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

Nicolas Audet,Céline Galés,Élodie Archer-Lahlou,Marc Vallières,Peter W Schiller,Michel Bouvier,Graciela Pineyro

doi:10.1074/jbc.m707941200

Abstract

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 delta-opioid receptors (DORs). DORs and alpha(i1)beta(1)gamma(2) 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.

Highlights

Heptahelical receptors are versatile membrane proteins that play an important role in cellular communication
We sought to determine whether DORs occupied by different ligands would differ in the way they interact with heterotrimeric G proteins, the rationale being that if DORs were stabilized in ligand-specific conformations, each of these receptor states should distinctively interact with its immediate signaling partners
Comparison of ligand rank order of efficacy in cyclase (Fig. 1A) and ERK (Fig. 1B) cascades revealed that DOR ligands did not maintain their ordinal positions in the two assays, an observation that has been classically associated with the existence of ligand-specific receptor states [4, 7]

Summary

Introduction

Heptahelical receptors are versatile membrane proteins that play an important role in cellular communication. The fact that the highly efficacious DOR agonist SNC-80 (10 ␮M; 2 min) modified BRET generated by DOR-GFP1⁄7␣i1-Luc91 but not that corresponding to CD8-GFP, further indicates that the spontaneous transfer of energy obtained by co-expressing DOR1⁄7␣i1 pairs was due to their specific interaction and not to their overexpression.

Results

Conclusion