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• W1521524023 abstract "In this study, we establish that the V1a vasopressin receptor (V1aR) is palmitoylated, and we show that this modification has an important functional role. Palmitoylation of the V1aR occurs within the Cys371/Cys372 couplet located in the proximal C-terminal tail domain. Substitution of these residues in a [C371G/C372G]V1aR construct effectively disrupted receptor palmitoylation. Our data also indicate an additional palmitoylation site at another locus in the receptor, as yet undefined. [3H]Palmitate incorporation was agonist-sensitive and increased following exposure to [Arg8]vasopressin (AVP). Given the hydrophobic nature of the acyl chain, palmitoylation of the C terminus of G-protein-coupled receptors has been proposed to form an additional intracellular loop. Consequently, palmitoylation/depalmitoylation will have a profound effect on the local conformation of this domain. The V1aR palmitoylation status regulated both phosphorylation and sequestration of the receptor, and furthermore, palmitoylation, phosphorylation, and sequestration were all regulated by AVP. The palmitoylation-defective construct [C371G/C372G]V1aR exhibited decreased phosphorylation compared to wild-type V1aR, under both basal and AVP-stimulated conditions, and was sequestered at a faster rate. In contrast, the binding of four different classes of ligand and intracellular signaling were not affected by palmitoylation. This study therefore establishes that there are different conformational requirements for signaling, agonist-induced phosphorylation, and sequestration of the V1aR. In this study, we establish that the V1a vasopressin receptor (V1aR) is palmitoylated, and we show that this modification has an important functional role. Palmitoylation of the V1aR occurs within the Cys371/Cys372 couplet located in the proximal C-terminal tail domain. Substitution of these residues in a [C371G/C372G]V1aR construct effectively disrupted receptor palmitoylation. Our data also indicate an additional palmitoylation site at another locus in the receptor, as yet undefined. [3H]Palmitate incorporation was agonist-sensitive and increased following exposure to [Arg8]vasopressin (AVP). Given the hydrophobic nature of the acyl chain, palmitoylation of the C terminus of G-protein-coupled receptors has been proposed to form an additional intracellular loop. Consequently, palmitoylation/depalmitoylation will have a profound effect on the local conformation of this domain. The V1aR palmitoylation status regulated both phosphorylation and sequestration of the receptor, and furthermore, palmitoylation, phosphorylation, and sequestration were all regulated by AVP. The palmitoylation-defective construct [C371G/C372G]V1aR exhibited decreased phosphorylation compared to wild-type V1aR, under both basal and AVP-stimulated conditions, and was sequestered at a faster rate. In contrast, the binding of four different classes of ligand and intracellular signaling were not affected by palmitoylation. This study therefore establishes that there are different conformational requirements for signaling, agonist-induced phosphorylation, and sequestration of the V1aR. G-protein-coupled receptors [Arg8]vasopressin Dulbecco's modified Eagle's medium fetal bovine serum InsP2, InsP3, and InsP4, inositol mono-, bis-, tris, and tetrakisphosphates, respectively hemagglutinin μ-opioid receptor phenylacetyl thyrotropin-stimulating hormone thyrotropin-releasing hormone receptors vasopressin V1a receptor vasopressin V1b receptor vasopressin V2 receptor Chinese hamster ovary polyacrylamide gel electrophoresis phosphate-buffered saline Post-translational modifications such as palmitoylation are important to the structure and function of a variety of proteins involved in cell signaling (reviewed in Refs. 1Casey P.J. Science. 1995; 268: 221-225Crossref PubMed Scopus (729) Google Scholar and 2Dunphy J.T. Linder M.E. Biochim. Biophys. Acta. 1998; 1436: 245-261Crossref PubMed Scopus (316) Google Scholar). Palmitoylation is a reversible covalent lipid modification, occurring in the post-endoplasmic reticulum or pre-Golgi compartment, involving the attachment of a C16 fatty acyl chain to cysteinyl residues of a nascent protein via a thioester bond (3Bonatti S. Migliaccio G. Simons K. J. Biol. Chem. 1989; 264: 12590-12595Abstract Full Text PDF PubMed Google Scholar). Investigating the role of palmitoylation has been undertaken for several G-protein-coupled receptors (GPCRs).1 However, the functional ramifications of palmitoylation are not predictable. Although no reliable consensus sequence for palmitoylation has been identified as yet, it has been found that many GPCRs contain one or more modified cysteine residues in the proximal region of the C-terminal tail (Fig. 1). Exceptions do occur. For example, the metabotropic glutamate mGluR1α receptor is not acylated (4Alaluf S. Mulvihill E.R. McIlhinney R.A.J. J. Neurochem. 1995; 64: 1548-1555Crossref PubMed Scopus (33) Google Scholar), and in the human α2C-adrenergic receptor a phenylalanine replaces the conserved cysteine (5Regan J.W. Kobilka T.S. Yang-Feng T.L. Caron M.G. Lefkowitz R.J. Kobilka B.K. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 6301-6305Crossref PubMed Scopus (375) Google Scholar). Moreover, the human gonadotropin-releasing hormone receptor completely lacks a C-terminal domain (6Davidson J.S. Flanagan C.A. Becker I.I. Illing N. Sealfon S.C. Millar R.P. Mol. Cell. Endocrinol. 1994; 100: 9-14Crossref PubMed Scopus (31) Google Scholar). To determine the precise role of palmitoylation for each GPCR, it is important to establish which cysteinyl residues are actually modified. Palmitoylation has been studied for a number of GPCRs, including those for biogenic amines, peptides, glycoproteins, and metabotropic receptors (Fig. 1). These studies can be divided into three main categories dictated by the rigor of the investigation: (i) GPCRs that have been shown directly to be palmitoylated by [3H]palmitate labeling and the locus of the modification established by mutagenesis of defined cysteinyl residues; (ii) GPCRs that have been shown to be palmitoylated but the precise locus of the modification has not been defined; and (iii) mutagenesis of cysteinyl residues within GPCRs that are merely putative palmitoylation sites, without establishing that they are actually derivatized. GPCRs that have been shown to be palmitoylated at specific cysteine(s) include rhodopsin (7Ovchinnikov Y.A. Abdulaev N.G. Bogachuk A.S. FEBS Lett. 1988; 230: 1-5Crossref PubMed Scopus (303) Google Scholar), α2A-adrenergic receptor (8Kennedy M.E. Limbird L.E. J. Biol. Chem. 1994; 269: 31915-31922Abstract Full Text PDF PubMed Google Scholar), β2-adrenergic receptor (9O'Dowd B.F. Hinatowich M. Caron M.G. Lefkowitz R.J. Bouvier M. J. Biol. Chem. 1989; 264: 7564-7569Abstract Full Text PDF PubMed Google Scholar), endothelin ETB(10Okamoto Y. Ninomiya H. Tanioka M. Sakamoto A. Miwa S. Masaki T. J. Biol. Chem. 1997; 272: 21589-21596Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar), human TSH (11Tanaka K. Nagayama Y. Nishihara E. Namba H. Yamashit S. Niwa M. Endocrinology. 1998; 139: 803-806Crossref PubMed Scopus (53) Google Scholar), luteinizing hormone/human chorionic gonadotropin (12Kawate N. Menon K.M.J. J. Biol. Chem. 1994; 269: 30651-30658Abstract Full Text PDF PubMed Google Scholar, 13Zhu H. Wang H. Ascoli M. Mol. Endocrinol. 1995; 9: 141-150Crossref PubMed Google Scholar), human m2 mAChR (14Hayashi M.K. Haga T. Arch. Biochem. Biophys. 1997; 340: 367-382Crossref Scopus (62) Google Scholar), vasopressin V2(15Sadeghi H.M. Innamorati G. Dagarag M. Birnbaumer M. Mol. Pharmacol. 1997; 52: 21-29Crossref PubMed Scopus (63) Google Scholar), dopamine D1 (16Jin H. Xie Z. George S. O'Dowd B.F. Eur. J. Pharmacol. 1999; 386: 305-312Crossref PubMed Scopus (34) Google Scholar), and the metabotropic glutamate mGluR4 and mGluR6 receptors (4Alaluf S. Mulvihill E.R. McIlhinney R.A.J. J. Neurochem. 1995; 64: 1548-1555Crossref PubMed Scopus (33) Google Scholar, 17Pickering D.S. Taverna F.A. Salter M.W. Hampson D.R. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 12090-12094Crossref PubMed Scopus (72) Google Scholar). Other GPCRs that have been demonstrated to be palmitoylated at an undefined locus are dopamine D2 (18Ng G.Y.K. Mouillac B. George S.R. Caron M. Dennis M. Bouvier M. O'Dowd B.F. Eur. J. Pharmacol. 1994; 267: 7-19Crossref PubMed Scopus (157) Google Scholar, 19Grünewald S. Haase W. Reiländer H. Michel H. Biochemistry. 1990; 35: 15149-15161Crossref Scopus (72) Google Scholar), serotonin 5-HT1B and 5-HT4(a) (20Ng G.Y.K. George S.R. Zastawny R.L. Caron M. Bouvier M. Dennis M. O'Dowd B.F. Biochemistry. 1993; 32: 11727-11733Crossref PubMed Scopus (137) Google Scholar, 21Ponimaskin E.G. Schmidt M.F.G. Heine M. Bickmeyer U. Richter D.W. Biochem. J. 2001; 353: 627-634Crossref PubMed Scopus (44) Google Scholar), μ-opioid (MOR) (22Chen C. Shahabi V. Xu W. Liu-Chen L.-Y. FEBS Lett. 1998; 441: 148-152Crossref PubMed Scopus (27) Google Scholar), and endothelin ETA receptors (23Horstmeyer A. Cramer H. Sauer T. Müller-Esterl W. Schroeder C. J. Biol. Chem. 1996; 271: 20811-20819Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar). The porcine m2 mAChR (24Van Koppen C.J. Nathanson N.M. J. Neurochem. 1991; 57: 1873-1877Crossref PubMed Scopus (25) Google Scholar), rat thyrotropin receptor (TSH) (25Kosugi S. Mori T. Biochem. Biophys. Res. Commun. 1996; 221: 636-640Crossref PubMed Scopus (21) Google Scholar), and thyrotropin-releasing hormone receptors (TRH) (26Nussenzveig D.R. Heinflink M. Gershengorn M.C. J. Biol. Chem. 1993; 268: 2389-2392Abstract Full Text PDF PubMed Google Scholar) have been studied following mutation of putative palmitoylation sites. The hydrophobic nature of the acyl chain suggests that the palmitate moiety of GPCRs would embed into the membrane to form a fourth receptor intracellular loop. The use of fluorescent fatty acid analogs with rhodopsin has provided direct evidence that this is indeed the case (27Moench S.J. Moreland J. Stewart D.H. Dewey T.G. Biochemistry. 1994; 33: 5791-5796Crossref PubMed Scopus (60) Google Scholar). This additional membrane anchorage point does not fulfill a common role throughout the receptor protein family as palmitoylation has been reported to regulate diverse aspects of GPCR function including the following: (i) receptor:G-protein coupling (9O'Dowd B.F. Hinatowich M. Caron M.G. Lefkowitz R.J. Bouvier M. J. Biol. Chem. 1989; 264: 7564-7569Abstract Full Text PDF PubMed Google Scholar, 10Okamoto Y. Ninomiya H. Tanioka M. Sakamoto A. Miwa S. Masaki T. J. Biol. Chem. 1997; 272: 21589-21596Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar, 23Horstmeyer A. Cramer H. Sauer T. Müller-Esterl W. Schroeder C. J. Biol. Chem. 1996; 271: 20811-20819Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar,28Morrison D.F. O'Brien P.J. Pepperberg D.R. J. Biol. Chem. 1991; 266: 20118-20123Abstract Full Text PDF PubMed Google Scholar); (ii) receptor expression (15Sadeghi H.M. Innamorati G. Dagarag M. Birnbaumer M. Mol. Pharmacol. 1997; 52: 21-29Crossref PubMed Scopus (63) Google Scholar, 24Van Koppen C.J. Nathanson N.M. J. Neurochem. 1991; 57: 1873-1877Crossref PubMed Scopus (25) Google Scholar); (iii) receptor phosphorylation (29Moffett S. Mouillac B. Bonin H. Bouvier M. EMBO J. 1993; 12: 349-356Crossref PubMed Scopus (142) Google Scholar); and (iv) agonist-induced desensitization/down-regulation (11Tanaka K. Nagayama Y. Nishihara E. Namba H. Yamashit S. Niwa M. Endocrinology. 1998; 139: 803-806Crossref PubMed Scopus (53) Google Scholar, 12Kawate N. Menon K.M.J. J. Biol. Chem. 1994; 269: 30651-30658Abstract Full Text PDF PubMed Google Scholar, 13Zhu H. Wang H. Ascoli M. Mol. Endocrinol. 1995; 9: 141-150Crossref PubMed Google Scholar,30Eason M.G. Jacinto M.T. Theiss C.T. Ligget S.B. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 11178-11182Crossref PubMed Scopus (96) Google Scholar, 31Schülein R. Liebenhoff U. Müller H. Birnbaumer M. Rosenthal W. Biochem. J. 1996; 313: 611-616Crossref PubMed Scopus (69) Google Scholar). The neurohypophysial peptide hormone [Arg8]vasopressin (AVP) generates a plethora of physiological responses including increasing blood pressure, antidiuresis, glycogenolysis, adrenocorticotropin secretion and regulating testis function, insulin secretion, and mitogenesis in some cells (32Howl J. Wheatley M. Gen. Pharmacol. 1995; 26: 1143-1152Crossref PubMed Scopus (55) Google Scholar). The majority of these effects are mediated by the vasopressin V1areceptor subtype (V1aR) with the exception of antidiuresis (V2 receptor subtype, V2R) and adrenocorticotropin secretion (V1b receptor subtype, V1bR). In this study, we demonstrate that the V1aR is palmitoylated at the Cys371/Cys372 couplet within the C-terminal domain, and we establish that this modification is agonist-regulated and has biological significance. Disruption of V1aR palmitoylation resulted in decreased receptor phosphorylation and increased the rate of agonist-induced internalization but did not affect ligand binding, intracellular signaling, or receptor expression. [[3,4,5-3H]Phe3]AVP, specific activity 37–68.5 Ci/mmol, 2-myo-[3H]inositol, specific activity 22.3 Ci/mmol, and [9,10-3H]palmitic acid, specific activity 52 Ci/mmol, were obtained from PerkinElmer Life Sciences; [32P]orthophosphate was obtained from Amersham Pharmacia Biotech. The linear peptide antagonist PhAcD-Tyr(Me)2Arg6Tyr(NH2)9AVP and cyclic antagonist d(CH2)5Tyr(Me)2AVP were from Bachem (Torrance, CA), and the non-peptide antagonist (SR 49059) was a gift from Dr. C. Serradiel-Le Gal (Sanofi Recherche, France). Dulbecco's modified Eagle's medium (DMEM), inositol-free DMEM, Iscove's DMEM, hypoxanthine/thymidine supplement, fetal bovine serum (FBS), trypsin/EDTA, and penicillin/streptomycin were from Life Technologies, Inc. The restriction enzymes Psp5II and Eco130I were obtained from MBI Fermentas (Sunderland, UK). All other reagents were of analytical grade. The two Cys residues (Cys371 and Cys372) corresponding to the putative palmitoylation site (Fig. 1) were mutated both individually and in combination. The Cys residues were mutated to Gly using a polymerase chain reaction approach (33Hawtin S.R. Wesley V.J. Parslow R.A. Patel S. Wheatley M. Biochemistry. 2000; 39: 13524-13533Crossref PubMed Scopus (28) Google Scholar) to generate the V1aR mutants [C371G]V1aR, [C372G]V1aR, and [C371G/C372G]V1aR. Mutagenic sense primers (incorporating the single base changes T→G (bold) for the Cys → Gly substitution (underlined) were 5′-C-CAA-AGT-TTC-CCA-GGC-TGC-CAC-AGC-3′, 5′-C-CCA-TGC-GGC-CAC-AGC-ATG-G-3′, and 5′-C-CAA-AGT-TTC-CCA-GGC-GGC-CAC-AGC-ATG-G-3′ (custom synthesized by Alta Bioscience, University of Birmingham, UK) for the C371G, C372G, and C371G/C372G mutations, respectively. For each mutation, Psp5II/Eco130I-digested fragments were subcloned into the pBluescript KSII vector containing an HA-epitope tagged V1aR at the N terminus (34Hawtin S.R. Wheatley M. Biochem. Soc. Trans. 1997; 25: 437SCrossref PubMed Google Scholar). Verification of each mutation and absence of possible Taq DNA polymerase-introduced errors was confirmed by automated sequencing (Alta Bioscience, University of Birmingham, UK). For mammalian expression, the wild-type and mutant V1aRs were subcloned into the expression vector pcDNA3 (Invitrogen) utilizing unique 5′-BamHI and 3′-EcoRI restriction sites. HEK 293T cells were cultured in DMEM supplemented with 10% (v/v) FBS, penicillin (100 units/ml), streptomycin (100 μg/ml). CHO-K1 cells were cultured in Iscove's DMEM containing hypoxanthine (100 μm), thymidine (16 μm), 10% (v/v) FBS, penicillin (100 units/ml) and streptomycin (100 μg/ml). Cells were maintained in a humidified 5% v/v CO2 incubator at 37 °C. HEK 293T cells were seeded at a density of ∼1 × 106 cells/100-mm dish and transfected using calcium phosphate precipitation. Briefly, the DNA-calcium phosphate co-precipitate, containing 10 μg of plasmid cDNA for each dish, was prepared 30 min before use. After incubation for 16 h, the medium was replaced by growth medium, and cells were incubated for a further 48 h before harvesting. HEK 293T cell membranes were prepared as described previously (35Wheatley M. Howl J. Yarwood N.J. Davies A.R.L. Parslow R.A. Methods Mol. Biol. 1997; 73: 305-332PubMed Google Scholar), and the protein concentration was determined using the Pierce BCA protein assay kit with BSA as standard. Binding assays were essentially as described previously (36Howl J. Wheatley M. Biochem. J. 1996; 317: 577-582Crossref PubMed Scopus (36) Google Scholar,37Howl J. Langel Ü. Hawtin S.R. Valkna A. Yarwood N.J. Saar K. Wheatley M. FASEB J. 1997; 11: 582-590Crossref PubMed Scopus (26) Google Scholar) comprising membranes (50–300 μg of protein) diluted in binding buffer (20 mm HEPES, 1 mm EGTA, 10 mm Mg(CH3COO2)2, 1 mg/ml BSA; pH 7.4), [[3,4,5-3H]Phe3]AVP (0.3–0.7 nm), and competing ligand at the concentration indicated in a final volume of 0.5 ml. Nonspecific binding was defined by a saturating concentration of unlabeled ligand (1 μm). After incubation for 90 min at 30 °C to establish equilibrium, bound and free ligands were separated by centrifugation at 12,000 ×g for 10 min. Membrane pellets were washed twice with water and solubilized with 50 μl of Soluene 350 (Packard Instrument Co.) prior to addition of 1 ml of HiSafe3TM (Wallac) liquid scintillation mixture for counting. HEK 293T cells were seeded at a density of 2.5 × 106cells/well in poly-d-lysine-treated 6-well plates and transfected as described above. AVP-induced accumulation of inositol phosphates was assayed as described previously (38Howl J. Rudge S.A. Lavis R.A. Davies A.R.L. Parslow R.A. Hughes P.J. Kirk C.J. Michell R.H. Wheatley M. Endocrinology. 1995; 136: 2206-2213Crossref PubMed Scopus (42) Google Scholar, 39Hawtin S.R. Howard H.C. Wheatley M. Biochem. J. 2001; 354: 272-465Crossref Google Scholar). Essentially, 24 h post-transfection, medium was replaced with serum-free inositol-free DMEM containing 2.5 μCi/ml 2-myo-[3H]inositol. After 36 h at 37 °C, cells were washed with PBS and incubated with medium containing 10 mm LiCl for 30 min, followed by the addition of AVP at the concentration indicated. Incubations were terminated by the adding 0.5 ml of 5% (v/v) perchloric acid containing 1 mm EDTA and 1 mg/ml phytic acid hydrolysate. Cells were pelleted at 12,000 × g for 5 min, supernatants neutralized on ice with 1.2 m KOH, 10 mm EDTA, and 50 mm HEPES, and loaded onto 0.8-ml Bio-Rad AG1-X8 columns (formate form). After elution of inositol (10 ml water) and glycerophosphoinositol (10 ml of 25 mmNH4COOH), a mixed inositol phosphate fraction containing inositol mono-, bis-, tris-, and tetrakisphosphates was eluted (10 ml of 1.25 m NH4COOH containing 0.1 mHCOOH). Radioactivity was determined by the addition of 10 ml of Ultima-FloTM AF scintillation fluid (Packard Instrument Co.). CHO-K1 cells were transfected using the calcium-phosphate precipitation method as described above for HEK 293T cells. After 48 h, cells were trypsinized, diluted with selection medium containing 2 mg/ml G418 (Life Technologies, Inc.), and distributed in 100-mm plates. After 10–14 days, G418-resistant clones were picked using cloning rings (Sigma) and seeded into 48-well plates. Clones were expanded in 6-well plates and screened for the presence of the V1aR by ligand binding and second messenger assays. Clones containing the V1aR were expanded in 96-well plates to obtain single cell clones and re-screened. CHO cells were plated onto 24-well plates at a density of 1 × 105 cells/well. After 16 h, cells were washed twice with ice-cold PBS, after which each well received 0.5 ml of binding buffer (described above) containing 2% (w/v) BSA, 1–2 nm[3H]AVP in the presence (nonspecific) or absence (total) of 1 μm AVP. Plates were incubated for 6 h on ice in the cold room before removal of the medium by aspiration. After two rinses with ice-cold PBS, 0.5 ml of 0.1 m NaOH was added to each well to extract radioactivity. After 15 min of incubation at 37 °C, the fluid from the plates was transferred to scintillation vials containing 8 ml of HiSafe3 scintillant mixture for counting. CHO cells were labeled with 400 μCi of [3H]palmitic acid in 1 ml of DMEM containing 1% (v/v) FBS for 16 h. The palmitic acid was initially dried under a stream of N2 before sequential dissolution in a minimum volume of Me2SO and finally in DMEM (1% v/v final concentration). Immunoprecipitation and analysis of vasopressin receptors was essentially as described previously (40Tobin A.B. Nahorski S.R. J. Biol. Chem. 1993; 268: 9817-9823Abstract Full Text PDF PubMed Google Scholar). Briefly, cells were solubilized by the addition of 0.7 ml of RIPA buffer (500 mm NaCl, 10 mm Tris, pH 8.0, 10 mmEDTA, 1% v/v Nonidet P-40, 0.1% v/v SDS, 0.5% w/v deoxycholic acid) to each well on ice for 15 min. Solubilized receptors (600 μl) were immunoprecipitated by incubation with the mouse monoclonal anti-HA antibody (12CA5, Roche Molecular Biochemicals) at a final concentration of 10 μg/ml for 1 h at 4 °C. The antigen-antibody complexes were separated by incubating the mixture with protein A-Sepharose beads (180 μl) for 15 min at 4 °C. The beads were centrifuged and washed three times with ice-cold TE buffer (10 mm Tris, pH 7.4, 2.5 mm EDTA). Samples were adjusted for equivalent receptor number by whole cell binding assay (described above) to CHO cells under identical conditions. Samples were denatured by incubation at 65 °C for 2 min in Laemmli sample buffer containing 50 mmβ-mercaptoethanol and resolved by 10% (w/v) SDS-PAGE. The polyacrylamide gel was treated with EnhanceTM (Packard Instrument Co.) and dried, and radioactive bands were visualized by exposure to Kodak X-Omat film for 5 weeks at −70 °C. To examine the effects of AVP on palmitic acid incorporation, pre-labeled cells were incubated with 10 μm AVP for 15 min at 37 °C prior to receptor solubilization. CHO cells were plated onto 24-well plates at a density of 1 × 105cells/well. After 16 h, cells were treated with 1 μmAVP at 37 °C to promote agonist-induced receptor internalization. After appropriate time intervals, cells were washed twice with ice-cold PBS on ice to remove excess ligand and terminate sequestration. Ligand was stripped from receptors by exposing cells for two rapid 40-s acid washes, pH 5.0, with 5 mm acetic acid, 150 mmNaCl, followed by three washes with ice-cold PBS to restore physiological pH to 7.4. The number of receptors at the cell surface was assayed by whole cell ligand binding as described above. CHO cells were plated onto 6-well plates at a density of 3 × 105 cells/well. After 16 h, cells were washed twice with phosphate-free Krebs buffer, followed by the addition of 50 μCi/well of [32P]H3PO4. After 2 h at 37 °C, cells were exposed to 10 μmAVP for 15 min and then chilled on ice and washed with PBS. The receptor proteins were extracted and immunoprecipitated as described above and analyzed by 10% (w/v) SDS-PAGE. The gels were dried and subjected to autoradiography. Radioligand binding results are expressed as a percentage of the control (i.e. specific binding in the absence of the competing ligand). IC50 values were determined by non-linear regression after the fitting of Langmuir binding isotherms to experimentally determined data using the Fig. P. Program (Biosoft, Milltown, NJ). Dissociation constants (Kd) and concentration of receptor sites (Bmax) were calculated from IC50values according to the method of Cheng and Prusoff (41Cheng Y. Prusoff W.H. Biochem. Pharmacol. 1973; 22: 3099-3108Crossref PubMed Scopus (12266) Google Scholar), using the experimentally determined value of affinity of [[3,4,5-3H[Phe3]AVP for each construct. EC50 values were determined by non-linear regression after fitting of logistic sigmoidal curves to experimental data. All data are quoted as the mean of three or more experiments ± S.E. unless otherwise stated. The V1aR possesses a cysteine couplet in the proximal region of the intracellular C-terminal tail domain. By analogy with other GPCRs, the location of these residues close to the seventh transmembrane domain suggests that Cys371 and Cys372 are putative palmitoylation sites (Figs. 1 and2). To assess the role of these cysteines in V1aR function, we constructed a series of mutant receptors in which the cysteines were replaced by glycine, individually and in combination. The C371G, C372G, and C371G/C372G receptor constructs generated were pharmacologically characterized after transient expression in HEK 293T cells and compared with wild-type V1aR. All receptor constructs were expressed at the same level of ∼0.5–1.5 pmol/mg protein (TableI). Four different classes of ligand were used to probe the pharmacology of the mutant receptors: (i) the cyclic natural agonist, AVP; (ii) cyclic antagonist, d(CH2)5Tyr(Me)2AVP; (iii) linear antagonist, PhAcD-Tyr(Me)2Arg6Tyr(NH2)9AVP; and (iv) non-peptide antagonist, SR 49059 (42Serradeil-Le Gal C. Wagnon J. Garcia C. Lacour C. Guiraudou P. Christophe B. Villanova G. Nisato D. Maffrand J.P. Le Fur G. J. Clin. Invest. 1993; 92: 224-231Crossref PubMed Scopus (268) Google Scholar). The competition binding curves from these radioligand binding studies are shown in Fig.3. The Kd values are presented in Table I, corrected for radioligand occupancy. These data established that substitution of Cys371 and Cys372, either individually or in combination, produced only minor effects on the affinity of all four classes of ligand tested.Table IPharmacological profile of wild-type and mutant vasopressin receptorsLigandBinding affinities Kd(nm)V1aRC371GC372GC371G/C372GAVP0.5 ± 0.11.5 ± 0.11.1 ± 0.12.1 ± 0.7PhAcD-Tyr(Me)2Arg6Tyr(NH2)9AVP0.3 ± 0.10.7 ± 0.10.2 ± 0.10.2 ± 0.1d(CH2)5Tyr(Me)2AVP0.6 ± 0.10.8 ± 0.20.7 ± 0.30.7 ± 0.2SR 490591.3 ± 0.51.2 ± 0.30.5 ± 0.11.4 ± 0.5Receptor expression (Bmax) (pmol/mg protein)0.8 ± 0.21.2 ± 0.10.5 ± 0.11.5 ± 0.4Wild-type and mutant V1aRs were expressed in HEK 293T cells and tested for their ability to bind AVP receptor ligands. [3H]AVP was used as radioligand tracer. IC50 values derived from competition binding experiments were determined by non-linear regression after fitting Langmuir binding isotherms using the Fig. P program. The affinity (Kd) and abundance (Bmax) of receptor constructs were calculated from IC50 values and corrected for radioligand occupancy as described under “Experimental Procedures.” Data shown are the mean ± S.E. (n = 3) of three replicates. Open table in a new tab Figure 3Ligand binding profile of wild-type and mutant V1aRs. Competition ligand binding curves are shown for wild-type V1aR (●), [C371G]V1aR (■), [C372G]V1aR (Δ), and [C371G/C372G]V1aR (▿) using agonist (a), cyclic peptide antagonist (b), linear peptide antagonist (c), and non-peptide antagonist (d). Data shown are the mean ± S.E. of three separate experiments each performed in triplicate. Values are expressed as % specific binding where nonspecific binding was defined by AVP (1 μm). A theoretical Langmuir isotherm competition curve was fitted to the experimental data as described under “Experimental Procedures.”View Large Image Figure ViewerDownload Hi-res image Download (PPT) Wild-type and mutant V1aRs were expressed in HEK 293T cells and tested for their ability to bind AVP receptor ligands. [3H]AVP was used as radioligand tracer. IC50 values derived from competition binding experiments were determined by non-linear regression after fitting Langmuir binding isotherms using the Fig. P program. The affinity (Kd) and abundance (Bmax) of receptor constructs were calculated from IC50 values and corrected for radioligand occupancy as described under “Experimental Procedures.” Data shown are the mean ± S.E. (n = 3) of three replicates. It was also important to establish if disruption of the cysteine couplet in the C-terminal tail of the V1aR affected the intracellular signaling capability of the receptor. The C371G, C372G, and C371G/C372G mutant V1aRs were characterized with respect to AVP-induced accumulation of InsP-InsP4. From the dose-response curves presented in Fig.4a, it can be seen that following challenge by AVP, all of the mutant V1aRs activated phosphoinositidase C in a manner comparable with wild-type receptor. In each case, maximum stimulation was observed at 10 nm AVP with EC50 values ranging from 0.5 to 1.0 nm (Fig. 4b). Consequently, ablation of the cysteine couplet in the proximal domain of the C terminus of the V1aR did not perturb either the ligand binding or intracellular signaling characteristics of the receptor. Prior to preparation of stable cell lines, a V1aR construct was engineered that incorporated a 9-residue influenza hemagglutinin (HA) epitope tag (YPYDVPDYA) inserted immediately after the initiation methionine (Fig. 2). This sequence is recognized by the 12CA5 antibody and was introduced to facilitate downstream analysis. We have established previously that introduction of this sequence at this locus in the V1aR does not perturb ligand recognition or intracellular signaling (34Hawtin S.R. Wheatley M. Biochem. Soc. Trans. 1997; 25: 437SCrossref PubMed Google Scholar). CHO-K1 cell lines were prepared exhibiting (i) stable expression of the HA epitope-tagged wild-type V1aR, and (ii) stable expression of the HA epitope-tagged C371G/C372G mutant. These were screened for both [3H]AVP binding and second messenger generation. The stable cell lines selected expressed the two receptor constructs at the same expression level of ∼2.5 pmol/mg protein. The binding profile for a range of ligands and the AVP-induced second messenger respo" @default.
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• W1521524023 date "2001-10-01" @default.
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• W1521524023 title "Palmitoylation of the Vasopressin V1a Receptor Reveals Different Conformational Requirements for Signaling, Agonist-induced Receptor Phosphorylation, and Sequestration" @default.
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• W1521524023 doi "https://doi.org/10.1074/jbc.m106142200" @default.
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