Mutations in the NPxxY motif stabilize pharmacologically distinct conformational states of the α- and β-adrenoceptors.

G protein-coupled receptors (GPCRs) convert extracellular stimuli to intracellular responses that regulate numerous physiological processes. Crystallographic and biophysical advances in GPCR structural analysis have aided investigations of structure-function relationships that clarify our understanding of these dynamic receptors, but the molecular mechanisms associated with activation and signaling for individual GPCRs may be more complex than was previously appreciated. Here, we investigated the proposed water-mediated, hydrogen-bonded activation switch between the conserved NPxxY motif on transmembrane helix 7 (TMH7) and a conserved tyrosine in TMH5, which contributes to α-adrenoceptor (α-AR) and β-AR activation. Disrupting this bond by mutagenesis stabilized the α-AR and the β-AR in inactive-state conformations, which displayed decreased agonist potency for stimulating downstream IP and cAMP signaling, respectively. Compared to that for wild-type receptors, agonist-mediated β-arrestin recruitment was substantially reduced or abolished for all α-AR and β-AR inactive-state mutants. However, the inactive-state β-ARs exhibited decreased agonist affinity, whereas the inactive-state α-ARs had enhanced agonist affinity. Conversely, antagonist affinity was unchanged for inactive-state conformations of both α-AR and β-AR. Removing the influence of agonist affinity on agonist potency gave a measure of signaling efficacy, which was markedly decreased for the α-AR mutants but little altered for the β-AR mutants. These findings highlight the pharmacological heterogeneity of inactive-state GPCR conformations, which may facilitate the rational design of drugs that target distinct conformational states of GPCRs.