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• W2074601908 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. 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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 was palmitoylated on Cys-2 and Cys-12 at the NH2 terminus (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). Mutation of Cys-2 and Cys-12 to alanine, and the resulting loss of palmitoylation on these residues, reduced the inhibition of both Gi- and Gq-coupled signaling normally observed in cells expressing RGS16. In the accompanying article (33Hiol A. Davey P.C. Osterhout J.L. Waheed A.A. Fischer E.R. Chen C.K. Milligan G. Druey K.M. Jones T.L.Z. J. Biol. Chem. 2002; 278: 19301-19308Abstract Full Text Full Text PDF Scopus (63) Google Scholar), we observed that mutation of Cys-2 and Cys-12 virtually eliminated palmitate incorporation into RGS16, reduced GAP activity toward Gα in a membrane-based assay, and mistargeted RGS16 away from lipid rafts. However, this abnormal localization could not explain the lack of RGS16 function, as disruption of lipid rafts with methyl-β-cyclodextrin increased membrane GTPase activity in the absence of RGS16 while preserving the ability of transfected RGS16 to further increase the agonist-evoked GTPase rate. Instead, we found that NH2-terminal palmitoylation permitted palmitoylation of RGS16 on an internal cysteine residue in the RGS box (Cys-98), possibly by retaining the protein in the lipid rafts, which are enriched in protein acyltransferase activity. Because previous studies have implicated a role for palmitoylation of an internal cysteine residue in the GAP activity of other RGS proteins (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), we investigated palmitoylation of RGS16 at Cys-98 to determine its importance for membrane localization, protein-protein interactions, and enzymatic activity. We found that mutation of this Cys to alanine reduced RGS16 GAP activity toward the 5-HT1A/Gαo1 fusion protein in membranes but did not substantially alter membrane or lipid raft localization. The analogous mutation in RGS4 resulted in a similar reduction in GAP activity. Most importantly, direct enzymatic palmitoylation of RGS16 or RGS4 by a protein acyltransferase followed by membrane preincubation markedly increased GAP activity over time. The C98A mutation also abrogated the ability of RGS16 or RGS4 to regulate Gi-mediated adenylyl cyclase inhibition, confirming the significance of this internal palmitoylation site for RGS function in mammalian cells. Reagents—Isoproterenol, somatostatin, EDTA, ATP, GTP, AMP-PNP, creatine phosphate, clostripain, and creatine phosphokinase were obtained from Sigma. Superfect transfection reagent was purchased from Qiagen, and pertussis toxin from Calbiochem. Cell Culture, Proteins, and Plasmids—HEK293 and COS-7 cells were obtained from ATCC. Cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum, 5 mm glutamine, and antibiotics in a humidified 5% CO2 incubator. HEK293 cells stably expressing a fusion protein between the human 5-HT1A receptor and Gαo1 containing a C351G mutation that renders the G-protein resistant to pertussis toxin were generated as previously described (34Kellett E. Carr I.C. Milligan G. Mol. Pharmacol. 1999; 56: 684-692PubMed Google Scholar). GST-RGS16 and His6RGS4 were produced in Escherichia coli and purified as described (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, 35Druey K.M. Kehrl J.H. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 12851-12856Crossref PubMed Scopus (48) Google Scholar). Purified proteins were dialyzed against 50 mm Tris, pH 8, 100 mm NaC1, 1 mm EDTA, and 5% glycerol and stored at -80 °C until use. Recombinant, myristoylated rat Gαo and Gαi1 were purchased from Calbiochem. Plasmids directing expression of human HA-RGS4 and RGS16 WT and HA-RGS16 (C2A/C12A) have been described elsewhere (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, 34Kellett E. Carr I.C. Milligan G. Mol. Pharmacol. 1999; 56: 684-692PubMed Google Scholar). HA-RGS16 (C98A) and HA-RGS4 (C95A) were generated using the QuikChange mutagenesis kit (Stratagene). Polyclonal antiserum against mouse RGS16 (CT265), which also recognizes human RGS16, has been previously described (36Chen C.K. Wieland T. Simon M.I. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 12885-12889Crossref PubMed Scopus (124) Google Scholar). Immunoblotting—Cell lysates were resolved on 10–20% Tris glycine gels and transferred to polyvinylidene difluroride membranes. After a 2-h blocking step in Tris-buffered saline plus 0.01% Tween 20 + 10% milk, membranes were incubated with primary antibody (CT265, 1:1000) or anti-HA (12CA5, Roche Diagnostics) for an additional 2 h. Blots were then equilibrated with horseradish peroxidase-conjugated goat anti-rabbit or anti-mouse IgG for 1 h. Signal was detected by enhanced chemiluminescence (Super Signal, Pierce) according to the manufacturer's instructions. Adenylyl Cyclase and Single Turnover GAP Assays—These assays were performed exactly as described previously (11Derrien A. Druey K.M. J. Biol. Chem. 2001; 276: 48532-48538Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). Plasma Membrane and Detergent-resistant Membrane (DRM) Fractionation, High Affinity GTPase Assays—These procedures were carried out as described in the accompanying article (33Hiol A. Davey P.C. Osterhout J.L. Waheed A.A. Fischer E.R. Chen C.K. Milligan G. Druey K.M. Jones T.L.Z. J. Biol. Chem. 2002; 278: 19301-19308Abstract Full Text Full Text PDF Scopus (63) Google Scholar). Purification of Protein Acyltransferase—Purification of pPAT was performed as described in detail elsewhere. 2A. Hiol, J. M. Caron, C. D. Smith, and T. L. Z. Jones, manuscript in preparation. Briefly, the plasma membrane from rat livers was extracted with 0.15% Triton X-100 and the PAT activity was purified over several chromatography steps, octyl-Sepharose, cerulenin analogue affinity, Q-Sepharose, and palmitoyl CoA-agarose. Enzymatic Palmitoylation of RGS Proteins—Recombinant RGS16 or RGS4 was diluted in incubation buffer (IB) (20 mm Tris-HCl, pH 7.4, 150 mm KCl, 1 mm EDTA) at a protein concentration of 0.4–1 mg/ml and incubated with the pPAT preparation (10 μg of protein), 200 μm CoA, 2 mm ATP, and 1 μl of [9,10-3H]palmitate (5 mCi/ml, 30–60 Ci/mmol, ARC Inc.). The final reaction volume was adjusted to 100 μl with IB. The reaction was stopped by the addition of sample buffer after 45 min at 30 °C. Proteins were subjected to one-dimensional PAGE and stained by MicrowaveBlue (Protiga). Acylation was determined by fluorography using the Wax technique (ISC BioExpress) following the instructions of the manufacturer. Stoichiometry of Palmitoylation—Recombinant RGS16 (260 pmol) incubated with pPAT was diluted into IB buffer that contained 0.1% bovine serum albumin and 1% sucrose. The solutions were loaded into an Ultrafree filtration unit (Amicon) previously equilibrated with IB. The samples were centrifuged at 2000 × g for 20 min and the filters washed once with 400 μl of 70% ethanol, then twice with IB. The insert cups were counted by liquid scintillation spectrometry, and palmitate incorporation was calculated based on observed counts and the specific activity of [3H]palmitate. Stoichiometry of labeling was determined as the ratio of picomole of palmitate to picomole of RGS16 per reaction. Clostripain Cleavage—This procedure was carried out as in the accompanying article (33Hiol A. Davey P.C. Osterhout J.L. Waheed A.A. Fischer E.R. Chen C.K. Milligan G. Druey K.M. Jones T.L.Z. J. Biol. Chem. 2002; 278: 19301-19308Abstract Full Text Full Text PDF Scopus (63) Google Scholar) except that recombinant RGS16 (10 μg) was used as the substrate. Statistical Analysis—Two tailed p values were determined by one-way analysis of variance followed by Tukey-Kramer multiple comparisons test using Graph Pad Instat software. p values <0.05 were considered significant. Sigma Plot 8.0 software was used for curve fitting. A Cysteine Residue in the RGS Box Is Critical for RGS16 GAP Activity—We tested the role of the conserved RGS box cysteine for the function of RGS16 and because it appeared to be a site of palmitoylation (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, 33Hiol A. Davey P.C. Osterhout J.L. Waheed A.A. Fischer E.R. Chen C.K. Milligan G. Druey K.M. Jones T.L.Z. J. Biol. Chem. 2002; 278: 19301-19308Abstract Full Text Full Text PDF Scopus (63) Google Scholar). We transfected a cell line stably expressing a 5-HT1A/Gαo1 (C351G) fusion protein with HA-RGS16 WT or RGS16 (C98A) plasmids. Cells were treated with pertussis toxin to block receptor coupling to endogenous Gαi/o proteins, and membranes were prepared. Both basal and agonist-induced GTPase activity in membranes incubated with a range of 5-HT concentration were increased in the presence of WT RGS16 or compared with membranes transfected with a vector control plasmid (LacZ) as expected (37Welsby P.J. Kellet E. Wilkinson G. Milligan G. Mol. Pharm. 2002; 61: 1211-1221Crossref PubMed Scopus (26) Google Scholar). However, GTPase activity in membranes expressing RGS16 (C98A) was decreased nearly 50% in comparison to membranes expressing WT RGS16 (FIG. 1A, upper panel). Addition of maximal 5-HT doses (10-6 to 10-4m) resulted in a ∼3.5-fold increase in activity in membranes containing WT RGS16 while the same dose resulted in only a 2.3-fold increase in activity in membranes containing RGS16 (C98A). Mutation of Cys-95 in RGS4 to alanine resulted in a similar reduction in GAP activity in this assay (FIG. 1A, lower panel). Decreased RGS16 or RGS4 levels in the membranes could not explain the loss of function of the cysteine mutants, because the amount of immunodetectable proteins was comparable (Fig. 1B). A previous study showed that the GAP activity of RGS4 was unaffected by mutation of Cys-95 to Val in a single turnover assay (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). To exclude the possibility that the reduction in transfected RGS16 (C98A) activity could be a result of mutation of cysteine to alanine independent of palmitoylation, we purified WT or C98A recombinant RGS16 from E. coli, which would not undergo posttranslational modification, and measured agonist-stimulated GTPase activity of the fusion protein in membranes in the presence of purified RGS16. Addition of WT RGS16 or C98A to membranes from cells expressing the 5-HT1A/Gαo1 protein enhanced GTPase activity equally in response to 5-HT even at subsaturating concentrations (Fig. 1C). In addition, we found no significant difference in the abilities of RGS16 WT or C98A to promote GTP hydrolysis by purified Gαi in a single turnover assay (Fig. 1D). These results suggest that the loss of a cysteine thiol group was not by itself responsible for the decreased activity of RGS16 (C98A) expressed in mammalian cell membranes. Cysteine 98 Is Essential for RGS16 and RGS4 Inhibition of Gi-regulated Adenylyl Cyclase Activity—We tested the ability of the cysteine mutants to regulate Gi signaling in HEK293 cells. Somatostatin decreases adenylyl cyclase activity through Gαi activation. Because decreases in basal levels of adenylyl cyclase activity are difficult to detect, we treated the cells concurrently with isoproterenol, which stimulates adenylyl cyclase through endogenous β-adrenergic receptors coupled to Gαs. Consistent with previous studies (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, 38Huang C. Hepler J.R. Gilman A.G. Mumby S.M. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 6159-6163Crossref PubMed Scopus (151) Google Scholar), expression of WT RGS16 or WT RGS4 inhibited the negative regulation of adenylyl cyclase activity induced by somatostatin compared with vector-transfected cells (Fig. 2). In contrast, expression of RGS16 (C98A), RGS16 (C2A/C12A), or the analogous RGS4 mutants did not alter adenylyl cyclase activity compared with control cells. These results demonstrate that neither mutant was able to regulate Gi-coupled adenylyl cyclase inhibition effectively in HEK293 cells. Cysteine 98 Is Not Required for RGS16 Plasma Membrane or Lipid Raft Localization—We delineated the membrane localization of RGS16 (C98A) by cellular fractionation and immunoblotting as previously described (33Hiol A. Davey P.C. Osterhout J.L. Waheed A.A. Fischer E.R. Chen C.K. Milligan G. Druey K.M. Jones T.L.Z. J. Biol. Chem. 2002; 278: 19301-19308Abstract Full Text Full Text PDF Scopus (63) Google Scholar) to determine whether redistribution of the protein in membranes could account for its lack of function. The amounts of RGS16 WT or C98A found in the total membrane fraction (T) as well as the plasma membrane fraction (P) were comparable (Fig. 3A). The integrity of the fractions was verified by enrichment of the marker Na+/K+-ATPase in the plasma membrane fraction. We then assessed the distribution of RGS16 (C98A) within the membrane fraction by OptiPrep gradient centrifugation. We found RGS16 (C98A) in fraction 1, which contains DRMs, indicated by caveolin and Gαi immunoreactivity (Fig. 3B). Because the distribution of the C98A mutant was similar to WT RGS16 (33Hiol A. Davey P.C. Osterhout J.L. Waheed A.A. Fischer E.R. Chen C.K. Milligan G. Druey K.M. Jones T.L.Z. J. Biol. Chem. 2002; 278: 19301-19308Abstract Full Text Full Text PDF Scopus (63) Google Scholar), this result suggests that the C98A mutation did not substantially alter the distribution of RGS16 in the plasma membrane or its localization to lipid rafts. Enzymatic Palmitoylation of RGS16—The aforementioned results suggest that the poor function of RGS16 (C98A) was not because of the cysteine to alanine mutation itself or a decrease in RGS16 found at the membrane. Therefore, we investigated whether palmitoylation on the RGS box cysteine could affect function of RGS16 in vitro. In the accompanying article (33Hiol A. Davey P.C. Osterhout J.L. Waheed A.A. Fischer E.R. Chen C.K. Milligan G. Druey K.M. Jones T.L.Z. J. Biol. Chem. 2002; 278: 19301-19308Abstract Full Text Full Text PDF Scopus (63) Google Scholar), we metabolically labeled cells expressing RGS16 WT or C98A with [3H]palmitate and immunoprecipitated RGS16. We then treated immunoprecipitates with the protease clostripain, which was expected to yield a 5-kDa fragment containing Cys-98. Consistent with this prediction, we observed palmitate incorporation in a 5-kDa band only in immunoprecipitates of RGS16 WT but not C98A. To assess whether this residue underwent enzymatic palmitoylation directly, we chemically acylated recombinant RGS16 with partially purified PAT from rat liver membranes. Incubation of RGS16 WT or C98A with pPAT and [3H]palmitoyl-CoA led to incorporation of tritium in a band with a molecular mass consistent with RGS16 (∼30 kDa, Fig. 4A, left panel), confirming that the NH2-terminal cysteines were the major sites of palmitoylation. RGS4 demonstrated a similar pattern of palmitoylation after incubation with the pPAT preparation (FIG. 4A, right panel). After clostripain cleavage of WT RGS16, we observed [3H]palmitate incorporation in a 5-kDa band only in samples containing RGS16 WT but not C98A. Coomassie Blue staining revealed an identical pattern of cleavage of RGS16 WT, C2A/C12A, and C98A, indicating that the 5-kDa band was present in the digest of C98A but was not palmitoylated (Fig. 4B). The stoichiometry of palmitate incorporation was ∼2.6 ± 0.1 mol of palmitate to 1 mol of RGS16 (WT), which correlates well with the 3 predicted palmitoylation sites (mean ± S.E. of three independent experiments). Moreover, each mutant demonstrated incorporation of [3H]palmitate congruent with the number of target cysteine residues present (0.7 ± 0.1 mol of palmitate/mol of RGS16 C2A/C12A; 1.5 ± 0.03 mol of palmitate/mol of RGS16 (C98A)). These results suggest that Cys-98 is a direct site of palmitoylation. Effect of Enzymatic Palmitoylation on RGS16 GAP Activity—We first assessed GAP activity of RGS16 preincubated with pPAT in a solution assay measuring single turnover GTP hydrolysis by purified Gαo. We treated RGS16 or RGS4 with pPAT or boiled pPAT and found that boiling the enzyme dramatically reduced the amount of RGS16 palmitoylation, suggesting that the process was predominantly enzymatic (data not shown). We observed no significant difference in GAP activity toward Gαo (RGS16) or Gαi (RGS4) between RGS proteins treated with pPAT and untreated proteins (Fig. 5A) or RGS proteins preincubated with boiled pPAT in the single turnover assay (data not shown). We next measured high affinity GTPase activity of the 5-HT1A/Gαo1 fusion protein in membranes incubated with recombinant RGS16 treated with pPAT. When assessed with a single time point (representing a 20-min incubation with 10-6m 5-HT), there was little difference in GAP activity between RGS16 and pPAT-treated RGS16 (data not shown). However, because recent studies suggest that RGS proteins may bind to membranes in a time-dependent fashion and that this process may be affected by palmitoylation (22Tu 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 recombinant RGS16 or RGS4 for various times before agonist stimulation to determine whether palmitoylation affected RGS membrane binding and GAP activity. The GAP activity of RGS16 or RGS4 that had been treated with pPAT and palmitate increased significantly with longer membrane preincubation times (Fig. 5, B–C). In contrast, membranes incubated with either the pPAT preparation alone or untreated RGS proteins failed to exhibit any significant time-dependent increase in GTPase activity. Although the GAP activity of untreated RGS16 did not increase over time (Fig. 5B), RGS16-containing membranes displayed significantly higher absolute GTPase activity compared with membranes incubated with pPAT alone (2.05 ± 0.21-fold). In contrast, activity of membranes containing pPAT was equivalent to buffer-treated membranes (0.98 ± 0.19-fold), indicating that the pPAT preparation lacked intrinsic GTPase or GAP activity. These results suggest that palmitoylation of RGS16 and RGS4 augments GAP activity toward a membrane-bound Gα subunit and that this enhancement is facilitated by RGS-membrane interaction over time. To confirm the sites of RGS16 palmitoylation and their relative roles in GAP activity, we first measured incorporation of [3H]palmitate into pPAT-treated, recombinant RGS16 WT or C2A/C12A and C98A mutants with or without cleavage with clostripain (Fig. 5C, right panel). Levels of tritium incorporation into full-length RGS16 (30 kDa) were compatible with the number of palmitoylated residues. No palmitoylation of the ∼5-kDa band containing Cys-98 was observed with the C98A mutant after clostripain treatment, whereas the palmitoylated fragment containing Cys-98 was clearly visualized with the C2A/C12A mutant. Palmitoylation of Cys-98 in the C2A/C12A mutant was expected in this assay because recombinant RGS16 was incubated with pPAT in vitro. To determine which site of palmitoylation was responsible for increased RGS16 GAP activity induced by membrane interaction, we treated recombinant RGS16 WT, C2A/C12A, and C98A with pPAT and then preincubated proteins with 5-HT1A/Gαo1 me" @default.
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• W2074601908 date "2003-05-01" @default.
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• W2074601908 title "Palmitoylation Regulates Regulator of G-protein Signaling (RGS) 16 Function" @default.
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