title: Palmitoylation of the V2 vasopressin receptor carboxyl tail enhances beta-arrestin recruitment leading to efficient receptor endocytosis and ERK1/2 activation.
abstract: 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 beta-arrestin which inhibit receptor endocytosis also attenuated vasopressin-stimulated mitogen-activated protein kinase activity, suggesting that the scaffolding protein, beta-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 beta-arrestin to the receptor. Interestingly, the changes in beta-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 beta-arrestin to the activated V2 vasopressin receptor thus facilitating processes requiring the scaffolding action of beta-arrestin.
authors.affiliation: Department of Biochemistry and Groupe de Recherche sur le Système Nerveux Autonome, Université de Montréal, Montréal, Québec H3C 3J7, Canada.
authors.forename: Pascale G
authors.initials: PG
authors.lastname: Charest
authors.forename: Michel
authors.initials: M
authors.lastname: Bouvier
mesh_terms: D000818:Animals; D019390:Arrestins / Q000378:metabolism*; D002460:Cell Line; D000242:Cyclic AMP / Q000378:metabolism; D003545:Cysteine / Q000737:chemistry; D004305:Dose-Response Relationship, Drug; D004705:Endocytosis; D005799:Genes, Dominant; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins; D006801:Humans; D015151:Immunoblotting; D008164:Luminescent Proteins / Q000378:metabolism; D020935:MAP Kinase Signaling System; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism*; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D019308:Palmitic Acid / Q000378:metabolism*; D010766:Phosphorylation; D010957:Plasmids / Q000378:metabolism; D011233:Precipitin Tests; D017434:Protein Structure, Tertiary; D051381:Rats; D017483:Receptors, Vasopressin / Q000378:metabolism*; D013997:Time Factors; D000071557:beta-Arrestins
chemical_list: D019390:Arrestins; D008164:Luminescent Proteins; D017483:Receptors, Vasopressin; D000071557:beta-Arrestins; D049452:Green Fluorescent Proteins; D019308:Palmitic Acid; D000242:Cyclic AMP; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases; D003545:Cysteine