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J. Biol. Chem., Vol. 283, Issue 22, 15078-15088, May 30, 2008

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Bioluminescence Resonance Energy Transfer Assays Reveal Ligand-specific Conformational Changes within Preformed Signaling Complexes Containing -Opioid Receptors and Heterotrimeric G Proteins^*

Nicolas Audet^¶, Céline Galés^||, Élodie Archer-Lahlou^¶1, Marc Vallières, Peter W. Schiller^**, Michel Bouvier^¶2, and Graciela Pineyro^¶3

From the Centre de Recherche Fernand-Seguin, Hôpital Louis-H. Lafontaine, Montréal, Quebec H1N 3V2, Canada, the Département de Pharmacologie, Département de Biochimie, and Département de Psychiatrie, Faculté de Médecine, Universitéde Montréal, Montréal, Quebec H3C 3J7, Canada, ^¶Groupe de Recherche Universitaire sur le Médicament, Montréal, Quebec H3C 3J7, Canada, ^||INSERM Unité 858, Équipe 8, Institut Louis Bugnard, Toulouse 31432, France, and ^**Laboratory of Chemical Biology and Peptide Research, Institut de Recherches Cliniques de Montréal, Montréal, Quebec 2W 1R7, Canada

Heptahelical receptors communicate extracellular information to the cytosolic compartment by binding an extensive variety of ligands. They do so through conformational changes that propagate to intracellular signaling partners as the receptor switches from a resting to an active conformation. This active state has been classically considered unique and responsible for regulation of all signaling pathways controlled by a receptor. However, recent functional studies have challenged this notion and called for a paradigm where receptors would exist in more than one signaling conformation. This study used bioluminescence resonance energy transfer assays in combination with ligands of different functional profiles to provide in vivo physical evidence of conformational diversity of -opioid receptors (DORs). DORs and _i1β₁₂ G protein subunits were tagged with Luc or green fluorescent protein to produce bioluminescence resonance energy transfer pairs that allowed monitoring DOR-G protein interactions from different vantage points. Results showed that DORs and heterotrimeric G proteins formed a constitutive complex that underwent structural reorganization upon ligand binding. Conformational rearrangements could not be explained by a two-state model, supporting the idea that DORs adopt ligand-specific conformations. In addition, conformational diversity encoded by the receptor was conveyed to the interaction among heterotrimeric subunits. The existence of multiple active receptor states has implications for the way we conceive specificity of signal transduction.

Received for publication, September 21, 2007 , and in revised form, February 26, 2008.

^* This work was supported by a grant from the National Sciences and Engineering Research Council of Canada and Grant MOP7 9432 from the Canadian Institutes of Health Research (to G. P.). 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 Fig. 1.

¹ Recipient of a Fond de Recherche en Santé du Québec (FRSQ) postdoctoral fellowship.

² Holder of a Canada Research Chair in molecular pharmacology and signal transduction.

³ Recipient of an FRSQ young investigator award. To whom correspondence should be addressed: Dépt. de Psychiatrie, Université de Montréal, 7331 Rue Hochelaga, Montréal, Quebec H1N 3V2, Canada. Tel.: 514-251-4015; Fax: 514-251-2617; E-mail: graciela.pineyro.filpo{at}umontreal.ca.

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