Agonist binding to G protein-coupled receptors is

The purpose of the present study was to develop an approach to directly monitor structural changes in a G protein-coupled receptor in response to drug binding. Purified human ␤ 2 adrenergic receptor was covalently labeled with the cysteine-reactive, fluorescent probe N,N -dimethyl-N-(iodoacetyl)-N -(7-nitrobenz-2-oxa-1,3-diazol-4-yl)ethylenediamine (IANBD). IANBD is characterized by a fluorescence which is highly sensitive to the polarity of its environment. We found that the full agonist, isoproterenol, elicited a stereoselective and dosedependent decrease in fluorescence from IANBD-labeled ␤ 2 receptor. The change in fluorescence could be plotted against the concentration of isoproterenol as a simple hyperbolic binding isotherm demonstrating interaction with a single binding site in the receptor. The ability of several adrenergic antagonists to reverse the response confirmed that this binding site is identical to the well described binding site in the ␤ 2 receptor. Comparison of the response to isoproterenol with a series of adrenergic agonists, having different biological efficacies, revealed a linear correlation between biological efficacy and the change in fluorescence. This suggests that the agonist-mediated decrease in fluorescence from IANBD-labeled ␤ 2 receptor is due to the same conformational change as involved in receptor activation and G protein coupling. In contrast to agonists, negative antagonists induced a small but significant increase in base-line fluorescence. Despite the small amplitude of this response, it supports the notion that antagonists by themselves may alter receptor structure. In conclusion, our data provide the first direct evidence for ligandspecific conformational changes occurring in a G protein-coupled receptor. Furthermore, the data demonstrate the potential of fluorescence spectroscopy as a tool for further delineating the molecular mechanisms of drug action at G protein-coupled receptors.The ␤ 2 adrenergic receptor is a prototype member of the G protein-coupled receptor family (1). The receptor family constitutes the largest group of plasma membrane receptors, which are characterized by a remarkable diversity in the chemical structure of their endogenous ligands (1-3). The receptors are all believed to share a common topology with seven ␣-helical, transmembrane segments; however, the helical arrangement and actual three-dimensional structure of the receptors remain unknown (1-3). Mutagenesis studies in the ␤ 2 adrenergic receptor as well as in many other G protein-coupled receptors have been able to assign distinct receptor functions, such as ligand binding and G protein coupling, to specific receptor domains (1-3). Several molecular models of these receptors have also been generated based on the structure of bacteriorhodopsin and rhodopsin for which more detailed structural information is available (3-5). Nevertheless, very little is known about the molecular events and structural changes in the receptor that provide the important link between ligand binding and transmissi...

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