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Serine 447 in the Carboxyl Tail of Human VPAC₁ Receptor Is Crucial for Agonist-Induced Desensitization but Not Internalization of the Receptor

Unité 410 (J.C-.M., C.R.-F., A.C., P.N., H.D., M.L.) and 479 (J.E.B.), Institut National de la Santé et de la Recherche Médicale, Faculté de Médecine Xavier Bichat, Paris, France

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.

Address correspondence to: Jean-Claude Marie, INSERM U410, Faculté de Médecine Xavier Bichat, 75018 Paris, France. E-mail: ugh{at}bichat.inserm.fr

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