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J. Biol. Chem., Vol. 280, Issue 30, 28034-28043, July 29, 2005

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Contribution of the Carboxyl Terminus of the VPAC₁ Receptor to Agonist-induced Receptor Phosphorylation, Internalization, and Recycling^*

Christelle Langlet, Ingrid Langer¶, Pascale Vertongen, Nathalie Gaspard, Jean-Marie Vanderwinden||**, and Patrick Robberecht

From the Laboratoire de Chimie Biologique et de la Nutrition and ^||Laboratoire de Neurophysiologie, Faculté de Médecine, Université Libre de Bruxelles, Bruxelles B-1070, Belgium

When exposed to vasoactive intestinal peptide (VIP), the human wild type VPAC₁ receptor expressed in Chinese hamster ovary (CHO) cells is rapidly phosphorylated, desensitized, and internalized in the endosomal compartment and is not re-expressed at the cell membrane within 2 h after agonist removal. The aims of the present work were first to correlate receptor phosphorylation level to internalization and recycling, measured by flow cytometry and in some cases by confocal microscopy using a monoclonal antibody that did not interfere with ligand binding, and second to identify the phosphorylated Ser/Thr residues. Combining receptor mutations and truncations allowed identification of Ser²⁵⁰ (in the second intracellular loop), Thr⁴²⁹, Ser⁴³⁵, Ser⁴⁴⁸ or Ser⁴⁴⁹, and Ser⁴⁵⁵ (all in the distal part of the C terminus) as candidates for VIP-stimulated phosphorylation. The effects of single mutations were not additive, suggesting alternative phosphorylation sites in mutated receptors. Replacement of all of the Ser/Thr residues in the carboxyl-terminal tail and truncation of the domain containing these residues completely inhibited VIP-stimulated phosphorylation and receptor internalization. There was, however, no direct correlation between receptor phosphorylation and internalization; in some truncated and mutated receptors, a 70% reduction in phosphorylation had little effect on internalization. In contrast to results obtained on the wild type and all of the mutated or truncated receptors that still underwent phosphorylation, internalization of the severely truncated receptor was reversed within 2 h of incubation in the absence of the agonist. Receptor recovery was blocked by monensin, an endosome inhibitor.

Received for publication, January 13, 2005 , and in revised form, May 26, 2005.

^* This work was supported by FRSM Grants 3.4504.99 and 3.4507.98, by an "Action de Recherche Concertée" from the Communauté Française de Belgique, and by an "Interuniversity Poles of Attraction Program-Belgian State, Prime Minister's Office, Federal Office for Scientific, Technical and Cultural Affairs." This work was also supported by grants from the Fondation Médicale Reine Elisabeth, the Fondation Universitaire David et Alice Van Buuren, Fonds National de la Recherche Scientifique (Fonds de la Recherche Scientifique Médicale) (Belgium), and Télévie (to J.-M. V.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Recipient of a doctoral fellowship from the Communauté Française de Belgique and a grant from the Fonds Lekime-Ropsy.

^¶ "Chargée de Recherches" from FNRS (Belgium).

^** Senior Research Associate of FNRS (Belgium).

To whom correspondence should be addressed: Dept. of Biochemistry and Nutrition, School of Medicine, Université Libre de Bruxelles, Bt G/E, CP 611, 808 Route de Lennik, B-1070 Bruxelles, Belgium. Tel.: 32-2-5556228; Fax: 32-2-5556230; E-mail: probbe{at}ulb.ac.be .