FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2117918340> ?p ?o ?g. }

• W2117918340 endingPage "38267" @default.
• W2117918340 startingPage "38260" @default.
• W2117918340 abstract "RGS4 and RGS10 expressed in Sf9 cells are palmitoylated at a conserved Cys residue (Cys95 in RGS4, Cys66 in RGS10) in the regulator of G protein signaling (RGS) domain that is also autopalmitoylated when the purified proteins are incubated with palmitoyl-CoA. RGS4 also autopalmitoylates at a previously identified cellular palmitoylation site, either Cys2 or Cys12. The C2A/C12A mutation essentially eliminates both autopalmitoylation and cellular [3H]palmitate labeling of Cys95. Membrane-bound RGS4 is palmitoylated both at Cys95 and Cys2/12, but cytosolic RGS4 is not palmitoylated. RGS4 and RGS10 are GTPase-activating proteins (GAPs) for the Gi and Gq families of G proteins. Palmitoylation of Cys95 on RGS4 or Cys66 on RGS10 inhibits GAP activity 80–100% toward either Gαi or Gαzin a single-turnover, solution-based assay. In contrast, when GAP activity was assayed as acceleration of steady-state GTPase in receptor-G protein proteoliposomes, palmitoylation of RGS10 potentiated GAP activity ≥20-fold. Palmitoylation near the N terminus of C95V RGS4 did not alter GAP activity toward soluble Gαz and increased Gz GAP activity about 2-fold in the vesicle-based assay. Dual palmitoylation of wild-type RGS4 remained inhibitory. RGS protein palmitoylation is thus multi-site, complex in its control, and either inhibitory or stimulatory depending on the RGS protein and its sites of palmitoylation. RGS4 and RGS10 expressed in Sf9 cells are palmitoylated at a conserved Cys residue (Cys95 in RGS4, Cys66 in RGS10) in the regulator of G protein signaling (RGS) domain that is also autopalmitoylated when the purified proteins are incubated with palmitoyl-CoA. RGS4 also autopalmitoylates at a previously identified cellular palmitoylation site, either Cys2 or Cys12. The C2A/C12A mutation essentially eliminates both autopalmitoylation and cellular [3H]palmitate labeling of Cys95. Membrane-bound RGS4 is palmitoylated both at Cys95 and Cys2/12, but cytosolic RGS4 is not palmitoylated. RGS4 and RGS10 are GTPase-activating proteins (GAPs) for the Gi and Gq families of G proteins. Palmitoylation of Cys95 on RGS4 or Cys66 on RGS10 inhibits GAP activity 80–100% toward either Gαi or Gαzin a single-turnover, solution-based assay. In contrast, when GAP activity was assayed as acceleration of steady-state GTPase in receptor-G protein proteoliposomes, palmitoylation of RGS10 potentiated GAP activity ≥20-fold. Palmitoylation near the N terminus of C95V RGS4 did not alter GAP activity toward soluble Gαz and increased Gz GAP activity about 2-fold in the vesicle-based assay. Dual palmitoylation of wild-type RGS4 remained inhibitory. RGS protein palmitoylation is thus multi-site, complex in its control, and either inhibitory or stimulatory depending on the RGS protein and its sites of palmitoylation. regulator of G protein signaling palmitoyl guanosine 5′-O-thiotriphosphate polyacrylamide gel electrophoresis dithiothreitol m2 muscarinic cholinergic receptor GTPase-activating protein Palmitoylation is increasingly recognized as a frequent and important modification of eukaryotic signaling proteins. Palmitoylated proteins include G protein α subunits and monomeric GTP-binding proteins (e.g. p21 ras), G protein-coupled receptors, RGS1 proteins, and protein kinases. Unlike prenylation and myristoylation, palmitoylation is reversible, allowing for its regulation. Protein-bound palmitate turns over continuously in cells (see Ref. 1Dunphy J.T. Linder M.E. Biochim. Biophys. Acta. 1998; 1436: 245-261Crossref PubMed Scopus (316) Google Scholar for review). Knowledge of the mechanism and control of protein palmitoylation remain rudimentary. Depalmitoylation may be the rate-limiting event in palmitate turnover. Duncan and Gilman (2Duncan J.A. Gilman A.G. J. Biol. Chem. 1998; 273: 15830-15837Abstract Full Text Full Text PDF PubMed Scopus (316) Google Scholar) recently identified a palmitoyl-protein thioesterase that is probably the enzyme that depalmitoylates Gα subunits and which may also depalmitoylate other proteins. The enzyme that transfers palmitate to proteins has not been clearly identified. Dunphy et al. (3Dunphy J.T. Greentree W.K. Manahan C.L. Linder M.E. J. Biol. Chem. 1996; 271: 7154-7159Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar) partially purified an activity from hepatic membranes that accelerates palmitoylation of Gα using Pal-CoA as donor, potentially a protein-palmitoyl transferase. However, Gα subunits can autopalmitoylate in vitro at the physiologically correct Cys residues (4Duncan J.A. Gilman A.G. J. Biol. Chem. 1996; 271: 23594-23600Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar), and G protein-coupled receptors may also autopalmitoylate (5O'Brien P.J. St. Jules R.S. Reddy T.S. Bazan N.G. Zatz M. J. Biol. Chem. 1987; 262: 5210-5215Abstract Full Text PDF PubMed Google Scholar, 6St. Jules R.S. O'Brien P.J. Exp. Eye Res. 1986; 43: 929-940Crossref PubMed Scopus (37) Google Scholar). Autopalmitoylation is somewhat slower than palmitoylation in vivo, but the correlation between Cys residues that autopalmitoylate in vitro and those that are naturally palmitoylated suggests that autopalmitoylation is at least involved in the physiological process. The Cys residues that selectively autopalmitoylate may do so because their thiol groups have an unusually low pI, because they are at relatively hydrophobic surfaces and are thus exposed to Pal-CoA, or because these proteins contain ancillary residues that catalyze the reaction. The ability of a specific residue to autopalmitoylate would provide selectivity to the palmitoylation process, and the protein studied by Dunphy et al. (3Dunphy J.T. Greentree W.K. Manahan C.L. Linder M.E. J. Biol. Chem. 1996; 271: 7154-7159Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar) might act non-selectively either as a Pal-CoA carrier protein or as a non-selective transferase. The functions of protein palmitoylation is an active area of study. In the case of p21ras and Gα subunits, palmitoylation helps anchor the proteins to the plasma membrane (1Dunphy J.T. Linder M.E. Biochim. Biophys. Acta. 1998; 1436: 245-261Crossref PubMed Scopus (316) Google Scholar, 7Mumby S.M. Curr. Opin. Cell Biol. 1997; 9: 148-154Crossref PubMed Scopus (238) Google Scholar, 8Ross E.M. Curr. Biol. 1995; 5: 107-109Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar). Palmitoylation of Gα subunits also enhances their binding to Gβγ and desensitizes them to the GAP activity of RGS proteins (9Iiri T. Backlund Jr., P.S. Jones T.L.Z. Wedegaertner P.B. Bourne H.R. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 14592-14597Crossref PubMed Scopus (106) Google Scholar, 10Tu Y. Wang J. Ross E.M. Science. 1997; 278: 1132-1135Crossref PubMed Scopus (130) Google Scholar). Palmitoylation has been reported to enhance the activity of a G protein-coupled receptor kinase (11Stoffer R.H. Inglese J. Macrae A.D. Lefkowitz R.J. Premont R.T. Biochemistry. 1998; 37: 16053-16059Crossref PubMed Scopus (41) Google Scholar), and mutations of the palmitoylated Cys residue in the C-terminal region of G protein-coupled receptors themselves have resulted in a variety of effects (8Ross E.M. Curr. Biol. 1995; 5: 107-109Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar). In none of these cases is it completely clear whether palmitoylation alters the structure of the palmitoylated protein itself, whether the palmitoyl group forms or blocks part of a protein-protein interface, or whether it only increases binding to hydrophobic surfaces. It has recently been reported that several RGS proteins are also palmitoylated in cells (12De 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, 13Srinivasa 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, 14Druey 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). RGS proteins are a family of variably selective GAPs for members of the Gi and Gqfamilies of heterotrimeric G proteins (15Berman D.M. Gilman A.G. J. Biol. Chem. 1998; 273: 1269-1272Abstract Full Text Full Text PDF PubMed Scopus (445) Google Scholar). RGS proteins inhibit G protein signaling in fungi and roundworms (16Koelle M.R. Horvitz H.R. Cell. 1996; 84: 115-125Abstract Full Text Full Text PDF PubMed Scopus (480) Google Scholar, 17Hajdu-Cronin Y.M. Chen W.J. Patikoglou G. Koelle M.R. Sternberg P.W. Genes Devel. 1999; 13: 1780-1793Crossref PubMed Scopus (150) Google Scholar, 18Dohlman H.G. Thorner J. J. Biol. Chem. 1997; 272: 3871-3874Abstract Full Text Full Text PDF PubMed Scopus (450) Google Scholar) and their overexpression can inhibit signaling in mammalian cells (see Ref. 15Berman D.M. Gilman A.G. J. Biol. Chem. 1998; 273: 1269-1272Abstract Full Text Full Text PDF PubMed Scopus (445) Google Scholarfor review). RGS proteins are also important in modulating the decay kinetics of G protein signaling (19Doupnik C.A. Davidson N. Lester H.A. Kofuji P. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 10461-10466Crossref PubMed Scopus (297) Google Scholar, 20Zerangue N. Jan L.Y. Curr. Biol. 1998; 8: 313-316Abstract Full Text Full Text PDF PubMed Google Scholar, 21Chuang H.-H. Yu M. Jan Y.N. Jan L.Y. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 11727-11732Crossref PubMed Scopus (108) Google Scholar, 22He W. Cowan C.W. Wensel T.G. Neuron. 1998; 20: 95-102Abstract Full Text Full Text PDF PubMed Scopus (309) Google Scholar, 23Arshavsky V.Y. Pugh Jr., E.N. Neuron. 1998; 20: 11-14Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar). The closely related proteins RGS4 and RGS16 are palmitoylated at Cys2 and/or Cys12 (13Srinivasa 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, 14Druey 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), and mutation of these residues diminished the ability of RGS16 to inhibit cellular signaling (14Druey 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). Membrane-bound GAIP is also palmitoylated near its N terminus, but in a cysteine string that characterizes a separate RGS protein subfamily (12De 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). We report here that RGS4 can autopalmitoylate stoichiometrically in vitro at two sites, one near the N terminus and the other in the conserved RGS box domain. RGS10, which lacks the N-terminal site, also autopalmitoylates at the conserved Cys residue. Autopalmitoylation of both proteins correlates well with their palmitoylation in cells. We report further that dual palmitoylation can either inhibit or potentiate GAP activity depending on the site of palmitoylation, the assay medium and on the identity of the RGS protein. Wild-type and mutant RGS4 and RGS10 were purified from Escherichia coli as described (24Wang J. Ducret A. Tu Y. Kozasa T. Aebersold R. Ross E.M. J. Biol. Chem. 1998; 273: 26014-26025Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar). RGS4 was purified from Sf9 cells by essentially the same procedure. Gαz, (25Kozasa T. Gilman A.G. J. Biol. Chem. 1995; 270: 1734-1741Abstract Full Text Full Text PDF PubMed Scopus (297) Google Scholar, 26Wang J. Tu Y. Woodson J. Song X. Ross E.M. J. Biol. Chem. 1997; 272: 5732-5740Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar), Gβ1γ2 (25Kozasa T. Gilman A.G. J. Biol. Chem. 1995; 270: 1734-1741Abstract Full Text Full Text PDF PubMed Scopus (297) Google Scholar), and m2AChR (27Parker E.M. Kameyama K. Higashijima T. Ross E.M. J. Biol. Chem. 1991; 266: 519-527Abstract Full Text PDF PubMed Google Scholar) were purified from Sf9 cells. Myristoylated Gαi1 was purified from E. coli (28Mumby S.M. Linder M.E. Methods Enzymol. 1994; 237: 254-268Crossref PubMed Scopus (112) Google Scholar). [γ-32P]GTP and [3H]Pal-CoA were synthesized and purified as described (4Duncan J.A. Gilman A.G. J. Biol. Chem. 1996; 271: 23594-23600Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar, 29Biddlecome G.H. Berstein G. Ross E.M. J. Biol. Chem. 1996; 271: 7999-8007Abstract Full Text Full Text PDF PubMed Scopus (212) Google Scholar). [9,10-3H]Palmitic acid was purchased from NEN Life Science Products. The Cys mutants C2A/C12A/C33A and C2A/C12A RGS4 were prepared by sequential polymerase chain reaction reactions. C33A RGS4 cDNA was first prepared by substituting codon 33 by GCG. The product and the wild-type cDNA were cut withNcoI and BamHI and ligated in-frame into a modified pQE60 (Qiagen) that encodes the sequence MGH6MG before the cloning site (30Popov S. Yu K. Kozasa T. Wilkie T.M. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 7216-7220Crossref PubMed Scopus (149) Google Scholar). In subsequent reactions, codons 2 and 12 were replaced by GCC. The final cDNA products, C2A/C12A and C2A/C12A/C33A RGS4, were cloned into the modified pQE60 vector as described above. The ΔN57 RGS4 deletion mutation was generated by polymerase chain reaction with the primer 5′-GATCCATGGGCAAATGGGCTGAATCGCTGGAA. The product was cut withNcoI and BamHI and cloned into the corresponding sites of the modified pQE60 (30Popov S. Yu K. Kozasa T. Wilkie T.M. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 7216-7220Crossref PubMed Scopus (149) Google Scholar). C95V RGS4 was generated using the QuikChange mutagenesis kit (Stratagene) to change codon 95 to GTT. All wild-type or mutant RGS proteins contained N-terminal His6. The accuracy of all constructions was checked by DNA sequencing. For expression in Sf9 cells, cDNAs were removed from pQE60 withNcoI and BamHI and inserted into pVL1392 modified to include a NcoI site at the 5′ end of the multiple cloning site. Recombinant baculoviruses were produced as described previously (27Parker E.M. Kameyama K. Higashijima T. Ross E.M. J. Biol. Chem. 1991; 266: 519-527Abstract Full Text PDF PubMed Google Scholar). The plasmid that encodes RGS10 box was prepared as described (30Popov S. Yu K. Kozasa T. Wilkie T.M. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 7216-7220Crossref PubMed Scopus (149) Google Scholar). For labeling with [3H]palmitate and small scale preparation, Sf9 cells (4 × 106) were infected with RGS4 or RGS10 virus for 32 h prior to metabolic labeling. Sodium [3H]palmitate (1 mCi) was suspended in 2 ml of IPL-41 that contained 1% ethanol and 2% heat-inactivated fetal calf serum. Cells were incubated in this medium for 1 h. The cells were harvested by centrifugation, washed with 2 ml of phosphate-buffered saline, and suspended in 0.5 ml of lysis buffer (20 mm NaHepes (pH 8.0), 2 mm MgCl2, 1 mm 2-mercaptoethanol, 1 mm phenylmethylsulfonyl fluoride, 20 μg/ml leupeptin, 10 μg/ml aprotinin). Cells were lysed by 5 freeze/thaw cycles, followed 10 passes through a 25-gauge needle. The lysates were centrifuged at 100,000 × g at 4 °C for 20 min in a Beckman TL100.3 rotor. The supernatant (cytosolic fraction) was removed, and the pellet (crude membrane) was resuspended in lysis buffer. Both fractions could then be analyzed by SDS-PAGE. For purification of active RGS protein, the pellet was solubilized by resuspension and stirring for 30 min at 4 °C in 100 mmNaCl, 50 mm NaHepes (pH 8.0), 0.5% deoxycholate, 0.5% Triton X-100, 0.1% SDS, 1 mm 2-mercaptoethanol, and the protease inhibitors listed above. After centrifugation for 30 min as described above, the supernatant was diluted 5-fold with 50 mm NaHepes (pH 8.0) and applied to nitrilotriacetic acid-Ni2+-agarose and purified as described for RGS proteins expressed in E. coli (24Wang J. Ducret A. Tu Y. Kozasa T. Aebersold R. Ross E.M. J. Biol. Chem. 1998; 273: 26014-26025Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar). RGS4 from the cytoplasmic fraction was purified similarly. Autopalmitoylation of RGS proteins was performed as described previously for Gα subunits with slight modification (4Duncan J.A. Gilman A.G. J. Biol. Chem. 1996; 271: 23594-23600Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar, 10Tu Y. Wang J. Ross E.M. Science. 1997; 278: 1132-1135Crossref PubMed Scopus (130) Google Scholar). Routinely, 5 μM RGS protein was incubated with 100 μM radioactive or non-radioactive Pal-CoA for up to 6 h at 30 °C in 50 mm NaHepes (pH 8.0), 0.005% Lubrol PX, and 100 μM 2-mercaptoethanol. Residual free Pal-CoA was removed by adsorbing the palmitoylated RGS protein to nitrilotriacetic acid-Ni2+-agarose followed by elution as described (24Wang J. Ducret A. Tu Y. Kozasa T. Aebersold R. Ross E.M. J. Biol. Chem. 1998; 273: 26014-26025Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar). To assess the degree of palmitoylation, the preparation was either precipitated with 10% trichloroacetic acid and isolated either on a glass fiber filter (10Tu Y. Wang J. Ross E.M. Science. 1997; 278: 1132-1135Crossref PubMed Scopus (130) Google Scholar) or by SDS-PAGE before liquid scintillation counting. Gels were stained with Coomassie Blue to detect proteins and appropriate slices were solubilized with 30% H2O2 for 16 h at 60 °C. RGS concentrations were determined by Amido Black binding (31Schaffner W. Weissmann C. Anal. Biochem. 1973; 56: 502-514Crossref PubMed Scopus (1953) Google Scholar) using bovine serum albumin as standard. SDS-PAGE was performed as described (10Tu Y. Wang J. Ross E.M. Science. 1997; 278: 1132-1135Crossref PubMed Scopus (130) Google Scholar). Gradient gels (15–22%) were used for CNBr-cleaved samples. Samples for PAGE were prepared as described (10Tu Y. Wang J. Ross E.M. Science. 1997; 278: 1132-1135Crossref PubMed Scopus (130) Google Scholar) to maintain palmitoylation during denaturation. Proteins were transferred to nitrocellulose as described (10Tu Y. Wang J. Ross E.M. Science. 1997; 278: 1132-1135Crossref PubMed Scopus (130) Google Scholar) and immunoblots were developed according to instructions in the ECL kit (Amersham Pharmacia Biotech). RGS4 (10 μg/ml, 50–100 pmol) in 70% (v/v) formic acid was mixed with 100 mg/ml CNBr and 1 mg/ml tryptophan and was incubated under argon in the dark at room temperature for 20 h. Products were diluted in water and dried under vacuum. Palmitoylated or non-palmitoylated RGS4 was alkylated by incubation with 15 mm N-ethylmaleimide to block free thiol groups and digested with CNBr. The mixture was resolved with SDS-PAGE, the gels were stained by Copper Stain (Bio-Rad), and appropriate bands were cut and extracted with 17% formic acid, 33% 2-propanol (32Cohen S.L. Chait B.T. Anal. Biochem. 1997; 247: 257-267Crossref PubMed Scopus (165) Google Scholar). The molecular masses of peptides were determined by matrix-assisted laser desorption ionization/time of flight spectrometry using a matrix of 3,5-dimethoxy-4-hydroxycinnamic acid on a Voyager-DE spectrometer (PE Biosystems). GAP activity was assayed in two formats. In the simpler assay, purified Gα is first bound to [γ -32P]GTP, and the rate of hydrolysis of the [γ-32P]GTP-Gα is measured in detergent solution in the presence and absence of the GAP. In this assay, GAP activity is defined as the increase in the first-order hydrolysis rate constant or is approximated as an increase in the initial rate of hydrolysis (26Wang J. Tu Y. Woodson J. Song X. Ross E.M. J. Biol. Chem. 1997; 272: 5732-5740Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar,33Wang J. Tu Y. Mukhopadhyay S. Chidiac P. Biddlecome G.H. Ross E.M. Manning D.R. G Proteins: Techniques of Analysis. CRC Press, Boca Raton, FL1999: 123-151Google Scholar). Such single-turnover GAP assays, using either ∼2 nmGαz-[γ-32P]GTP at 15 °C or ∼10 nm Gαi1-[γ-32P]GTP at 8 °C, were performed as described (10Tu Y. Wang J. Ross E.M. Science. 1997; 278: 1132-1135Crossref PubMed Scopus (130) Google Scholar, 26Wang J. Tu Y. Woodson J. Song X. Ross E.M. J. Biol. Chem. 1997; 272: 5732-5740Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). A more sensitive and presumably more physiological assay for GAP activity monitors the enhancement of agonist-stimulated steady-state GTPase activity in proteoliposomes reconstituted with receptor and heterotrimeric G protein. Reconstitution of purified m2AChR with either Gior Gz and was performed as described (24Wang J. Ducret A. Tu Y. Kozasa T. Aebersold R. Ross E.M. J. Biol. Chem. 1998; 273: 26014-26025Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar). RGS proteins were usually incubated with the vesicles for 1 h at 30 °C prior to assay. Conditions for measuring carbachol-stimulated GTPase activity in this system have been described (24Wang J. Ducret A. Tu Y. Kozasa T. Aebersold R. Ross E.M. J. Biol. Chem. 1998; 273: 26014-26025Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar). When RGS10 or RGS4 were incubated with [3H]Pal-CoA under the conditions originally described by Duncan and Gilman (4Duncan J.A. Gilman A.G. J. Biol. Chem. 1996; 271: 23594-23600Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar) for autopalmitoylation of Gα subunits, they incorporated 3H through a bond that was sensitive to both NH2OH and DTT (Fig. 1 A), presumably a palmitoyl thioester. Both RGS4 (13Srinivasa 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) and RGS10 are also naturally palmitoylated in Sf9 cells, as indicated by labeling with [3H]palmitic acid (Fig. 1 B). In vivo [3H]palmitoylation was essentially limited to the RGS protein found in the particulate fraction. Little label was found on soluble RGS protein despite the fact that about 30% of RGS4 and almost all of the RGS10 were found in the cytoplasmic fraction. The extent of in vitro autopalmitoylation of RGS10 was approximately 1 mol of palmitate/mol (Fig. 1 A). A truncated RGS10 consisting only of the conserved RGS box was also labeled to about 1 mol/mol with approximately similar kinetics. Because Cys66 is the only Cys residue in the RGS10 box, its palmitoylation accounts for that observed in full-length RGS10. Intact RGS4 incorporated 2 mol of palmitate/mol, but an N-terminal truncation mutant of RGS4 was labeled to only 1 mol/mol. This finding suggests that one palmitoylation site lies in the RGS4 box and that the other lies in the N-terminal region. Because Cys66 in RGS10 is the only highly conserved Cys residue in the RGS protein family, its palmitoylation suggests that the corresponding Cys residue in RGS4, Cys95, might be the site of palmitoylation in the RGS4 box. Both Cys2 and Cys12 are candidate sites for the N-terminal palmitoylation site (13Srinivasa 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, 14Druey 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), and we did not try to distinguish between them in this study. We analyzed the palmitoylation sites of RGS4 by a combination of CNBr peptide mapping and mutagenesis. As shown in Fig. 2 A, autopalmitoylated RGS4 contained [3H]palmitate in two CNBr-generated peptides, one of about 15,000 Da and the other of about 2000 Da. Both peptides were also labeled in RGS4 that had been palmitoylated in Sf9 cells. The palmitoylated peptides were identified both by Edman sequencing and mass spectrometry as Lys20–Met141, which includes most of the RGS4 box, and Cys2–Met19. Comparison of the masses of these peptides in samples prepared from palmitoylated and non-palmitoylated samples indicated that each incorporated 1 palmitoyl group. Mutation of Cys95 decreased palmitoylation of intact RGS4 that was labeled either in vitro or in vivo, and essentially eliminated incorporation of palmitate into the ∼15-kDa Lys20–Met141 peptide (Fig. 2 B). This finding, coupled with the unique palmitoylation of the homologous Cys66 residue in RGS10, indicates that Cys95 is the site of palmitoylation in the RGS4 box. In autopalmitoylated RGS4, Cys95 and the more N-terminal site were each labeled to approximately 1 mol/mol. In Sf9 cells, however, more [3H]palmitate was incorporated into the N-terminal site than into Cys95 under our standard conditions for Sf9 cell growth. This difference might reflect either relatively more incorporation of palmitate into the N-terminal site or more complete turnover at that site, although net incorporation of label into RGS4 was essentially complete within 1 h. To study the interdependence of palmitoylation of the N-terminal region and the RGS box, we mutated Cys2 and Cys12, which are probable sites of cellular palmitoylation in both RGS4 and RGS16 (13Srinivasa 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, 14Druey 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). Surprisingly, mutation of these sites eliminated all RGS4 autopalmitoylation, including autopalmitoylation of Cys95 (Fig. 3 B). To determine whether N-terminal palmitoylation precedes Cys95 autopalmitoylation in wild-type RGS4, we monitored autopalmitoylation of both sites in vitro. As shown in Fig. 3 C, autopalmitoylation at Cys95 lagged somewhat behind autopalmitoylation at the N terminus. While the lag was not great, it was reproducible in three similar experiments. These findings indicate that palmitoylation of the N-terminal site is kinetically favored over Cys95 palmitoylation. Consistent with this idea, the ΔN57 mutant of RGS4 and RGS10 (which is not N-terminally palmitoylated) both autopalmitoylate very slowly (t 1/2 ∼ 2 h, compared with at 1/2 of 30 min for wild-type RGS4). These results suggest that initial N-terminal palmitoylation promotes subsequent palmitoylation in the RGS4 box. N-terminal palmitoylation is not absolutely required for autopalmitoylation within the RGS box because both Cys95 in the RGS4 box construct and Cys66 in RGS10 do slowly autopalmitoylate. Coupling of palmitoylation in the RGS box with that in the N-terminal region also appears to hold true in cells, where the C2A/C12A mutation eliminates all RGS4 palmitoylation (13Srinivasa 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). Autopalmitoylation of RGS4 inhibited its GAP activity essentially completely when activity was measured in a single-turnover assay in detergent solution (Fig. 3 A). Upon incubation with Pal-CoA, GAP activity declined with a time course similar to that of incorporation of palmitate (data not shown; see Fig. 4 for similar data for RGS10). Incubation of autopalmitoylated RGS4 with DTT largely reversed inhibition (Fig. 3 A), and incubation of RGS4 without Pal-CoA was without effect. The GAP assays shown in Fig. 3 A used Gαz-GTP as substrate, but similar results were obtained in similar experiments that used Gαi1-GTP as substrate and Triton X-100 instead of Lubrol PX. Mutation of Cys95 in RGS4 blocked inhibition of GAP activity by incubation with Pal-CoA, suggesting that inhibition is mediated by palmitoylation of Cys95 (Fig. 3 B; see also Fig. 7 A). As would be predicted by its inability to incorporate palmitate at Cys95, Both C2A/C12A and C2A/C12A/C33A RGS4 were also not inhibited by incubation with Pal-CoA (Fig. 3 B). Palmitoylation also inhibited the GAP activity of RGS10 when activity was measured in the solution-based assay (Fig. 4). Again, fractional inhibition of GAP activity paralleled fractional palmitoylation and suggested that stoichiometrically palmitoylated RGS10 is inhibited 80–90% at this substrate concentration. Control incubations without Pal-CoA did not cause inhibition (Fig. 4), and inhibition was reversed by incubation with DTT (not shown). Because RGS10 is palmitoylated only on Cys66, these data combine with those of Fig. 3 to indicate that palmitoylation of the conserved Cys residue in the RGS box blocks the GAP activity of these RGS proteins in the single-turnover assay. In contrast to the inhibition described above, palmitoylation of RGS10 markedly stimulated its GAP activity as measured during receptor-stimulated, steady-state GTP hydrolysis. The agonist-stimulated GTPase activity of unilamellar phospholipid vesicles that contained heterotrimeric Gi and m2AChR was measured in the presence of increasing concentrations of RGS10 (Fig. 5). When agonist-bound receptor drives GDP/GTP exchange in these vesicles, hydrolysis of Gi-bound GTP becomes rate-limiting and a GAP increases steady-state hydrolysis until the overall reaction again approaches the rate of receptor-catalyzed GDP/GTP exchange (29Biddlecome G.H. Berstein G. Ross E.M. J. Biol. Chem. 1996; 271: 7999-8007Abstract Full Text Full Text PDF PubMed Scopus (212) Google Scholar, 34Posner B.A. Mukhopadhyay S. Tesmer J.J. Gilman A.G. Ross E.M. Biochemistry. 1999; 38: 7773-7779Crossref PubMed Scopus (68) Google Scholar, 35Mukhopadhyay S. Ross E.M. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 9539-9544Crossref PubMed Scopus (154) Google Scholar). In m2AChR-Gi vesicles, RGS10 increases agonist-stimulated GTPase activity about 10-fold at 5 μM, the highest concentration tested (Fig. 5). Palmitoylation at Cys66 markedly potentiated the GAP activity of RGS10. Although we wer" @default.
• W2117918340 created "2016-06-24" @default.
• W2117918340 creator A5036589276 @default.
• W2117918340 creator A5040281068 @default.
• W2117918340 creator A5046801159 @default.
• W2117918340 creator A5057979687 @default.
• W2117918340 date "1999-12-01" @default.
• W2117918340 modified "2023-10-18" @default.
• W2117918340 title "Palmitoylation of a Conserved Cysteine in the Regulator of G Protein Signaling (RGS) Domain Modulates the GTPase-activating Activity of RGS4 and RGS10" @default.
• W2117918340 cites W136758419 @default.
• W2117918340 cites W1510104405 @default.
• W2117918340 cites W1540505454 @default.
• W2117918340 cites W1554226168 @default.
• W2117918340 cites W1593507398 @default.
• W2117918340 cites W1965307343 @default.
• W2117918340 cites W1966434597 @default.
• W2117918340 cites W1968129691 @default.
• W2117918340 cites W1968536472 @default.
• W2117918340 cites W1976395664 @default.
• W2117918340 cites W1979746078 @default.
• W2117918340 cites W1985420287 @default.
• W2117918340 cites W1989347792 @default.
• W2117918340 cites W1993485378 @default.
• W2117918340 cites W1995686520 @default.
• W2117918340 cites W1999175477 @default.
• W2117918340 cites W2013741213 @default.
• W2117918340 cites W2020438797 @default.
• W2117918340 cites W2022391435 @default.
• W2117918340 cites W2041611018 @default.
• W2117918340 cites W2050307742 @default.
• W2117918340 cites W2053069121 @default.
• W2117918340 cites W2053217010 @default.
• W2117918340 cites W2064096152 @default.
• W2117918340 cites W2067207344 @default.
• W2117918340 cites W2071242185 @default.
• W2117918340 cites W2073209349 @default.
• W2117918340 cites W2082497364 @default.
• W2117918340 cites W2091240754 @default.
• W2117918340 cites W2093331733 @default.
• W2117918340 cites W2110458673 @default.
• W2117918340 cites W2118656164 @default.
• W2117918340 cites W2124367646 @default.
• W2117918340 cites W2126828900 @default.
• W2117918340 cites W2133453479 @default.
• W2117918340 cites W2147353099 @default.
• W2117918340 cites W2148310842 @default.
• W2117918340 cites W2162884305 @default.
• W2117918340 cites W2165888364 @default.
• W2117918340 cites W2170755436 @default.
• W2117918340 cites W2172052207 @default.
• W2117918340 cites W2187773005 @default.
• W2117918340 cites W2413435377 @default.
• W2117918340 doi "https://doi.org/10.1074/jbc.274.53.38260" @default.
• W2117918340 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/10608901" @default.
• W2117918340 hasPublicationYear "1999" @default.
• W2117918340 type Work @default.
• W2117918340 sameAs 2117918340 @default.
• W2117918340 citedByCount "97" @default.
• W2117918340 countsByYear W21179183402012 @default.
• W2117918340 countsByYear W21179183402014 @default.
• W2117918340 countsByYear W21179183402015 @default.
• W2117918340 countsByYear W21179183402016 @default.
• W2117918340 countsByYear W21179183402017 @default.
• W2117918340 countsByYear W21179183402018 @default.
• W2117918340 countsByYear W21179183402019 @default.
• W2117918340 countsByYear W21179183402020 @default.
• W2117918340 countsByYear W21179183402021 @default.
• W2117918340 countsByYear W21179183402023 @default.
• W2117918340 crossrefType "journal-article" @default.
• W2117918340 hasAuthorship W2117918340A5036589276 @default.
• W2117918340 hasAuthorship W2117918340A5040281068 @default.
• W2117918340 hasAuthorship W2117918340A5046801159 @default.
• W2117918340 hasAuthorship W2117918340A5057979687 @default.
• W2117918340 hasBestOaLocation W21179183401 @default.
• W2117918340 hasConcept C104317684 @default.
• W2117918340 hasConcept C146727910 @default.
• W2117918340 hasConcept C163181125 @default.
• W2117918340 hasConcept C181199279 @default.
• W2117918340 hasConcept C185592680 @default.
• W2117918340 hasConcept C207332259 @default.
• W2117918340 hasConcept C2777993845 @default.
• W2117918340 hasConcept C2779201268 @default.
• W2117918340 hasConcept C2780298669 @default.
• W2117918340 hasConcept C4963665 @default.
• W2117918340 hasConcept C55493867 @default.
• W2117918340 hasConcept C62478195 @default.
• W2117918340 hasConcept C6929976 @default.
• W2117918340 hasConcept C80631254 @default.
• W2117918340 hasConcept C86803240 @default.
• W2117918340 hasConcept C95444343 @default.
• W2117918340 hasConceptScore W2117918340C104317684 @default.
• W2117918340 hasConceptScore W2117918340C146727910 @default.
• W2117918340 hasConceptScore W2117918340C163181125 @default.
• W2117918340 hasConceptScore W2117918340C181199279 @default.
• W2117918340 hasConceptScore W2117918340C185592680 @default.
• W2117918340 hasConceptScore W2117918340C207332259 @default.
• W2117918340 hasConceptScore W2117918340C2777993845 @default.
• W2117918340 hasConceptScore W2117918340C2779201268 @default.

• next