) Google Scholar). In addition to the classical RGS family, an additional group of proteins exists termed the RhoGEF RGSs (rgRGS) (12.Chen Z. Wells C.D. Sternweis P.C. Sprang S.R. Nat. Struct. Biol. 2001; 8: 805-809Crossref PubMed Scopus (50) Google Scholar, 13.Longenecker K.L. Lewis M.E. Chikumi H. Gutkind J.S. Derewenda Z.S. Structure. 2001; 9: 559-569Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar). These proteins, which contain a highly diverged RGS homology domain, act as GAPs specifically for Gα12/13 and also stimulate GTP binding by Rho family members. Although all RGS proteins contain the conserved RGS box, they display significant variability in sequences outside of this region. For example, a cysteine string found in RGS-GAIP and RGS20 is a site of palmitoylation, which may assist in membrane anchorage (14.De Vries L. Elenko E. Hubler L. Jones T.L. Farquhar M.G. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 15203-15208Crossref PubMed Scopus (156) Google Scholar). The Gαs GAP RGS-PX1 also contains a Phox domain that may be involved in intracellular trafficking (11.Zheng B. Ma Y.C. Ostrom R.S. Lavoie C. Gill G.N. Insel P.A. Huang X.Y. Farquhar M.G. Science. 2001; 294: 1939-1942Crossref PubMed Scopus (195) Google Scholar). Unique motifs in certain RGSs may modify the G protein GTPase cycle or G protein effector stimulation in additional ways. RGS14, for example, contains a “GoLoco” motif that inhibits guanine nucleotide dissociation on Gαi (15.Siderovski D.P. Strockbine B. Behe C.I. Crit. Rev. Biochem. Mol. Biol. 1999; 34: 215-251Crossref PubMed Scopus (96) Google Scholar). Most RGS proteins do not exhibit GAP activity toward Gαs; however, a short form of RGS3 (RGS3T) blocks calcitonin gene-related peptide-induced cAMP generation (16.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). More recently, it was shown that RGS2 directly inhibits some isoforms of adenylyl cyclase, independently of Gαs (17.Sinnarajah S. Dessauer C.W. Srikumar D. Chen J. Yuen J. Yilma S. Dennis J.C. Morrison E.E. Vodyanoy V. Kehrl J.H. Nature. 2001; 409: 1051-1055Crossref PubMed Scopus (209) Google Scholar), suggesting that RGS proteins can affect signaling through interactions with proteins downstream of Gα. Here we report the characterization of RGS13, the smallest RGS found in mammalian tissues. Human RGS13 was cloned from lung cDNA and is expressed most prominently in immune tissues such as tonsil, thymus, lymph node, and spleen. As with other RGS proteins, recombinant RGS13 exhibits GAP activity toward Gαi family members and not Gαs. RGS13 binds Gαq in the presence of AMF, implying that it acts as a GAP for Gαq as well. Transfected RGS13 blunts MAPK activation evoked by either Gαq- or Gαi-coupled receptors. In addition, it blocks GαqQL-induced CRE-dependent transcription, consistent with a potential role for RGS13 as an effector antagonist of Gαq. RGS13 inhibits receptor-stimulated cAMP generation, suggesting that it regulates Gαs signaling despite its lack of Gαs GAP activity. Thus, RGS13 may regulate G protein-mediated processes in the lung and immune system. DISCUSSIONThis study represents the initial characterization of RGS13 expression and function. RGS13 is most abundant in human tonsil followed by thymus, lymph node, lung, and spleen with low levels or a lack of expression in various other tissues. Ectopically expressed GFP-RGS13 localizes in both membrane and nuclear fractions of 293T cells with a very small portion of RGS13 in the cytosol. Similar to other classical RGS proteins, RGS13 possesses GAP activity toward Gαi family members and inhibits signaling evoked by Gαi-coupled receptors. RGS13 binds to Gαqin the presence of AMF, suggesting that RGS13 is a GAP for Gαq. In addition to its GAP activity, RGS13 likely blocks Gαq signaling by effector antagonism as well because GTPase-deficient GαqQL-mediated CRE stimulation is attenuated by RGS13. RGS13 is not a GAP for Gαs, although it inhibits cAMP generation induced by stimulation of a Gαs-coupled receptor.The expression pattern of RGS family members is highly variable and may contribute to physiological specificity of RGS proteins that share similar biochemical properties. RGS16 is expressed predominantly in the retina, pituitary, and liver (22.Chen C. Zheng B. Han J. Lin S.C. J. Biol. Chem. 1997; 272: 8679-8685Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar, 23.Chen C.K. Wieland T. Simon M.I. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 12885-12889Crossref PubMed Scopus (124) Google Scholar, 24.Snow B.E. Antonio L. Suggs S. Siderovski D.P. Gene. 1998; 206: 247-253Crossref PubMed Scopus (32) Google Scholar). RGS18 is expressed in hematopoietic tissues and lung (25.Park I.K. Klug C.A. Li K. Jerabek L. Li L. Nanamori M. Neubig R.R. Hood L. Weissman I.L. Clarke M.F. J. Biol. Chem. 2001; 276: 915-923Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar, 26.Yowe D. Weich N. Prabhudas M. Poisson L. Errada P. Kapeller R. Yu K. Faron L. Shen M. Cleary J. Wilkie T.M. Gutierrez-Ramos C. Hodge M.R. Biochem. J. 2001; 359: 109-118Crossref PubMed Scopus (51) Google Scholar), whereas RGS2 and RGS3 are ubiquitously expressed (20.Druey K.M. Blumer K.J. Kang V.H. Kehrl J.H. Nature. 1996; 379: 742-746Crossref PubMed Scopus (404) Google Scholar, 27.Siderovski D.P. Heximer S.P. Forsdyke D.R. DNA Cell Biol. 1994; 13: 125-147Crossref PubMed Scopus (107) Google Scholar). RGS13 expression is highest in tonsil (about 6.8 × 106 times greater than CD4+ cells). There is 10-fold less RGS13 in thymus, lymph node, lung, and spleen, with much lower levels of RGS13 detected in other tissues such as brain, which is a reservoir for many of the RGS family members (28.Gold S.J. Ni Y.G. Dohlman H.G. Nestler E.J. J. Neurosci. 1997; 17: 8024-8037Crossref PubMed Google Scholar,29.Grafstein-Dunn E. Young K.H. Cockett M.I. Khawaja X.Z. Brain Res. Mol. Brain Res. 2001; 88: 113-123Crossref PubMed Scopus (74) Google Scholar). In contrast to earlier reports describing RGS13 expression in brain (29.Grafstein-Dunn E. Young K.H. Cockett M.I. Khawaja X.Z. Brain Res. Mol. Brain Res. 2001; 88: 113-123Crossref PubMed Scopus (74) Google Scholar, 30.Norlin E.M. Berghard A. Mol. Cell. Neurosci. 2001; 17: 872-882Crossref PubMed Scopus (28) Google Scholar), we failed to detect any RGS13 in brain cDNA. This discrepancy could be explained by localization of RGS proteins within the brain; that is, certain areas of the brain enriched with RGS13 may be underrepresented in total brain cDNA.Using the RGS13 peptide antibodies, we evaluated endogenous RGS13 expression in human lung, spleen, liver, pancreas, kidney, heart, and brain (data not shown). We concluded that although the peptide antibodies were able to detect recombinant RGS13 protein and not other closely related RGS proteins of similar size, the sensitivity of the peptide antibodies was insufficient to identify endogenous protein in tissue lysates by immunoblotting. To address this issue, we have initiated generation of knockout mice in which a LacZreporter gene is placed under control of the RGS13 promoter, which should allow us to better define the anatomical and developmental expression of RGS13.Transfected RGS13 localizes in the cell membrane and the nucleus. Several RGS proteins have been localized to the cell membrane (31.Heximer S.P. Lim H. Bernard J.L. Blumer K.J. J. Biol. Chem. 2001; 276: 14195-14203Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar) and have undergone post-translational modifications such as palmitoylation, which may assist in membrane anchorage (32.Peppelenbosch M.P. Tertoolen L.G. Vries-Smits A.M. Qiu R.G. M'Rabet L. Symons M.H. de Laat S.W. Bos J.L. J. Biol. Chem. 1996; 271: 7883-7886Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar, 33.Druey K.M. Ugur O. Caron J.M. Chen C.K. Backland P.S. Jones T.L. J. Biol. Chem. 1999; 274: 18836-18842Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar). RGS13 lacks several of the N-terminal residues that adopt an α-helical conformation in closely related RGSs such as RGS4, 5, and 16 and facilitate direct interaction with anionic membrane phospholipids (34.Tu Y. Woodson J. Ross E.M. J. Biol. Chem. 2001; 276: 20160-20166Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar, 35.Bernstein L.S. Grillo A.A. Loranger S.S. Linder M.E. J. Biol. Chem. 2000; 275: 18520-18526Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar). It will be interesting to determine whether palmitoylation of RGS13 is required for its membrane localization or whether another mechanism of membrane targeting is responsible. It has been reported that RGS2 and RGS10 are localized in the nucleus (36.Chatterjee T.K. Fisher R.A. J. Biol. Chem. 2000; 275: 24013-24021Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar). We show here that transfected RGS13 is also located in the nucleus, although the functional significance of nuclear RGS expression has yet to be determined.RGS13 functions similarly to many RGS proteins in that it bears GAP activity toward members of the Gαi family and likely has GAP activity toward Gαq as well. RGS13 may be an effector antagonist for Gαq as demonstrated by its ability to block GαqQL-mediated CRE activation. Although one could postulate that the capacity of RGS13 to block cAMP generation may have influenced the CRE-Luciferase reporter assay, the lack of significant decrease in either basal cAMP levels or basal CRE-Luciferase activity suggests that this mechanism would not account for the inhibition of GαqQL-stimulated CRE-Luciferase by RGS13.RGS13 blunted isoproterenol-evoked increases in cAMP but exhibited no GAP activity toward Gαs. There are several possible explanations for this result. An interaction between RGS9 and a phosphodiesterase has been reported (37.He W. Cowan C.W. Wensel T.G. Neuron. 1998; 20: 95-102Abstract Full Text Full Text PDF PubMed Scopus (305) Google Scholar). RGS13 may enhance cAMP metabolism by stimulating a phosphodiesterase, resulting in a net decrease in cAMP concentration. In that case one would expect to see decreased basal levels of cAMP, which we failed to detect. A truncated form of RGS3 (RGS3T) was shown to decrease receptor-mediated cAMP generation without decreasing basal cAMP levels (16.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 authors suggested that it was unlikely that RGS3T blocked adenylyl cyclase directly. Given the similarity in our results, RGS13 and RGS3T may be acting via the same uncharacterized mechanism. Another possibility is that like RGS2 (17.Sinnarajah S. Dessauer C.W. Srikumar D. Chen J. Yuen J. Yilma S. Dennis J.C. Morrison E.E. Vodyanoy V. Kehrl J.H. Nature. 2001; 409: 1051-1055Crossref PubMed Scopus (209) Google Scholar) RGS13 may block adenylyl cyclase directly. It has been demonstrated that RGS1, RGS2, and RGS3 (but not RGS4 or RGS5) block GTPγS/odorant-mediated cAMP generation in olfactory epithelium membranes. In addition, RGS2 was shown to block forskolin-evoked increases in cAMP (17.Sinnarajah S. Dessauer C.W. Srikumar D. Chen J. Yuen J. Yilma S. Dennis J.C. Morrison E.E. Vodyanoy V. Kehrl J.H. Nature. 2001; 409: 1051-1055Crossref PubMed Scopus (209) Google Scholar), indicating that some RGS proteins regulate certain isoforms of adenylyl cyclase directly. Interestingly, RGS2 failed to attenuate cAMP production induced by stimulation of endogenous adenylyl cyclase in HEK-293 cells (17.Sinnarajah S. Dessauer C.W. Srikumar D. Chen J. Yuen J. Yilma S. Dennis J.C. Morrison E.E. Vodyanoy V. Kehrl J.H. Nature. 2001; 409: 1051-1055Crossref PubMed Scopus (209) Google Scholar). In contrast we found that RGS13 inhibited cAMP generation in these cells, suggesting that RGS2 and RGS13 may regulate different isoforms of adenylyl cyclase. Given that the only identified domain in RGS13 is the RGS box, it will be of interest to examine whether the RGS box is the region that confers the ability of RGS proteins to inhibit cAMP production. If so, why then do RGS4 and RGS5 lack the ability to regulate this process?Functional analysis of RGS13 reported here was derived from cells transfected with GFP-RGS13. We utilized GFP-RGS13 only after several failed attempts to express RGS13 in untagged or His-tagged forms. Although we used the GFP vector or other GFP-tagged RGS proteins as controls, the difficulty in expressing untagged RGS13 is of interest. Future experiments are required to determine whether the difficulty in detecting untagged RGS13 is a result of such anomalies as unstable transcription or translation. This explanation seems unlikely because we could easily detect in vitro-translated untagged RGS13 in rabbit reticulocyte lysates (data not shown). Alternatively, RGS13 may require a binding partner or modification for stable expression in mammalian cells. An example of this type of protein instability is Gβ5, which requires expression of RGS9 in the brain (18.Chen C.K. Burns M.E. He W. Wensel T.G. Baylor D.A. Simon M.I. Nature. 2000; 403: 557-560Crossref PubMed Scopus (330) Google Scholar). These studies might also explain the difficulty in detecting endogenous levels of RGS13 by immunoblotting.Through the characterization of RGS13 we have added another piece to the puzzle of the RGS family. Unlike many RGS proteins RGS13 has no identified domain other than the RGS box, but because of its high expression in the immune system and lung and its ability to block Gαi, Gαq, and cAMP generation, the biological niche of RGS13 might be to regulate specific G protein-dependent signal transduction pathways in these regions. The physiological relevance of RGS13 and other RGS proteins is slowly being evaluated through the use of targeted gene disruption and expression of transgenes in mice. Through these lines of experimentation we hope to better understand the function of RGS13 and its relevance to health and disease. This study represents the initial characterization of RGS13 expression and function. RGS13 is most abundant in human tonsil followed by thymus, lymph node, lung, and spleen with low levels or a lack of expression in various other tissues. Ectopically expressed GFP-RGS13 localizes in both membrane and nuclear fractions of 293T cells with a very small portion of RGS13 in the cytosol. Similar to other classical RGS proteins, RGS13 possesses GAP activity toward Gαi family members and inhibits signaling evoked by Gαi-coupled receptors. RGS13 binds to Gαqin the presence of AMF, suggesting that RGS13 is a GAP for Gαq. In addition to its GAP activity, RGS13 likely blocks Gαq signaling by effector antagonism as well because GTPase-deficient GαqQL-mediated CRE stimulation is attenuated by RGS13. RGS13 is not a GAP for Gαs, although it inhibits cAMP generation induced by stimulation of a Gαs-coupled receptor. The expression pattern of RGS family members is highly variable and may contribute to physiological specificity of RGS proteins that share similar biochemical properties. RGS16 is expressed predominantly in the retina, pituitary, and liver (22.Chen C. Zheng B. Han J. Lin S.C. J. Biol. Chem. 1997; 272: 8679-8685Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar, 23.Chen C.K. Wieland T. Simon M.I. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 12885-12889Crossref PubMed Scopus (124) Google Scholar, 24.Snow B.E. Antonio L. Suggs S. Siderovski D.P. Gene. 1998; 206: 247-253Crossref PubMed Scopus (32) Google Scholar). RGS18 is expressed in hematopoietic tissues and lung (25.Park I.K. Klug C.A. Li K. Jerabek L. Li L. Nanamori M. Neubig R.R. Hood L. Weissman I.L. Clarke M.F. J. Biol. Chem. 2001; 276: 915-923Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar, 26.Yowe D. Weich N. Prabhudas M. Poisson L. Errada P. Kapeller R. Yu K. Faron L. Shen M. Cleary J. Wilkie T.M. Gutierrez-Ramos C. Hodge M.R. Biochem. J. 2001; 359: 109-118Crossref PubMed Scopus (51) Google Scholar), whereas RGS2 and RGS3 are ubiquitously expressed (20.Druey K.M. Blumer K.J. Kang V.H. Kehrl J.H. Nature. 1996; 379: 742-746Crossref PubMed Scopus (404) Google Scholar, 27.Siderovski D.P. Heximer S.P. Forsdyke D.R. DNA Cell Biol. 1994; 13: 125-147Crossref PubMed Scopus (107) Google Scholar). RGS13 expression is highest in tonsil (about 6.8 × 106 times greater than CD4+ cells). There is 10-fold less RGS13 in thymus, lymph node, lung, and spleen, with much lower levels of RGS13 detected in other tissues such as brain, which is a reservoir for many of the RGS family members (28.Gold S.J. Ni Y.G. Dohlman H.G. Nestler E.J. J. Neurosci. 1997; 17: 8024-8037Crossref PubMed Google Scholar,29.Grafstein-Dunn E. Young K.H. Cockett M.I. Khawaja X.Z. Brain Res. Mol. Brain Res. 2001; 88: 113-123Crossref PubMed Scopus (74) Google Scholar). In contrast to earlier reports describing RGS13 expression in brain (29.Grafstein-Dunn E. Young K.H. Cockett M.I. Khawaja X.Z. Brain Res. Mol. Brain Res. 2001; 88: 113-123Crossref PubMed Scopus (74) Google Scholar, 30.Norlin E.M. Berghard A. Mol. Cell. Neurosci. 2001; 17: 872-882Crossref PubMed Scopus (28) Google Scholar), we failed to detect any RGS13 in brain cDNA. This discrepancy could be explained by localization of RGS proteins within the brain; that is, certain areas of the brain enriched with RGS13 may be underrepresented in total brain cDNA. Using the RGS13 peptide antibodies, we evaluated endogenous RGS13 expression in human lung, spleen, liver, pancreas, kidney, heart, and brain (data not shown). We concluded that although the peptide antibodies were able to detect recombinant RGS13 protein and not other closely related RGS proteins of similar size, the sensitivity of the peptide antibodies was insufficient to identify endogenous protein in tissue lysates by immunoblotting. To address this issue, we have initiated generation of knockout mice in which a LacZreporter gene is placed under control of the RGS13 promoter, which should allow us to better define the anatomical and developmental expression of RGS13. Transfected RGS13 localizes in the cell membrane and the nucleus. Several RGS proteins have been localized to the cell membrane (31.Heximer S.P. Lim H. Bernard J.L. Blumer K.J. J. Biol. Chem. 2001; 276: 14195-14203Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar) and have undergone post-translational modifications such as palmitoylation, which may assist in membrane anchorage (32.Peppelenbosch M.P. Tertoolen L.G. Vries-Smits A.M. Qiu R.G. M'Rabet L. Symons M.H. de Laat S.W. Bos J.L. J. Biol. Chem. 1996; 271: 7883-7886Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar, 33.Druey K.M. Ugur O. Caron J.M. Chen C.K. Backland P.S. Jones T.L. J. Biol. Chem. 1999; 274: 18836-18842Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar). RGS13 lacks several of the N-terminal residues that adopt an α-helical conformation in closely related RGSs such as RGS4, 5, and 16 and facilitate direct interaction with anionic membrane phospholipids (34.Tu Y. Woodson J. Ross E.M. J. Biol. Chem. 2001; 276: 20160-20166Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar, 35.Bernstein L.S. Grillo A.A. Loranger S.S. Linder M.E. J. Biol. Chem. 2000; 275: 18520-18526Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar). It will be interesting to determine whether palmitoylation of RGS13 is required for its membrane localization or whether another mechanism of membrane targeting is responsible. It has been reported that RGS2 and RGS10 are localized in the nucleus (36.Chatterjee T.K. Fisher R.A. J. Biol. Chem. 2000; 275: 24013-24021Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar). We show here that transfected RGS13 is also located in the nucleus, although the functional significance of nuclear RGS expression has yet to be determined. RGS13 functions similarly to many RGS proteins in that it bears GAP activity toward members of the Gαi family and likely has GAP activity toward Gαq as well. RGS13 may be an effector antagonist for Gαq as demonstrated by its ability to block GαqQL-mediated CRE activation. Although one could postulate that the capacity of RGS13 to block cAMP generation may have influenced the CRE-Luciferase reporter assay, the lack of significant decrease in either basal cAMP levels or basal CRE-Luciferase activity suggests that this mechanism would not account for the inhibition of GαqQL-stimulated CRE-Luciferase by RGS13. RGS13 blunted isoproterenol-evoked increases in cAMP but exhibited no GAP activity toward Gαs. There are several possible explanations for this result. An interaction between RGS9 and a phosphodiesterase has been reported (37.He W. Cowan C.W. Wensel T.G. Neuron. 1998; 20: 95-102Abstract Full Text Full Text PDF PubMed Scopus (305) Google Scholar). RGS13 may enhance cAMP metabolism by stimulating a phosphodiesterase, resulting in a net decrease in cAMP concentration. In that case one would expect to see decreased basal levels of cAMP, which we failed to detect. A truncated form of RGS3 (RGS3T) was shown to decrease receptor-mediated cAMP generation without decreasing basal cAMP levels (16.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 authors suggested that it was unlikely that RGS3T blocked adenylyl cyclase directly. Given the similarity in our results, RGS13 and RGS3T may be acting via the same uncharacterized mechanism. Another possibility is that like RGS2 (17.Sinnarajah S. Dessauer C.W. Srikumar D. Chen J. Yuen J. Yilma S. Dennis J.C. Morrison E.E. Vodyanoy V. Kehrl J.H. Nature. 2001; 409: 1051-1055Crossref PubMed Scopus (209) Google Scholar) RGS13 may block adenylyl cyclase directly. It has been demonstrated that RGS1, RGS2, and RGS3 (but not RGS4 or RGS5) block GTPγS/odorant-mediated cAMP generation in olfactory epithelium membranes. In addition, RGS2 was shown to block forskolin-evoked increases in cAMP (17.Sinnarajah S. Dessauer C.W. Srikumar D. Chen J. Yuen J. Yilma S. Dennis J.C. Morrison E.E. Vodyanoy V. Kehrl J.H. Nature. 2001; 409: 1051-1055Crossref PubMed Scopus (209) Google Scholar), indicating that some RGS proteins regulate certain isoforms of adenylyl cyclase directly. Interestingly, RGS2 failed to attenuate cAMP production induced by stimulation of endogenous adenylyl cyclase in HEK-293 cells (17.Sinnarajah S. Dessauer C.W. Srikumar D. Chen J. Yuen J. Yilma S. Dennis J.C. Morrison E.E. Vodyanoy V. Kehrl J.H. Nature. 2001; 409: 1051-1055Crossref PubMed Scopus (209) Google Scholar). In contrast we found that RGS13 inhibited cAMP generation in these cells, suggesting that RGS2 and RGS13 may regulate different isoforms of adenylyl cyclase. Given that the only identified domain in RGS13 is the RGS box, it will be of interest to examine whether the RGS box is the region that confers the ability of RGS proteins to inhibit cAMP production. If so, why then do RGS4 and RGS5 lack the ability to regulate this process? Functional analysis of RGS13 reported here was derived from cells transfected with GFP-RGS13. We utilized GFP-RGS13 only after several failed attempts to express RGS13 in untagged or His-tagged forms. Although we used the GFP vector or other GFP-tagged RGS proteins as controls, the difficulty in expressing untagged RGS13 is of interest. Future experiments are required to determine whether the difficulty in detecting untagged RGS13 is a result of such anomalies as unstable transcription or translation. This explanation seems unlikely because we could easily detect in vitro-translated untagged RGS13 in rabbit reticulocyte lysates (data not shown). Alternatively, RGS13 may require a binding partner or modification for stable expression in mammalian cells. An example of this type of protein instability is Gβ5, which requires expression of RGS9 in the brain (18.Chen C.K. Burns M.E. He W. Wensel T.G. Baylor D.A. Simon M.I. Nature. 2000; 403: 557-560Crossref PubMed Scopus (330) Google Scholar). These studies might also explain the difficulty in detecting endogenous levels of RGS13 by immunoblotting. Through the characterization of RGS13 we have added another piece to the puzzle of the RGS family. Unlike many RGS proteins RGS13 has no identified domain other than the RGS box, but because of its high expression in the immune system and lung and its ability to block Gαi, Gαq, and cAMP generation, the biological niche of RGS13 might be to regulate specific G protein-dependent signal transduction pathways in these regions. The physiological relevance of RGS13 and other RGS proteins is slowly being evaluated through the use of targeted gene disruption and expression of transgenes in mice. Through these lines of experimentation we hope to better understand the function of RGS13 and its relevance to health and disease. We thank Angela Daily, Emily Whipple, and Nicole Mammarella for technical assistance and Dean Metcalfe for support." @default.
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