The majority of extracellular physiologic signaling molecules act by stimulating GTP-binding protein (G-protein)-coupled receptors (GPCRs). To monitor directly the formation of the active state of a prototypical GPCR, we devised a method to site specifically attach fluorescein to an endogenous cysteine (Cys-265) at the cytoplasmic end of transmembrane 6 (TM6) of the ␤2 adrenergic receptor (␤2AR), adjacent to the G-protein-coupling domain. We demonstrate that this tag reports agonist-induced conformational changes in the receptor, with agonists causing a decline in the fluorescence intensity of fluorescein-␤ 2AR that is proportional to the biological efficacy of the agonist. We also find that agonists alter the interaction between the fluorescein at Cys-265 and fluorescence-quenching reagents localized to different molecular environments of the receptor. These observations are consistent with a rotation and͞or tilting of TM6 on agonist activation. Our studies, when compared with studies of activation in rhodopsin, indicate a general mechanism for GPCR activation; however, a notable difference is the relatively slow kinetics of the conformational changes in the ␤2AR, which may reflect the different energetics of activation by diffusible ligands.D espite diverse physiologic roles, the majority of GTPbinding protein (G-protein)-coupled receptors (GPCRs) are thought to share a common activation mechanism. Briefly, agonists induce conformational changes in receptors, which then stimulate heterotrimeric G proteins. Activated G proteins influence cellular physiology by modulating specific effector enzymes and ion channels involved in cardiovascular, neural, endocrine, and sensory signaling systems (1). GPCRs share a common structural motif consisting of seven TM helices with an extracellular amino terminus. For all known GPCRs, the site of ligand binding is distant from the site of G-protein regulation (2). Therefore, the overall structural effects of agonists binding to extracellular sequences or TM domains must physically converge at the cytoplasmic interface between the receptor and its G protein.We chose to characterize the conformational changes involved in G-protein activation by studying the human ␤ 2 adrenergic receptor (␤ 2 AR). The ␤ 2 AR is an important pharmaceutical target for pulmonary and cardiovascular diseases, and a wide spectrum of pharmacologically well-characterized agonists and antagonists is readily available (3). Moreover, extensive mutagenesis studies have identified amino acids involved in ligand binding and G-protein coupling (Fig. 1 A), thereby providing a basis for focusing our studies to domains likely to report conformational changes involved in receptor activation (4). We devised a means of labeling purified detergent-solubilized ␤ 2 AR at Cys-265 in the carboxyl-terminal region of IC3 with the sulfhydryl-reactive f luorescent probe f luorescein maleimide (FM). Cys-265 is found in the native receptor, and labeling at this position did not require alteration of the receptor's amino acid se...

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 ␤ 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 ␤ 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.G protein-coupled receptors (GPCRs) 1 are remarkably versatile biological sensors. They are responsible for the majority of cellular responses to hormones and neurotransmitters, as well as for the senses of sight, smell, and taste. Our current models of the mechanism of GPCR activation by diffusible agonists have been deduced from indirect measures of receptor conformation, such as G protein or second messenger activation (1-4). These indirect assays of GPCR activity provide only limited insight into the agonist-induced structural changes that define the active state of the receptor.To elucidate the mechanism of GPCR activation by diffusible agonists, we developed a means for directly monitoring the active conformation of purified, detergent-solubilized ␤ 2 adrenergic receptor (␤ 2 AR) by site-specific labeling of an endogenous cysteine (Cys 265 ) with fluorescein maleimide (FM-␤ 2 AR) (5). Based on homology with rhodopsin (6), Cys 265 is located in the third intracellular loop (IC3) at the cytoplasmic end of the transmembrane 6 (TM6) ␣ helix (Fig. 1A). Mutagenesis studies have shown this region of IC3 to be important for G protein coupling (7,8). An environmentally sensitive fluorophore covalently bound to Cys 265 is therefore well positioned to detect agonist-induced conformational changes relevant to G protein activation. The effect of agonists and partial agonists on the fluorescence intensity of FM-␤ 2 AR correlates well with their biological properties (5). Binding of the full agonist isoproterenol induces a conformational change that decreases the fluorescence intensity of FM bound to Cys 265 by ϳ15% (Fig. 1B), whereas binding of partial agonists results in smaller changes in intensity, and binding of an antagonist has no effect (5).Agonist-induced movement of FM bound to Cys 265 was characterized by examining the interaction between the fluorescein at Cys 265 and fluorescence quenching reagents localized to different ...