title: Serine 447 in the carboxyl tail of human VPAC1 receptor is crucial for agonist-induced desensitization but not internalization of the receptor.
abstract: The VPAC1 receptor for vasoactive intestinal peptide (VIP) belongs to the class II family of G protein-coupled receptors and is coupled to Gs protein/adenylyl cyclase. We assessed whether 10 different Ser/Thr residues in human VPAC1 receptor intracellular domains play a role in the process of VIP-induced desensitization/internalization by performing a site-directed mutagenesis study. The Ser/Thr residues mutated to Ala include potential G protein-coupled receptor kinase, protein kinase A and protein kinase C targets that are of particular interest for VPAC1 receptor desensitization. The data show that when Chinese hamster ovary cells expressing wild-type receptors were pretreated for 5 min with VIP (50 nM), receptor desensitization occurred with a 10-fold right shift of the ED50 for adenylyl cyclase activation. When the construct with the widest span of mutations was studied, there was no longer any short-term desensitization. By using constructs with fewer and fewer mutations, we identified Ser447 in the C-terminal tail to be crucial for rapid desensitization. We also showed that Ser447 plays an essential role for VIP-induced VPAC1 phosphorylation in Chinese hamster ovary cells. Furthermore, we demonstrated that none of the mutated Ser/Thr residues was involved in down-regulation after a 12-h treatment of cells with 50 nM VIP. Neither were they involved in VIP and VIP-induced receptor internalization as shown using a novel fluorescein-tagged VIP and VPAC1 receptor bearing a Flag epitope in the N-terminal domain and a green fluorescent protein at the C terminus. We conclude that Ser447, a likely G protein-coupled receptor kinase target, is crucial for VIP-induced phosphorylation and rapid desensitization of VPAC1 receptor.
authors.affiliation: INSERM U410, Faculté de Médecine Xavier Bichat, 75018 Paris, France. ugh@bichat.inserm.fr
authors.forename: Jean-Claude
authors.initials: JC
authors.lastname: Marie
authors.forename: Christiane
authors.initials: C
authors.lastname: Rouyer-Fessard
authors.forename: Alain
authors.initials: A
authors.lastname: Couvineau
authors.forename: Pascal
authors.initials: P
authors.lastname: Nicole
authors.forename: Hélène
authors.initials: H
authors.lastname: Devaud
authors.forename: Jamel
authors.initials: J
authors.lastname: El Benna
authors.forename: Marc
authors.initials: M
authors.lastname: Laburthe
mesh_terms: D000595:Amino Acid Sequence / Q000235:genetics; D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D004305:Dose-Response Relationship, Drug; D006801:Humans; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000235:genetics / Q000378:metabolism*; D017434:Protein Structure, Tertiary / Q000235:genetics; D018005:Receptors, Vasoactive Intestinal Peptide / Q000819:agonists* / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D051238:Receptors, Vasoactive Intestinal Polypeptide, Type I; D012694:Serine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014660:Vasoactive Intestinal Peptide / Q000378:metabolism / Q000494:pharmacology
chemical_list: D010446:Peptide Fragments; D018005:Receptors, Vasoactive Intestinal Peptide; D051238:Receptors, Vasoactive Intestinal Polypeptide, Type I; D014660:Vasoactive Intestinal Peptide; D012694:Serine