Portal de Periódicos da CAPES

Palmitoylation of the V2 Vasopressin Receptor Carboxyl Tail Enhances β-Arrestin Recruitment Leading to Efficient Receptor Endocytosis and ERK1/2 Activation

2003; Elsevier BV; Volume: 278; Issue: 42 Linguagem: Inglês

10.1074/jbc.m306589200

1083-351X

Pascale G. Charest, Michel Bouvier,

Neuropeptides and Animal Physiology

A large number of G protein-coupled receptors are palmitoylated on cysteine residues located in their carboxyl tail, but the general role of this post-translational modification remains poorly understood. Here we show that preventing palmitoylation of the V2 vasopressin receptor, by site-directed mutagenesis of cysteines 341 and 342, significantly delayed and decreased both agonist-promoted receptor endocytosis and mitogen-activated protein kinase activation. Pharmacological blockade of receptor endocytosis is without effect on the vasopressin-stimulated mitogen-activated protein kinase activity, excluding the possibility that the reduced kinase activation mediated by the palmitoylation-less mutant could result from altered receptor endocytosis. In contrast, two dominant negative mutants of β-arrestin which inhibit receptor endocytosis also attenuated vasopressin-stimulated mitogen-activated protein kinase activity, suggesting that the scaffolding protein, β-arrestin, represents the common link among receptor palmitoylation, endocytosis, and kinase activation. Coimmunoprecipitation and bioluminescence resonance energy transfer experiments confirmed that inhibiting receptor palmitoylation considerably reduced the vasopressin-stimulated recruitment of β-arrestin to the receptor. Interestingly, the changes in β-arrestin recruitment kinetics were similar to those observed for vasopressin-stimulated receptor endocytosis and mitogen-activated protein kinase activation. Taken together the results indicate that palmitoylation enhances the recruitment of β-arrestin to the activated V2 vasopressin receptor thus facilitating processes requiring the scaffolding action of β-arrestin. A large number of G protein-coupled receptors are palmitoylated on cysteine residues located in their carboxyl tail, but the general role of this post-translational modification remains poorly understood. Here we show that preventing palmitoylation of the V2 vasopressin receptor, by site-directed mutagenesis of cysteines 341 and 342, significantly delayed and decreased both agonist-promoted receptor endocytosis and mitogen-activated protein kinase activation. Pharmacological blockade of receptor endocytosis is without effect on the vasopressin-stimulated mitogen-activated protein kinase activity, excluding the possibility that the reduced kinase activation mediated by the palmitoylation-less mutant could result from altered receptor endocytosis. In contrast, two dominant negative mutants of β-arrestin which inhibit receptor endocytosis also attenuated vasopressin-stimulated mitogen-activated protein kinase activity, suggesting that the scaffolding protein, β-arrestin, represents the common link among receptor palmitoylation, endocytosis, and kinase activation. Coimmunoprecipitation and bioluminescence resonance energy transfer experiments confirmed that inhibiting receptor palmitoylation considerably reduced the vasopressin-stimulated recruitment of β-arrestin to the receptor. Interestingly, the changes in β-arrestin recruitment kinetics were similar to those observed for vasopressin-stimulated receptor endocytosis and mitogen-activated protein kinase activation. Taken together the results indicate that palmitoylation enhances the recruitment of β-arrestin to the activated V2 vasopressin receptor thus facilitating processes requiring the scaffolding action of β-arrestin. Among the molecular mechanisms regulating G protein-coupled receptor (GPCR) 1The abbreviations used are: GPCR, G protein-coupled receptor; AVP, arginine vasopressin; β2AR, β2-adrenergic receptor; BRET, bioluminescence resonance energy transfer; 2-BrP, 2-bromopalmitic acid; BSA, bovine serum albumin; DSP, dithiobis(succinimidyl propionate); ERK, extracellular signal-regulated kinase; FBS, fetal bovine serum; GFP, green fluorescent protein; HEK293, human embryonic kidney 293 cells; MAPK, mitogen-activated protein kinase; PBS, phosphate-buffered saline; P-ERK1/2, phosphorylated ERK1/2; V2R, V2 vasopressin receptor; wt, wild-type. function, post-translational modifications such as phosphorylation and palmitoylation have been the subject of numerous studies. The role of phosphorylation in receptor desensitization is now well established (1Ferguson S.S. Pharmacol. Rev. 2001; 53: 1-24PubMed Google Scholar). Upon activation, GPCRs become phosphorylated by both second messenger-activated and GPCR kinases on serine and threonine residues located in the third intracellular loop and/or carboxyl-terminal tail of the receptors. These phosphorylation events prevent further G protein interaction through the involvement of β-arrestin proteins preferentially binding to receptors phosphorylated by GPCR kinases. In addition to promoting receptor/G protein uncoupling, the β-arrestins target such desensitized receptors to clathrin-coated pits for endocytosis by functioning as adaptor proteins that link the receptors to components of the endocytic machinery. Furthermore, β-arrestin acts as a scaffolding protein directly linking the receptors to the mitogen-activated protein kinases (MAPK) signaling pathways, a process that has been shown to play an important role in GPCR-mediated MAPK activation (2Luttrell L.M. Lefkowitz R.J. J. Cell Sci. 2002; 115: 455-465Crossref PubMed Google Scholar). On the other hand, GPCR palmitoylation has also been shown to affect receptor function, but its precise role is not well defined. This post-translational modification, resulting from the covalent linkage of palmitic acid through a thioester bond, usually occurs at the level of conserved cysteine residues in the carboxyl tail of receptors. GPCR palmitoylation has been found to affect differentially a broad spectrum of biological processes, including G protein coupling efficiency and selectivity, receptor phosphorylation and desensitization, receptor endocytosis, cell surface expression and trafficking, and receptor down-regulation (3Qanbar R. Bouvier M. Pharmacol. Ther. 2003; 97: 1-33Crossref PubMed Scopus (201) Google Scholar). Despite the role that β-arrestin plays in several of these processes, the influence of receptor palmitoylation on the recruitment of the scaffolding protein has never been investigated. For the V2 vasopressin receptor (V2R), palmitoylation has been shown to occur on cysteine residues 341 and 342 in the carboxyl tail of the receptor (4Sadeghi H.M. Innamorati G. Dagarag M. Birnbaumer M. Mol. Pharmacol. 1997; 52: 21-29Crossref PubMed Scopus (63) Google Scholar). In two studies, mutation of these palmitoylation sites had no effect on the arginine vasopressin (AVP) binding affinity or agonist-stimulated adenylyl cyclase activity (4Sadeghi H.M. Innamorati G. Dagarag M. Birnbaumer M. Mol. Pharmacol. 1997; 52: 21-29Crossref PubMed Scopus (63) Google Scholar, 5Schülein R. Liebenhoff U. Muller H. Birnbaumer M. Rosenthal W. Biochem. J. 1996; 313: 611-616Crossref PubMed Scopus (69) Google Scholar). Similarly, no difference in agonist-promoted desensitization was observed between wild-type and the palmitoylation-less mutant V2R (4Sadeghi H.M. Innamorati G. Dagarag M. Birnbaumer M. Mol. Pharmacol. 1997; 52: 21-29Crossref PubMed Scopus (63) Google Scholar). In contrast, Schülein et al. (5Schülein R. Liebenhoff U. Muller H. Birnbaumer M. Rosenthal W. Biochem. J. 1996; 313: 611-616Crossref PubMed Scopus (69) Google Scholar) also reported that lack of palmitoylation was associated with a reduced rate of agonist-promoted endocytosis. However, this finding was not confirmed by Sadeghi et al. (4Sadeghi H.M. Innamorati G. Dagarag M. Birnbaumer M. Mol. Pharmacol. 1997; 52: 21-29Crossref PubMed Scopus (63) Google Scholar), where wild-type V2R and two palmitoylation-less V2R mutants (C341G/C342G, C341S/C342S) were reported to have identical AVP-promoted endocytosis. Although stimulation of adenylyl cyclase through Gs is the best characterized signaling cascade engaged by the V2R, extracellular signal-regulated kinases 1 and 2 (ERK1/2) have also been shown to be activated upon activation of the V2R by its natural ligand, AVP (6Thibonnier M. Conarty D.M. Preston J.A. Wilkins P.L. Berti-Mattera L.N. Mattera R. Adv. Exp. Med. Biol. 1998; 449: 251-276Crossref PubMed Scopus (127) Google Scholar). Among diverse mechanisms, β-arrestin recruitment and receptor endocytosis have often been proposed to be implicated in the activation of these MAPKs by GPCR. Despite the growing recognition that activation of MAPK by GPCR plays important roles in downstream signaling events (7Marinissen M.J. Gutkind J.S. Trends Pharmacol. Sci. 2001; 22: 368-376Abstract Full Text Full Text PDF PubMed Scopus (839) Google Scholar) and the putative role of palmitoylation in agonist-promoted endocytosis, very little is known of the potential role of receptor palmitoylation in MAPK activation. In the present study, we investigated the role of V2R palmitoylation on AVP-stimulated MAPK activation, receptor endocytosis, and β-arrestin recruitment. We report that the lack of palmitoylation at cysteines 341 and 342 significantly decreases the rate and extent of β-arrestin recruitment to the V2R, thus leading to slower and reduced receptor endocytosis and MAPK activation. Our results also indicate that although the scaffolding function of β-arrestin plays important roles in both AVP-stimulated endocytosis and MAPK activation, the latter can occur independently of receptor internalization. We thus suggest that V2R palmitoylation serves to induce and/or stabilize a particular receptor conformation, optimizing the interaction of β-arrestin with the receptor and facilitating β-arrestin-dependent downstream events such as ERK1/2 activation and endocytosis. Dulbecco's modified Eagle's medium, fetal bovine serum (FBS), penicillin, streptomycin, glutamine, Fungizone, G418, and phosphate-buffered saline (PBS) were all from Wisent, Inc. Cell culture plates and dishes were from Corning. Bovine serum albumin (BSA), AVP, 3-isobutylmethylxanthine, o-phenylenediamine dihydrochloride tablets (OPD peroxidase substrate), and tunicamycin were from Sigma. The Bio-Rad DC Protein Assay kit and the Bradford reagent were from Bio-Rad Laboratories. [3H]Adenine, [3H]palmitic acid, 32Pi, and ECL were from PerkinElmer Life Sciences. Antibodies recognizing ERK1/2 and their phosphorylated forms (P-ERK1/2), anti-myc mouse monoclonal 9E10, and rabbit polyclonal A14 IgGs were from Santa Cruz Biotechnology Inc. Anti-human (h)V2R AS435 antibody was a generous gift from W. Muller-Esterl (University of Frankfurt Medical School, Frankfurt). wtV2R and C341A/C342A-V2R—Wild-type hV2R was subcloned 3′ to the myc epitope sequence (MEQKLISEEDLNA) into the pBC12BI mammalian expression vector as described previously (8Morello J.P. Salahpour A. Laperriere A. Bernier V. Arthus M.F. Lonergan M. Petaja-Repo U. Angers S. Morin D. Bichet D.G. Bouvier M. J. Clin. Invest. 2000; 105: 887-895Crossref PubMed Scopus (477) Google Scholar). The myc epitope-tagged hV2R (mycV2R) was then subcloned into a pcDNA3 vector in which the cytomegalovirus promoter had been replaced by the Rous sarcoma virus (RSV) promoter. The V2R mutant lacking the palmitoylation sites cysteine 341 and cysteine 342 (C341A/C342A-V2R) was constructed by PCR site-directed mutagenesis of the cysteines into alanines using the wild-type pcDNA3-RSV-mycV2R. V2R-GFP and C341A/C342A-V2R-GFP—The coding sequence of the green fluorescent protein variant GFP10 (9Mercier J.F. Salahpour A. Angers S. Breit A. Bouvier M. J. Biol. Chem. 2002; 277: 44925-44931Abstract Full Text Full Text PDF PubMed Scopus (422) Google Scholar) was inserted in-frame 3′ of the wild-type V2R coding sequence into pcDNA3.1-V2R so that a 14-amino acid linker (GSGTAGPGSPPVAT) separated the carboxyl tail of the V2R and the initiator methionine of GFP10. The palmitoylation sites cysteine 341 and cysteine 342 were then replaced by alanine residues by PCR site-directed mutagenesis to generate the pcDNA3.1-C341A/C342A-V2R-GFP. β-Arrestin Constructs—For the β-arrestin-Rluc, the rat β-arrestin2 (arrestin3) coding sequence was inserted in-frame 5′ of the Renilla luciferase sequence in the pRluc vector (PerkinElmer Life Sciences) so that a 7-amino acid linker (GAGALAT) separated the carboxyl terminus of β-arrestin and the initiator methionine of Rluc. For the β-arrestin-GFP, the rat β-arrestin2 coding sequence was subcloned in-frame 5′ of the GFP10 coding sequence within the pcDNA3.1-V2R-GFP (see above) after removing the V2R coding sequence. This led to a construct in which the carboxyl terminus of the β-arrestin was separated from the initiator methionine of GFP10 by a 6-amino acid linker (GSGTGS). β-Arrestin (319–418) and β-arrestin V53D (10Krupnick J.G. Santini F. Gagnon A.W. Keen J.H. Benovic J.L. J. Biol. Chem. 1997; 272: 32507-32512Abstract Full Text Full Text PDF PubMed Scopus (180) Google Scholar) were generously provided by J. Benovic (Thomas Jefferson University, Philadelphia). All constructs were confirmed by sequencing. Human embryonic kidney 293 cells (HEK293) were cultured in Dulbecco's modified Eagle's medium supplemented with 10% FBS, 2 mm glutamine, 0.1 unit/ml penicillin, 0.1 mg/ml streptomycin, and 0.25 μg/ml Fungizone. Stable transfections of myc-tagged wt- or C341A/C342A-V2R were performed using the calcium phosphate precipitation method, and neomycin-resistant cells were selected in the presence of 450 μg/ml G418. Resistant clones were screened for V2R expression by radioligand binding assay. In cases where receptors and accessory proteins needed to be overexpressed, transient transfections were made, and cells were harvested 48 h after transfection. In bioluminescence resonance energy transfer (BRET) assays, the calcium phosphate precipitation method (11Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1989: 16.33-16.36Google Scholar) was used, whereas FuGENE 6 transfection reagent (Roche Applied Science) was utilized, according to the manufacturer's protocol, in all other cases. Radioligand binding assays were carried out in both whole cell and purified membrane preparations using [3H]AVP (PerkinElmer Life Sciences) as radioligand. In the case of membrane binding, cells were washed twice with cold PBS, lysed in 15 mm Tris-HCl, 2 mm MgCl2, 0.3 mm EDTA, pH 7.4, using a Polytron (three times for 5 s). The supernatant resulting from a 5-min 200 × g centrifugation was recentrifuged at 40,000 × g for 20 min. The pelleted membranes were washed once in 50 mm Tris-Cl, 5 mm MgCl2, pH 7.4, and a binding assay was carried out in the same buffer. 15 μg of membrane proteins were incubated for 30 min at room temperature in the presence of increasing concentrations of [3H]AVP (0.1–40 nm) and 1 mg/ml BSA, in a total volume of 300 μl. Nonspecific binding was determined in the presence of 10 μm cold AVP. For whole cell binding, cells were detached from 100-mm dishes using 5mm EDTA in PBS. Cells (40 μg of cell proteins) were then resuspended in ice-cold PBS and the radioligand binding initiated by adding a saturating concentration of [3H]AVP (20 nm) in the presence or absence 10 μm cold AVP for 2 h at 4 °C. In both cases, the reaction was stopped by rapid filtration over glass fiber (GF/C) filters (Whatman) and bound radioligand detected by scintillation counting. Cells were grown in 100-mm dishes, preincubated for 1 h in serum-free medium, and labeled with 0.4 mCi/ml [3H]palmitic acid for 2 h at 37 °C. Where indicated, cells were also pretreated for 16 h in the presence of 100 μm 2-bromopalmitic acid (2-BrP) (Fluka Chemie) or 4 h with 30 μm tunicamycin at 37 °C. After metabolic labeling, V2Rs were purified by immunoprecipitation using the AS435 antibody raised against the carboxyl-terminal portion of the human V2R (peptide ARG29, ARGRTPPSLGPQDESCTTASSSLAKDTSS). The selectivity of the antibody was confirmed by the fact that a band was identified only in cells transfected with the V2R and that the myc-tagged V2R was recognized by both AS435 and the anti-myc 9E10 antibodies. Labeled cells were washed twice with cold PBS and lysed by sonication (twice for 15 s) in 25 mm Tris-HCl, 2 mm EDTA, pH 7.4, in the presence of protease inhibitors (10 μg/ml benzamidine, 5 μg/ml soybean trypsin inhibitor, 5 μg/ml leupeptin) and 5 mm N-ethylmaleimide at 4 °C. The lysate was centrifuged for 5 min at 500 × g and then 20 min at 40,000 × g at 4 °C. The recovered membranes were then washed once in the same buffer and solubilized in RIPA buffer (150 mm NaCl, 50 mm Tris-HCl, pH 8.0, 5 mm EDTA, 1% Nonidet P-40, 0.5% deoxycholic acid, 0.1% SDS, with protease inhibitors and N-ethylmaleimide) for 1 h at 4 °C under gentle agitation. The solution was then centrifuged 1 h at 145,000 × g at 4 °C to get rid of insoluble material, and the supernatant was preincubated with a suspension of Staphylococcus aureus (Pansorbin; Calbiochem) for 30 min at 4 °C before incubation with 5 μl of AS435 antibody in the presence of 1% BSA for 16 h at 4 °C. Antigen-antibody complexes were then isolated by incubation with 40 μl of Pansorbin for 2 h at 4 °C followed by centrifugation at 4,000 × g for 2 min. Precipitates were washed five times in RIPA buffer and proteins eluted in 50 μl of SDS-PAGE loading buffer (125 mm Tris-HCl, pH 6.5, 4% SDS, 1 m urea, 5% glycerol, 0.1% bromphenol blue). Proteins were then resolved on SDS-PAGE in nonreducing conditions and transferred to nitrocellulose membranes. Tritium-labeled proteins were detected using EA-Wax (EABiotech Ltd., Interscience) and exposing the membranes to Kodak X-Omat film for 1 week. Detection of the receptors by immunoblots was performed using the anti-myc monoclonal 9E10 IgG as a primary antibody followed by an anti-mouse horseradish peroxidase-conjugated IgG (Amersham Biosciences) for chemiluminescence detection. Cells were grown in 6-well plates and incubated in the presence of 2 μCi/ml [3H]adenine in complete Dulbecco's modified Eagle's medium for 16 h. They were then washed twice with PBS containing 1 mm isobutylmethylxanthine before being incubated in the presence of increasing concentration of AVP for 15 min at 37 °C. The reaction was stopped by adding 1 ml of ice-cold 5% trichloroacetic acid and 1 mm unlabeled cAMP to decrease enzymatic degradation of [3H]cAMP. The cells were scraped off the plates and centrifuged at 800 × g for 20 min at 4 °C to clear the lysate. The [3H]cAMP was then separated by sequential chromatography over Dowex and Alumina columns as described previously (56Salomon Y. Londos C. Rodbell M. Anal. Biochem. 1974; 58: 541-548Crossref PubMed Scopus (3374) Google Scholar). The cAMP accumulation was calculated as ([3H]cAMP cpm/([3H]cAMP cpm + [3H]ATP cpm)) × 1,000 and expressed as a percentage of the maximal AVP-stimulated cAMP production for the wtV2R. Cells were grown in 6-well plates and rendered quiescent by serum starvation for 24 h prior to stimulation with 10% FBS or different concentrations of AVP for the indicated time. Cells were then placed on ice, washed twice with ice-cold PBS, and solubilized directly in 100 μlof Laemmli sample buffer containing 50 mm dithiothreitol. The samples were sonicated for 10 s, heated for 5 min at 95 °C, and microcentrifuged 5 min before fractionation of the proteins on SDS-PAGE. ERK1/2 phosphorylation was detected by protein immunoblotting using P-ERK1/2-specific antibodies and horseradish peroxidase-conjugated IgG as secondary antibody for chemiluminescence detection. After quantification of phosphorylation by autoradiography, nitrocellulose membranes were stripped of immunoglobulins and reprobed using anti-ERK1/2. ERK1/2 phosphorylation was normalized according to the loading of proteins by expressing the data as a ratio of P-ERK1/2 over total ERK1/2. Receptor internalization was measured as described previously (12Orsini M.J. Benovic J.L. J. Biol. Chem. 1998; 273: 34616-34622Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar). Cells were grown in 24-well plates and washed twice with 0.2 m Hepes in Dulbecco's modified Eagle's medium prior to incubation with AVP for the indicated time. Stimulations were stopped on ice, and cells were washed twice with cold PBS and blocked for 30 min with 1% BSA in PBS before being incubated for 1 h with the anti-myc 9E10 antibody. Cells were then washed three times with 1% BSA in PBS and fixed for 15 min at room temperature with 3% paraformaldehyde in PBS. They were then washed twice with PBS, reblocked for 15 min, and incubated with an anti-mouse horseradish peroxidase antibody. Antibody binding was then visualized after three additional washes by adding 0.5 ml of OPD peroxidase substrate diluted in PBS. Reactions were stopped by adding 0.1 ml of 3 n HCl, and the absorbance of the samples was read at 492 nm in a spectrophotometer. The BRET between the V2R-GFP and β-arrestin2-Rluc was measured as described previously (13Angers S. Salahpour A. Joly E. Hilairet S. Chelsky D. Dennis M. Bouvier M. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 3684-3689PubMed Google Scholar). Briefly, 48 h post-transfection, cells were detached and washed twice with PBS at room temperature. Cells (40 μg of proteins) were then distributed in a 96-well microplate (white Optiplate from Packard Bioscience) and incubated in the presence or absence of 1 μm AVP for the indicated time. DeepBlueC™ coelanterazine (Packard Bioscience) was added at a final concentration of 5 μm, and readings were collected using a modified top count apparatus (BRETCount) that allows the sequential integration of the signals detected in the 370–450 and 500–530 nm windows using filters with the appropriate band pass (Chroma). The BRET signal was determined by calculating the ratio of the light emitted by the receptor-GFP (500–530 nm) to the light emitted by the β-arrestin2-Rluc (370–450 nm). The values were corrected by subtracting the background signal detected when the β-arrestin2-Rluc construct was expressed alone. Where indicated, cells were pretreated for 16 h in the presence of 100 μm 2-BrP or 4 h with 30 μm tunicamycin at 37 °C before being detached. Coimmunoprecipitation of covalently cross-linked β-arrestin to V2Rs was performed as described previously (14Tohgo A. Choy E.W. Gesty-Palmer D. Pierce K.L. Laporte S. Oakley R.H. Caron M.G. Lefkowitz R.J. Luttrell L.M. J. Biol. Chem. 2003; 278: 6258-6267Abstract Full Text Full Text PDF PubMed Scopus (301) Google Scholar). Cells were transiently transfected with β-arrestin2-GFP and myc-tagged wt- or C341A/C342A-V2R. 48 h post-transfection, cells were incubated with or without 1 μm AVP in PBS for 15 min at 37 °C, and stimulations were terminated by the addition of the membrane-permeable and reversible cross-linking agent DSP at a final concentration of 2 mm. Cells were then incubated for 30 min at room temperature under gentle agitation; washed twice with 50 mm Tris-HCl, pH 7.4 in PBS to neutralize unreacted DSP; lysed in 0.5 ml of 50 mm Hepes, 50 mm NaCl, 10% (v/v) glycerol, 0.5% (v/v) Nonidet P-40, 2 mm EDTA, 100 μm Na3VO4, 1 mm phenylmethylsulfonyl fluoride, 10 μg/ml benzamidine, 5 μg/ml soybean trypsin inhibitor, 5 μg/ml leupeptin, and N-ethylmaleimide; and clarified by centrifugation. 25-μl aliquots of whole cell lysate was removed and mixed with an equal volume of 2× reducing loading buffer (with dithiothreitol at a final concentration of 50 mm). To isolate V2R-bound β-arrestin2-GFP, BSA was added to each lysate to a final concentration of 1%, and immunoprecipitation was performed for 16 h at 4 °C using the anti-myc monoclonal 9E10 antibody precoated on protein G-Sepharose beads (Amersham Biosciences). Immune complexes were washed three times with glycerol lysis buffer and eluted in 1× reducing loading buffer 15 min at 45 °C. Proteins were resolved on SDS-PAGE and transferred to nitrocellulose for immunoblotting. Immunoblotting of β-arrestin2-GFP was performed using rabbit polyclonal anti-GFP IgG (Clontech), and immunoblotting of mycV2Rs was performed using rabbit polyclonal anti-myc A14 IgG. Immune complexes were then visualized by chemiluminescence detection using anti-rabbit horseradish peroxidase-conjugated IgG. Cells were grown in 100-mm dishes, preincubated for 1 h in 0.2 m Hepes in phosphate-free medium, and labeled with 0.5 mCi/ml 32Pi for 2 h at 37 °C with or without 1 μm AVP for the last 15 min. Labeled cells were washed twice with cold PBS and incubated in RIPA buffer for 1 h under gentle agitation. The solution was then centrifuged for 1 h at 145,000 × g at 4 °C to get rid of insoluble material, and the V2Rs were purified by overnight immunoprecipitation at 4 °C using the anti-myc monoclonal 9E10 antibody precoated on protein G-Sepharose beads. Immune complexes were washed three times with RIPA buffer and eluted in 1× loading buffer 15 min at 45 °C. Proteins were then resolved on SDS-PAGE and transferred to nitrocellulose membranes where 32P-labeled proteins were detected using the Molecular Imager® FX PhosphorImager (Bio-Rad). Detection of the receptors by immunoblots was performed using rabbit polyclonal anti-myc A14 IgG. Immune complexes were then visualized by chemiluminescence detection using anti-rabbit horseradish peroxidase-conjugated IgG. Protein concentrations were determined using either the Bio-Rad DC Protein Assay or the Bradford quantification assay using BSA as standard. Dose-response curves and saturation experiments were analyzed by nonlinear regression using Prism (GraphPad Software), and the EC50 values were derived from the curves. Affinity constant (Kd) and maximal binding (B max) values of the radioligand were derived from the curve fitting. Immunoreactivities were determined by densitometric analysis of the films using NIH Image software. The extent of receptor phosphorylation was determined by digital analysis of the images generated by the PhosphorImager using the Quantity One software (Bio-Rad). The MAPK activation, internalization, and β-arrestin recruitment rates were evaluated by analyzing the linear portion of the curves with the following rate equation q(t)=q(t→∞)+q(t=0)e(-R)t(Eq. 1) where t is the time of incubation (in min), R is the rate, and q represents the level of MAPK activation, internalized receptors, or β-arrestin recruitment. The data in the initial part of the curves were plotted as in Equation 2. f(t)=ln(q(t)/q(0))(Eq. 2) The half-times were estimated as t where q(t) = 50% of the maximal effect in each data set. Statistical significances of the differences were carried out using unpaired Student's t test. p < 0.05 was considered statistically significant. Expression and Palmitoylation State of Wild-type and Mutant V2Rs—To study the role of palmitoylation of the V2Rs, HEK293 cells were stably transfected with myc-tagged wtV2Rs or mutant receptors in which the potential palmitoylation sites (cysteines 341 and 342) were replaced by alanines (C341A/C342A-V2R). Clonal cell lines expressing similar and physiological levels (wtV2R, 350 ± 20 fmol/mg; C341A/C342A, 290 ± 20 fmol/mg) of V2Rs as determined by [3H]AVP binding were selected and used in the various assays. Saturation binding experiments performed on isolated membranes of these clones confirmed, as reported previously (5Schülein R. Liebenhoff U. Muller H. Birnbaumer M. Rosenthal W. Biochem. J. 1996; 313: 611-616Crossref PubMed Scopus (69) Google Scholar), that the replacement of cysteines 341 and 342 by alanines does not affect the affinity of the receptor for AVP (Kd = wtV2R, 0.8 ± 0.1 nm versus C341A/C342A, 0.7 ± 0.1 nm). The palmitoylation state of the V2Rs was then verified by metabolic labeling of the cells with [3H]palmitic acid followed by immunoprecipitation of the receptors as described under "Experimental Procedures." Fig. 1 shows that mutation of cysteines 341 and 342 in the V2R greatly reduced the level of tritium incorporation in the receptor, confirming that these cysteines represent the major palmitoylation sites (4Sadeghi H.M. Innamorati G. Dagarag M. Birnbaumer M. Mol. Pharmacol. 1997; 52: 21-29Crossref PubMed Scopus (63) Google Scholar). Role of V2R Palmitoylation in Intracellular Signaling—To assess the importance of palmitoylation in receptor signaling, we investigated the effect of mutating cysteines 341 and 342 on the ability of the V2R to activate adenylyl cyclase and MAPK pathways. As reported previously (4Sadeghi H.M. Innamorati G. Dagarag M. Birnbaumer M. Mol. Pharmacol. 1997; 52: 21-29Crossref PubMed Scopus (63) Google Scholar, 5Schülein R. Liebenhoff U. Muller H. Birnbaumer M. Rosenthal W. Biochem. J. 1996; 313: 611-616Crossref PubMed Scopus (69) Google Scholar), replacing cysteines 341 and 342 with alanine residues did not affect AVP-stimulated adenylyl cyclase activity. Indeed, neither the efficacy nor the potency of AVP for this signaling pathway was affected by the mutation in HEK293 cells stably expressing comparable amounts of receptors (Fig. 2). In contrast, significant differences in the pattern of the ERK1/2 MAPK stimulation were observed between wt- and C341A/C342A-V2R (Fig. 3). Although an AVP-mediated increase in ERK1/2 activity was observed with both receptors, as assessed by immunoblotting using phospho-specific ERK1/2 antibodies, a delayed and reduced activation was found for the palmitoylation-less mutant (Fig. 3A). However, AVP was found to promote a more sustained ERK1/2 activation through the C341A/C342A-V2R. Quantitative assessment of these differences using total ERK1/2 immunoreactivity to normalize the phosphorylation levels is shown in Fig. 3B. The maximal level of wtV2R-mediated ERK1/2 phosphorylation was reached as early as 2 min after the addition of AVP and returned to basal level after 10 min, whereas the C341A/C342A-V2R-mediated phosphorylation of the kinases peaked after ∼4 min and only returned to basal level after 20 min. Kinetic analysis of the initial ERK1/2 activation curves revealed a significantly (p