Identification of an Agonist-induced Conformational Change Occurring Adjacent to the Ligand-binding Pocket of the M3 Muscarinic Acetylcholine Receptor

Sung-Jun Han,Fadi F Hamdan,Soo-Kyung Kim,Kenneth A Jacobson,Lanh M Bloodworth,Bo Li,Jürgen Wess

doi:10.1074/jbc.m506711200

Abstract

To study the conformational changes that convert G protein-coupled receptors (GPCRs) from their resting to their active state, we used the M(3) muscarinic acetylcholine receptor, a prototypical class A GPCR, as a model system. Specifically, we employed a recently developed in situ disulfide cross-linking strategy that allows the formation of disulfide bonds in Cys-substituted mutant M(3) muscarinic receptors present in their native membrane environment. At present, little is known about the conformational changes that GPCR ligands induce in the immediate vicinity of the ligand-binding pocket. To address this issue, we generated 11 Cys-substituted mutant M(3) muscarinic receptors and characterized these receptors in transfected COS-7 cells. All analyzed mutant receptors contained an endogenous Cys residue (Cys-532(7.42)) located within the exofacial segment of transmembrane domain (TM) VII, close to the agonist-binding site. In addition, all mutant receptors harbored a second Cys residue that was introduced into the exofacial segment of TM III, within the sequence Leu-142(3.27)-Asn-152(3.37). Disulfide cross-linking studies showed that muscarinic agonists, but not antagonists, promoted the formation of a disulfide bond between S151(3.36)C and Cys-532. A three-dimensional model of the inactive state of the M(3) muscarinic receptor indicated that Cys-532 and Ser-151 face each other in the center of the TM receptor core. Our cross-linking data therefore support the concept that agonist activation pulls the exofacial segments of TMs VII and III closer to each other. This structural change may represent one of the early conformational events triggering the more pronounced structural reorganization of the intracellular receptor surface. To the best of our knowledge, this is the first direct demonstration of a conformational change occurring in the immediate vicinity of the binding site of a GPCR activated by a diffusible ligand.

Highlights

G protein-coupled receptors (GPCRs)5 form one of the largest gene families found in nature
This study was undertaken to investigate whether diffusible ligands can induce conformational changes in the immediate vicinity of the ligandbinding pocket of a class A GPCR
ACh and other classic muscarinic agonists are predicted to bind to the M3 muscarinic receptor and other members of the muscarinic receptor family (M1–M5) within a central binding crevice formed by the ring-like arrangement of the seven transmembrane domain (TM) [9, 11]

Summary

Introduction

G protein-coupled receptors (GPCRs) form one of the largest gene families found in nature. The classic biogenic amine neurotransmitters bind to their target GPCRs within a cavity formed by the ring-like arrangement of the seven TMs (8 –11). In this case, the key residues contributing to ligand recognition are located primarily within the exofacial segments of TMs III and V–VII (8 –11). Biophysical and biochemical studies carried out with bovine rhodopsin [14, 15, 17, 18] and the ␤2-adrenergic receptor (16, 19 –24) have revealed several activity-dependent conformational changes involving the intracellular receptor surface. Several studies suggest that similar conformational changes occur in GPCRs activated by diffusible ligands such as the ␤2-adrenergic receptor (16, 19 –24)

Objectives

Results

Conclusion