Functionally Different Agonists Induce Distinct Conformations in the G Protein Coupling Domain of the β2Adrenergic Receptor

Pejman Ghanouni,Zygmunt Gryczynski,Jacqueline J Steenhuis,Tae Weon Lee,David L Farrens,Joseph R Lakowicz,Brian K Kobilka

doi:10.1074/jbc.c100162200

Abstract

G protein-coupled receptors represent the largest class of drug discovery targets. Drugs that activate G protein-coupled receptors are classified as either agonists or partial agonists. To study the mechanism whereby these different classes of activating ligands modulate receptor function, we directly monitored ligand-induced conformational changes in the G protein-coupling domain of the beta(2) adrenergic receptor. Fluorescence lifetime analysis of a reporter fluorophore covalently attached to this domain revealed that, in the absence of ligands, this domain oscillates around a single detectable conformation. Binding to an antagonist does not change this conformation but does reduce the flexibility of the domain. However, when the beta(2) adrenergic receptor is bound to a full agonist, the G protein coupling domain exists in two distinct conformations. Moreover, the conformations induced by a full agonist can be distinguished from those induced by partial agonists. These results provide new insight into the structural consequence of antagonist binding and the basis of agonism and partial agonism.

Highlights

From the §Department of Molecular and Cellular Physiology and **Division of Cardiovascular Medicine, ‡Howard Hughes Medical Institute, Stanford University Medical School, Stanford, California 94305, the ¶Department of Biochemistry and Molecular Biology, Center for Fluorescence Spectroscopy, School of Medicine, University of Maryland, Baltimore, Maryland 21201, and the ʈDepartments of Biochemistry and Molecular Biology, Oregon Health Sciences University, Portland, Oregon 97201
When the ␤2 adrenergic receptor is bound to a full agonist, the G protein coupling domain exists in two distinct conformations
To elucidate the mechanism of G protein-coupled receptors (GPCRs) activation by diffusible agonists, we developed a means for directly monitoring the active conformation of purified, detergent-solubilized ␤2 adrenergic receptor (␤2AR) by site-specific labeling of an endogenous cysteine (Cys265) with fluorescein maleimide (FM-␤2AR) [5]

Summary

Accelerated Publication

Different Agonists Induce Distinct Conformations in the G Protein Coupling Domain of the ␤2 Adrenergic Receptor*. Our findings suggest that the conformational changes associated with ␤2AR activation are similar to those in rhodopsin [9] and indicate a shared mechanism of GPCR activation These results provide insight into the nature of the structural changes that occur upon agonist binding. We observe no change in the fluorescence intensity from FM-␤2AR upon antagonist binding. This could indicate that antagonists do not alter receptor structure or that the structural changes are not detectable by FM bound to Cys265. These results help elucidate the structural mechanisms that underlie ligand efficacy

EXPERIMENTAL PROCEDURES

RESULTS AND DISCUSSION

TABLE I

No drug ALP ISO SAL DOB