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 ...

In most studies, coupling of the β 2 -adrenoceptor (β 2 AR) to the stimulatory, heterotrimeric GTP-binding protein of adenylyl cyclase the (G s ) is studied indirectly by measuring adenylyl cyclase activation. The aim of this study was to establish a model system in which β 2 AR-G s interactions can be studied directly at the level of the G-protein. We expressed the β 2 AR alone, in combination with the A-subunit of G s (G sA ), and as fusion protein with G sA (β 2 AR-G sA ) in Sf9 insect cells. The β 2 AR expressed alone couples poorly to the endogenous G sA -like G-protein of Sf9 cells since no high-affinity agonist binding could be detected, and the effects of agonist and inverse agonist on adenylyl cyclase, high-affinity GTPase and guanosine 5′-O-(3-thiotriphosphate) (GTP[S]) binding were small. β 2 AR-G sA reconstituted high-affinity agonist binding and regulated adenylyl cyclase more effectively than the β 2 AR co-expressed with a large excess of G sA. In membranes expressing β2AR-GsA, highly effective agonist-and inverse agonist regulation of high-affinity GTP hydrolysis and GTP[S] binding was observed. In contrast, agonist and inverse agonist regulation of GTP hydrolysis and GTP[S] binding in membranes expressing β 2 AR and G sA as separate proteins was difficult to detect. Our data show that the β2AR interacts with GsA more efficiently when expressed as a fusion protein than when expressed with an excess of non-fused G sA . The β 2 AR-G sA fusion protein provides a very sensitive model system to study the regulation of G s function by β 2AR agonists and inverse agonists directly at the level of the G-protein.

The ␤ 2 adrenergic receptor (␤ 2 AR) is a prototypical family A G protein-coupled receptor (GPCR) and an excellent model system for studying the mechanism of GPCR activation. The ␤ 2 AR agonist binding site is well characterized, and there is a wealth of structurally related ligands with functionally diverse properties. In the present study, we use catechol (1,2-benzenediol, a structural component of catecholamine agonists) as a molecular probe to identify mechanistic differences between ␤ 2 AR activation by catecholamine agonists, such as isoproterenol, and by the structurally related non-catechol partial agonist salbutamol. Using biophysical and pharmacologic approaches, we show that the aromatic ring of salbutamol binds to a different site on the ␤ 2 AR than the aromatic ring of catecholamines. This difference is important in receptor activation as it has been hypothesized that the aromatic ring of catecholamines plays a role in triggering receptor activation through interactions with a conserved cluster of aromatic residues in the sixth transmembrane segment by a rotamer toggle switch mechanism. Our experiments indicate that the aromatic ring of salbutamol does not activate this mechanism either directly or indirectly. Moreover, the non-catechol ring of partial agonists does not interact optimally with serine residues in the fifth transmembrane helix that have been shown to play an important role in activation by catecholamines. These results demonstrate unexpected differences in binding and activation by structurally similar agonists and partial agonists. Moreover, they provide evidence that activation of a GPCR is a multistep process that can be dissected into its component parts using agonist fragments.