Palmitoylation Regulates Regulator of G-protein Signaling (RGS) 16 Function

Abstract

Palmitoylation is a reversible post-translational modification used by cells to regulate protein activity. The regulator of G-protein signaling (RGS) proteins RGS4 and RGS16 share conserved cysteine (Cys) residues that undergo palmitoylation. In the accompanying article (Hiol, A., Davey, P. C., Osterhout, J. L., Waheed, A. A., Fischer, E. R., Chen, C. K., Milligan, G., Druey, K. M., and Jones, T. L. Z. (2003) J. Biol. Chem. 278, 19301–19308), we determined that mutation of NH2-terminal cysteine residues in RGS16 (Cys-2 and Cys-12) reduced GTPase accelerating (GAP) activity toward a 5-hydroxytryptamine (5-HT1A)/Gαo1 receptor fusion protein in cell membranes. NH2-terminal acylation also permitted palmitoylation of a cysteine residue in the RGS box of RGS16 (Cys-98). Here we investigated the role of internal palmitoylation in RGS16 localization and GAP activity. Mutation of RGS16 Cys-98 or RGS4 Cys-95 to alanine reduced GAP activity on the 5-HT1A/Gαo1 fusion protein and regulation of adenylyl cyclase inhibition. The C98A mutation had no effect on RGS16 localization or GAP activity toward purified G-protein α subunits. Enzymatic palmitoylation of RGS16 resulted in internal palmitoylation on residue Cys-98. Palmitoylated RGS16 or RGS4 WT but not C98A or C95A preincubated with membranes expressing 5-HT1a/Gαo1 displayed increased GAP activity over time. These results suggest that palmitoylation of a Cys residue in the RGS box is critical for RGS16 and RGS4 GAP activity and their ability to regulate Gi-coupled signaling in mammalian cells. Palmitoylation is a reversible post-translational modification used by cells to regulate protein activity. The regulator of G-protein signaling (RGS) proteins RGS4 and RGS16 share conserved cysteine (Cys) residues that undergo palmitoylation. In the accompanying article (Hiol, A., Davey, P. C., Osterhout, J. L., Waheed, A. A., Fischer, E. R., Chen, C. K., Milligan, G., Druey, K. M., and Jones, T. L. Z. (2003) J. Biol. Chem. 278, 19301–19308), we determined that mutation of NH2-terminal cysteine residues in RGS16 (Cys-2 and Cys-12) reduced GTPase accelerating (GAP) activity toward a 5-hydroxytryptamine (5-HT1A)/Gαo1 receptor fusion protein in cell membranes. NH2-terminal acylation also permitted palmitoylation of a cysteine residue in the RGS box of RGS16 (Cys-98). Here we investigated the role of internal palmitoylation in RGS16 localization and GAP activity. Mutation of RGS16 Cys-98 or RGS4 Cys-95 to alanine reduced GAP activity on the 5-HT1A/Gαo1 fusion protein and regulation of adenylyl cyclase inhibition. The C98A mutation had no effect on RGS16 localization or GAP activity toward purified G-protein α subunits. Enzymatic palmitoylation of RGS16 resulted in internal palmitoylation on residue Cys-98. Palmitoylated RGS16 or RGS4 WT but not C98A or C95A preincubated with membranes expressing 5-HT1a/Gαo1 displayed increased GAP activity over time. These results suggest that palmitoylation of a Cys residue in the RGS box is critical for RGS16 and RGS4 GAP activity and their ability to regulate Gi-coupled signaling in mammalian cells. Activation of G-protein-coupled receptors by peptides and hormones catalyzes the exchange of GDP with GTP on the α subunit of its associated heterotrimeric G protein. The active, GTP-bound form of the α subunit interacts with effectors, initiating a signaling cascade. Deactivation of this signaling pathway is mediated by the intrinsic GTPase activity of α subunits, which is in turn accelerated by cognate GTPase activating proteins (GAPs), 1The abbreviations used are: GAP, GTPase activating protein; RGS, regulator of G protein signaling; pPAT, partially purified protein acyltransferase; GST, glutathione S-transferase; DRMs, detergent-resistant membranes; WT, wild-type; 5-HT, 5-hydroxytryptamine (serotonin); AMP-PNP, adenosine 5′-(β,γ-imino)triphosphate; HA, hemagglutinin; HEK, human embryonic kidney; IB, incubation buffer. the regulators of G-protein signaling (RGS proteins) (reviewed in Ref. 1DeVries L. Zheng B. Fischer T. Elenko E. Farquhar M.G. Annu. Rev. Pharm. Toxicol. 2000; 40: 235-271Crossref PubMed Scopus (508) Google Scholar, 2Ross E.M. Wilkie T.M. Annu. Rev. Biochem. 2000; 69: 795-827Crossref PubMed Scopus (929) Google Scholar, 3Hollinger S. Hepler J.R. Pharmacol. Rev. 2002; 54: 527-559Crossref PubMed Scopus (601) Google Scholar). The RGS protein family is large (more than 30 proteins in mammalian cells), and its members share high sequence homology within the conserved RGS domain that confers GAP activity. Regulation of RGS proteins may determine their cell-to-cell specificity and preference for certain G-protein α subunits or G-protein-coupled receptor-linked signaling pathways. Modulation of RGS activity may occur by post-translational modifications such as phosphorylation (4Garrison T.R. Zhang Y. Pausch M. Apanovitch D. Aebersold R. Dohlman H.G. J. Biol. Chem. 1999; 274: 36387-36391Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar, 5Fischer T. Elenko E. Wan L. Thomas G. Farquhar M.G. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 4040-4045Crossref PubMed Scopus (41) Google Scholar, 6Ogier-Denis E. Pattingre S. El Benna J. Codogno P. J. Biol. Chem. 2000; 275: 39090-39095Abstract Full Text Full Text PDF PubMed Scopus (257) Google Scholar, 7Cunningham M.L. Waldo G.L. Hollinger S. Hepler J.R. Harden T.K. J. Biol. Chem. 2001; 276: 5438-5444Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar, 8Hu G. Jang G.F. Cowan C.W. Wensel T.G. Palczewski K. J. Biol. Chem. 2001; 276: 22287-22295Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar, 9Burgon P.G. Lee W.L. Nixon A.B. Peralta E.G. Casey P.J. J. Biol. Chem. 2001; 276: 32828-32834Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar, 10Chen C. Wang H. Fong C.W. Lin S.C. FEBS Lett. 2001; 504: 16-22Crossref PubMed Scopus (31) Google Scholar, 11Derrien A. Druey K.M. J. Biol. Chem. 2001; 276: 48532-48538Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar, 12Chikumi H. Fukuhara S. Gutkind J.S. J. Biol. Chem. 2002; 277: 12463-12473Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar, 13Nair K.S. Balasubramanian N. Slepak V.Z. Curr. Biol. 2002; 12: 421-425Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar, 14Niu J. Scheschonka A. Druey K.M. Davis A. Reed E. Kolenko V. Bodnar R. Voyno-Yasenetskaya T.M. Du X. Kehrl J.H. Dulin N.O. Biochem. J. 2002; 365: 677-684Crossref PubMed Google Scholar, 15Benzing T. Kottgen M. Johnson M. Schermer B. Zentgraf H. Walz G. Kim E. J. Biol. Chem. 2002; 277: 32954-32962Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar) or palmitoylation (16De 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, 17Chen 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, 18Druey K.M. Ugur O. Caron J.M. Chen C.K. Backlund P.S. Jones T.L.Z. J. Biol. Chem. 1999; 274: 18836-18842Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar, 19Tu Y. Popov S. Slaughter C. Ross E.M. J. Biol. Chem. 1999; 274: 38260-38267Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar, 20Popov S. Krishna U.M. Falck J.R. Wilkie T.M. J. Biol. Chem. 2000; 275: 18962-18968Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar, 21Rose J.J. Taylor J.B. Shi J. Cockett M.I. Jones P.G. Hepler J.R. J. Neurochem. 2000; 75: 2103-2112Crossref PubMed Scopus (71) Google Scholar, 22Tu Y. Woodson J. Ross E.M. J. Biol. Chem. 2001; 276: 20160-20166Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar, 23Castro-Fernandez C. Janovick J. Brothers S.P. Fisher R.A. Ji T.H. Conn M.P. Endocrinology. 2002; 143: 1310-1317Crossref PubMed Scopus (26) Google Scholar). Indeed, palmitoylation of many G-protein signaling pathway components, from the G-protein-coupled receptors itself to the RGS protein, affects their activity (24Chen C.A. Manning D.R. Oncogene. 2001; 20: 1643-1652Crossref PubMed Scopus (169) Google Scholar, 25Iiri T. Backlund P.S. Jones T.L. Wedegaertner P.B. Bourne H.R. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 14592-14597Crossref PubMed Scopus (106) Google Scholar, 26Hughes T.E. Zhang H. Logothetis D.E. Berlot C.H. J. Biol. Chem. 2001; 276: 4227-4235Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar, 27Bhattacharyya R. Wedegaertner P. J. Biol. Chem. 2000; 275: 14992-14999Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar). Although the NH2 and COOH termini were not observed in the crystal structure of RGS4 (28Tesmer J.J. Berman D.M. Gilman A.G. Sprang S.R. Cell. 1997; 89: 251-261Abstract Full Text Full Text PDF PubMed Scopus (687) Google Scholar), it is clear that in the case of both RGS4 and RGS16, the amino terminus is also required for function (29Xu X. Zeng W. 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, 30Srinivasa S.P. Bernstein L.S. Blumer K.J. Linder M.E. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 5584-5589Crossref PubMed Scopus (130) Google Scholar, 31Chen C. Lin S.C. FEBS Lett. 1998; 422: 359-362Crossref PubMed Scopus (36) Google Scholar, 32Bernstein 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 (109) Google Scholar). A short amphipathic α-helical region in the NH2-terminal region of RGS16, conserved in both RGS4 and RGS5, imparts membrane localization (17Chen 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, 32Bernstein 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 (109) Google Scholar). In a previous study, we found that RGS16 on and the NH2 terminus K.M. Ugur O. Caron J.M. Chen C.K. Backlund P.S. Jones T.L.Z. J. Biol. Chem. 1999; 274: 18836-18842Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). Mutation of and to and the of palmitoylation on reduced the of both and signaling observed in cells expressing In the accompanying article A. A.A. Fischer Chen C.K. G. Druey K.M. Jones T.L.Z. J. Biol. Chem. 2002; Full Text Full Text PDF Scopus Google Scholar), we observed that mutation of and RGS16, reduced GAP activity toward in a and RGS16 from this localization not the of RGS16 as of with increased membrane GTPase activity in the of RGS16 the ability of RGS16 to the GTPase we found that NH2-terminal palmitoylation permitted palmitoylation of RGS16 on internal cysteine residue in the RGS box by the protein in the which in protein activity. previous a role for palmitoylation of internal cysteine residue in the GAP activity of RGS proteins Y. Popov S. Slaughter C. Ross E.M. J. Biol. Chem. 1999; 274: 38260-38267Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar), we investigated palmitoylation of RGS16 Cys-98 to determine its for membrane and activity. found that mutation of this Cys to alanine reduced RGS16 GAP activity toward the 5-HT1A/Gαo1 fusion protein in membranes but not membrane or The mutation in RGS4 resulted in a in GAP activity. palmitoylation of RGS16 or RGS4 by a protein by membrane increased GAP activity over time. The C98A mutation also the ability of RGS16 or RGS4 to regulate adenylyl cyclase the of this internal palmitoylation for RGS function in mammalian cells. AMP-PNP, and were from from and from and and cells were from were in with and in a cells expressing a fusion protein the human receptor and a mutation that the G-protein to were as E. G. Pharmacol. 1999; Google Scholar). and were in and purified as K.M. Ugur O. Caron J.M. Chen C.K. Backlund P.S. Jones T.L.Z. J. Biol. Chem. 1999; 274: 18836-18842Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar, K.M. Kehrl J.H. Proc. Natl. Acad. Sci. U. S. A. 1997; PubMed Scopus Google Scholar). proteins were and and and were from of human and RGS16 WT and K.M. Ugur O. Caron J.M. Chen C.K. Backlund P.S. Jones T.L.Z. J. Biol. Chem. 1999; 274: 18836-18842Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar, E. G. Pharmacol. 1999; Google Scholar). and were the RGS16 which also human RGS16, C.K. T. M.I. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: PubMed Scopus Google Scholar). were on and to membranes. a in membranes were with or for were with or for by to the and GAP were as A. Druey K.M. J. Biol. Chem. 2001; 276: 48532-48538Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). and GTPase were as in the accompanying article A. A.A. Fischer Chen C.K. G. Druey K.M. Jones T.L.Z. J. Biol. Chem. 2002; Full Text Full Text PDF Scopus Google Scholar). of of as in J. M. C. D. and T. L. Z. Jones, in the membrane from with and the activity purified over and Enzymatic Palmitoylation of RGS RGS16 or RGS4 in incubation a protein of and with the of and of The to with The by the of 30 were to and by determined by the the of the of RGS16 with that and The were with The were for and the with of with The were by and on observed and the activity of of determined as the of of to of RGS16 as in the accompanying article A. A.A. Fischer Chen C.K. G. Druey K.M. Jones T.L.Z. J. Biol. Chem. 2002; Full Text Full Text PDF Scopus Google Scholar) that RGS16 used as the were determined by of by were used for A in the RGS for RGS16 GAP the role of the conserved RGS box cysteine for the function of RGS16 and it to a of palmitoylation Y. Popov S. Slaughter C. Ross E.M. J. Biol. Chem. 1999; 274: 38260-38267Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar, A. A.A. Fischer Chen C.K. G. Druey K.M. Jones T.L.Z. J. Biol. Chem. 2002; Full Text Full Text PDF Scopus Google Scholar). a cell expressing a 5-HT1A/Gαo1 fusion protein with WT or RGS16 were with to receptor to and membranes were and GTPase activity in membranes with a of were increased in the of WT RGS16 or with membranes with a as P.J. E. G. G. Pharm. 2002; PubMed Scopus (26) Google Scholar). GTPase activity in membranes expressing RGS16 in to membranes expressing WT RGS16 of to resulted in a in activity in membranes WT RGS16 the resulted in a in activity in membranes RGS16 Mutation of Cys-95 in RGS4 to alanine resulted in a in GAP activity in this RGS16 or RGS4 in the membranes not the of function of the cysteine the of proteins A previous that the GAP activity of RGS4 by mutation of Cys-95 to in a Y. Popov S. Slaughter C. Ross E.M. J. Biol. Chem. 1999; 274: 38260-38267Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar). the that the in RGS16 activity a of mutation of cysteine to alanine of we purified WT or C98A RGS16 from E. which not undergo and GTPase activity of the fusion protein in membranes in the of purified of WT RGS16 or C98A to membranes from cells expressing the 5-HT1A/Gαo1 protein GTPase activity in to In we found no in the of RGS16 WT or C98A to GTP by purified in a These results suggest that the of a cysteine not by itself for the activity of RGS16 in mammalian cell membranes. for RGS16 and RGS4 of the ability of the cysteine to regulate signaling in cells. adenylyl cyclase activity in of adenylyl cyclase activity to we the cells with which adenylyl cyclase receptors to with previous K.M. Ugur O. Caron J.M. Chen C.K. Backlund P.S. Jones T.L.Z. J. Biol. Chem. 1999; 274: 18836-18842Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar, C. Hepler J.R. Gilman A.G. Proc. Natl. Acad. Sci. U. S. A. 1997; PubMed Scopus Google Scholar), of WT RGS16 or WT RGS4 the regulation of adenylyl cyclase activity by with cells In of RGS16 RGS16 or the RGS4 not adenylyl cyclase activity with cells. These results that to regulate Gi-coupled adenylyl cyclase in cells. for RGS16 or the membrane localization of RGS16 by and as A. A.A. Fischer Chen C.K. G. Druey K.M. Jones T.L.Z. J. Biol. Chem. 2002; Full Text Full Text PDF Scopus Google Scholar) to determine of the protein in membranes for its of The of RGS16 WT or C98A found in the membrane as as the membrane were The of the by of the in the membrane the of RGS16 within the membrane by found RGS16 in which DRMs, by and the of the C98A to WT RGS16 A. A.A. Fischer Chen C.K. G. Druey K.M. Jones T.L.Z. J. Biol. Chem. 2002; Full Text Full Text PDF Scopus Google Scholar), this that the C98A mutation not the of RGS16 in the membrane or its localization to Enzymatic Palmitoylation of results suggest that the function of RGS16 not of the cysteine to alanine mutation itself or a in RGS16 found the we investigated palmitoylation on the RGS box cysteine function of RGS16 in In the accompanying article A. A.A. Fischer Chen C.K. G. Druey K.M. Jones T.L.Z. J. Biol. Chem. 2002; Full Text Full Text PDF Scopus Google Scholar), we cells expressing RGS16 WT or C98A with and with the which to a Cys-98. with this we observed in a in of RGS16 WT but not this residue palmitoylation we RGS16 with partially purified from membranes. of RGS16 WT or C98A with and to of in a with a with RGS16 that the NH2-terminal were the of palmitoylation. RGS4 a of palmitoylation incubation with the of WT RGS16, we observed in a in RGS16 WT but not of of RGS16 WT, and that the in the of C98A but not The of of to of RGS16 which with the palmitoylation of of with the of cysteine residues of of RGS16 of of RGS16 These results suggest that Cys-98 is a of palmitoylation. of Enzymatic Palmitoylation on RGS16 GAP GAP activity of RGS16 preincubated with in a GTP by purified RGS16 or RGS4 with or and found that the reduced the of RGS16 that the not observed no in GAP activity toward or RGS proteins with and proteins or RGS proteins preincubated with in the not high GTPase activity of the 5-HT1A/Gαo1 fusion protein in membranes with RGS16 with with a a incubation with in GAP activity RGS16 and RGS16 not suggest that RGS proteins may to membranes in a and that this may by palmitoylation Y. Woodson J. Ross E.M. J. Biol. Chem. 2001; 276: 20160-20166Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar), we preincubated membranes with RGS16 or RGS4 for to determine palmitoylation RGS membrane and GAP activity. The GAP activity of RGS16 or RGS4 that had with and increased with membrane In membranes with the or RGS proteins to in GTPase activity. Although the GAP activity of RGS16 not over membranes displayed GTPase activity with membranes with In activity of membranes to membranes that the intrinsic GTPase or GAP activity. These results suggest that palmitoylation of RGS16 and RGS4 GAP activity toward a subunit and that this is by over time. the of RGS16 palmitoylation and their in GAP we of RGS16 WT or and C98A with or with of RGS16 were with the of palmitoylation of the Cys-98 observed with the C98A the Cys-98 with the Palmitoylation of Cys-98 in the in this RGS16 with in determine which of palmitoylation for increased RGS16 GAP activity by membrane we RGS16 WT, and C98A with and preincubated proteins with 5-HT1A/Gαo1 membranes GTPase activity in to of the with to a in GAP activity that reduced with WT a with WT RGS16, GTPase activity to GTPase activity of membranes GTPase activity of membranes preincubated with the reduced GTPase activity of membranes preincubated with C98A for reduced with membranes preincubated with WT RGS16 the GTPase activity of membranes with WT RGS4 for activity in membranes RGS4 for the RGS16 C98A with the in palmitoylation increased the GAP activity of to a than of GAP activity were for with WT RGS and the activity of with not over time. we that NH2-terminal cysteine residues of RGS16 and RGS4 internal RGS box cysteine palmitoylation is critical for GAP activity. of residues of RGS proteins a effect of palmitoylation on RGS GAP activity K.M. Ugur O. Caron J.M. Chen C.K. Backlund P.S. Jones T.L.Z. J. Biol. Chem. 1999; 274: 18836-18842Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar, 19Tu Y. Popov S. Slaughter C. Ross E.M. J. Biol. Chem. 1999; 274: 38260-38267Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar, 22Tu Y. Woodson J. Ross E.M. J. Biol. Chem. 2001; 276: 20160-20166Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). In the accompanying article A. A.A. Fischer Chen C.K. G. Druey K.M. Jones T.L.Z. J. Biol. Chem. 2002; Full Text Full Text PDF Scopus Google Scholar), we that NH2-terminal palmitoylation required for RGS16 GAP activity in membranes as as its to Although NH2-terminal palmitoylation of RGS16 is not required for membrane it may a role in the of RGS16 within the membrane in the of RGS16 palmitoylation on we found activity in The that is this internal palmitoylation the function of investigated the and specificity of palmitoylation of cysteine for RGS16 GAP activity and found that RGS16 Cys-98 is to the internal palmitoylation on Cys-95 in RGS4 that Y. Popov S. Slaughter C. Ross E.M. J. Biol. Chem. 1999; 274: 38260-38267Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar). palmitoylation RGS16 and RGS4 GAP activity to a in a Mutation of Cys-98 to alanine not RGS16 localization in membrane or but resulted in a of of on a the Cys-98 the in RGS16 GAP activity by membrane mutation of Cys-98 in RGS16 or Cys-95 in RGS4 of signaling by RGS proteins to a as mutation of NH2-terminal cysteine residues (Cys-2 and In with mutation of the cysteine in which is the of its ability to signaling by of the receptor C. Janovick J. Brothers S.P. Fisher R.A. Ji T.H. Conn M.P. Endocrinology. 2002; 143: 1310-1317Crossref PubMed Scopus (26) Google Scholar). In of its GAP activity it is in both receptor and which GTPase Y. Popov S. Slaughter C. Ross E.M. J. Biol. Chem. 1999; 274: 38260-38267Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar). a role for NH2-terminal palmitoylation as as palmitoylation of a cysteine residue in the RGS box for the function of and and Y. Popov S. Slaughter C. Ross E.M. J. Biol. Chem. 1999; 274: 38260-38267Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar) that of RGS4 on Cys-95 GAP activity in or In GAP activity of RGS16 or RGS4 with mammalian cell membranes expressing a GTPase over time. The by Y. Popov S. Slaughter C. Ross E.M. J. Biol. Chem. 1999; 274: 38260-38267Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar) from the in that partially for the Y. Popov S. Slaughter C. Ross E.M. J. Biol. Chem. 1999; 274: 38260-38267Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar) the RGS box cysteine to in RGS4 we used cysteine to alanine of both RGS4 and is a residue than as a this membrane of palmitoylation. we palmitoylation for a short of which not to the intrinsic GAP activity in In required G. Jang G.F. Cowan C.W. Wensel T.G. Palczewski K. J. Biol. Chem. 2001; 276: 22287-22295Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar) and incubation to The of palmitoylation the is in the of WT RGS16 to of palmitoylation In the of may not in mammalian cells. the of used in the also RGS to a RGS4 GAP which is on the NH2 terminus of RGS4 Y. S. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). Y. Popov S. Slaughter C. Ross E.M. J. Biol. Chem. 1999; 274: 38260-38267Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar) of in a of is that in a effect on RGS4 the NH2 proteins in mammalian membrane in with to RGS or activity. RGS16 interacts with a membrane the NH2-terminal region of the RGS domain Cockett M.I. W. Jones P.G. K. S. R. 2000; PubMed Scopus (36) Google Scholar). Although the effect of on RGS16 GAP activity not this also to RGS16 or within the In RGS16 phosphorylation on both and residues C. Wang H. Fong C.W. Lin S.C. FEBS Lett. 2001; 504: 16-22Crossref PubMed Scopus (31) Google Scholar, 11Derrien A. Druey K.M. J. Biol. Chem. 2001; 276: 48532-48538Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). RGS16 interacts with the receptor for which RGS16 phosphorylation and RGS16 GAP activity. and palmitoylation may to the GAP activity of RGS16 in membranes. Palmitoylation the internal cysteine may in the of RGS16 or RGS4 within the membrane Y. Woodson J. Ross E.M. J. Biol. Chem. 2001; 276: 20160-20166Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar) found that RGS4 its NH2 terminus in a but which the GAP activity of RGS4 WT but not RGS4 over time. Although the effect of palmitoylation not in the results with that the internal palmitoylation in RGS16 and RGS4 is critical for the in GAP activity by membrane with this which the NH2-terminal and NH2-terminal palmitoylation that also GAP activity over to RGS4 Y. Woodson J. Ross E.M. J. Biol. Chem. 2001; 276: 20160-20166Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar), or palmitoylation a in RGS16 or RGS4 that G-protein and palmitoylation not RGS GAP activity in it may to a in the protein. RGS palmitoylation occur to the structure of RGS16 or RGS4 in the membrane may and G-protein Cys-95 in RGS4 on in to the and which of the with region A on Cys-95 in RGS4 or Cys-98 in RGS16 the residues and in human to which to GAP activity (28Tesmer J.J. Berman D.M. Gilman A.G. Sprang S.R. Cell. 1997; 89: 251-261Abstract Full Text Full Text PDF PubMed Scopus (687) Google Scholar, B. Chen D. Farquhar M.G. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: PubMed Scopus Google Scholar) A that mutation of to not GAP activity toward in but GAP activity toward a in membranes. In RGS16 to regulate of activity in T. N. C. M.I. J. Biol. Chem. 2000; 275: Full Text Full Text PDF PubMed Scopus Google Scholar). These results suggest a RGS16 and that may in membranes. by palmitoylation of the cysteine residue in this the activity or RGS16 in its localization to to the that localization of RGS16 or for their of activity. results that palmitoylation role in the GAP activity of both RGS16 and In the it of to the regulation and activity of RGS proteins in their which