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J. Biol. Chem., Vol. 279, Issue 49, 50904-50914, December 3, 2004

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A Cyclic Peptide Mimicking the Third Intracellular Loop of the V₂ Vasopressin Receptor Inhibits Signaling through Its Interaction with Receptor Dimer and G Protein^*

Sébastien Granier, Sonia Terrillon¶, Robert Pascal||, Hélène Déméné**, Michel Bouvier, Gilles Guillon, and Christiane Mendre

From the Unité INSERM U469, CCIPE, 141 rue de la Cardonille, 34094 Montpellier Cedex, France, Département Biochimie, Université de Montréal, Montréal, Québec H3C 3J7, Canada, ^||UMR 5073, Université Montpellier 2, CC017, Place E. Bataillon, 34095 Montpellier Cedex, and ^**UMR 5048 CNRS-UM1/UMR 554 INSERM-UM1, Centre de Biochimie Structurale, 29 rue de Navacelles, 34060 Montpellier Cedex, France

In this study, we investigated the mechanism by which a peptide mimicking the third cytoplasmic loop of the vasopressin V2 receptor inhibits signaling. This loop was synthesized as a cyclic peptide (i3 cyc) that adopted defined secondary structure in solution. We found that i3 cyc inhibited the adenylyl cyclase activity induced by vasopressin or a nonhydrolyzable analog of GTP, guanosine 5'-O-(3-thio)triphosphate. This peptide also affected the specific binding of [³H]AVP by converting vasopressin binding sites from a high to a low affinity state without any effect on the global maximal binding capacity. The inhibitory actions of i3 cyc could also be observed in the presence of maximally uncoupling concentration of guanosine 5'-O-(3-thio)triphosphate, indicating a direct effect on the receptor itself and not exclusively on the interaction between the Gs protein and the V₂ receptor (V₂-R). Bioluminescence resonance energy-transfer experiments confirmed this assumption, because i3 cyc induced a significant inhibition of the bioluminescence resonance energy-transfer signal between the Renilla reniformis luciferase and the enhanced yellow fluorescent protein fused V₂-R. This suggests that the proper arrangement of the dimer could be an important prerequisite for triggering Gs protein activation. In addition to its effect on the receptor itself, the peptide exerted some of its actions at the G protein level, because it could also inhibit guanosine 5'-O-(3-thio)triphosphate-stimulated AC activity. Taken together, the data demonstrate that a peptide mimicking V₂-R third intracellular loop affects both the dimeric structural organization of the receptor and has direct inhibitory action on Gs.

Received for publication, May 7, 2004 , and in revised form, September 9, 2004.

^* This work was supported by the PCV00–86 grant from the "Programme CNRS Physique Chimie du Vivant" and a grant from the Canadian Institute for Health Research, the Kidney Foundation of Canada, and travel grant from the Fonds de la Recherche en Santé du Québec (to M. B.). 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.

The on-line version of this article (available at http://www.jbc.org) contains Supplemental Figs. S1–S3.

^¶ Supported by a studentship from the Fondation pour la Recherche Médicale.

Holder of the Hans Selye Chair in Molecular and Cell Biology as well as a Canada Research Chair in Signal Transduction and Molecular Pharmacology.

To whom correspondence should be addressed. Tel.: 33-4-67-14-29-84; Fax: 33-4-67-54-24-32; E-mail: christiane.mendre{at}ccipe.cnrs.fr.

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