Allosteric regulation of Gs on agonist, antagonist and inverse agonist binding to the beta2AR

G protein-coupled receptors are seven transmembrane domain proteins that regulate a diverse array of cellular functions primarily through G protein-mediated signaling. GPCR function can be regulated by different ligands that are classified as agonists, partial agonists, antagonists or inverse agonists. However, many GPCRs exhibit a degree of basal or ligand-independent activity. Full and partial agonists stabilize a conformation of the receptor that can couple to G proteins. Neutral antagonists are believed to bind to all receptor conformations indiscriminately and block the binding of other ligands, thus having no effect on second messenger signal transduction pathways. In contrast, inverse agonists bind to and stabilize the inactive conformation causing a decrease in basal activity. However, the molecular mechanisms for basal activity, ligand binding and their activation and inhibition of G proteins are poorly understood, largely because receptors exist in a dynamic ensemble of conformations that are difficult to isolate. Here we investigate the activation of the G protein (Gs) by the beta 2Adrenergic Receptor (beta2AR) by using a purified receptor reconstituted into recombinant HDL particles with a stoichiometric excess of Gs. The beta2AR was site-specifically labeled with a small, environmentally sensitive fluorophore allowing direct monitoring of ligand- and Gs induced conformational changes. In the absence of an agonist, the beta2AR and Gs can be trapped in a complex that is nucleotide-free. Formation of this complex is enhanced by the agonist isoproterenol, and disrupted by the addition of saturating amounts of guanine nucleotides. The inverse agonist ICI prevents the formation of the complex but it is unable to disrupt the pre-formed complex. Saturation and ligand inhibition assays used to further characterize this complex revealed unexpectedly that the nucleotide-free beta2AR-Gs complex does not bind to the antagonist alprenolol. Moreover, binding is recovered by addition of guanine nucleotides. These results provide insights into G protein-induced conformational changes in the beta2AR and the structural basis for ligand efficacy. More importantly, we provide evidence that argues against the existence of "neutral" antagonists. Thus a re-evaluation of current GPCR binding theory and experimental design is required.