Mutations in the NPxxY motif stabilize pharmacologically distinct conformational states of the α1B- and β2-adrenoceptors

Abstract

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_1B-adrenoceptor (α_1B-AR) and β₂-AR activation. Disrupting this bond by mutagenesis stabilized the α_1B-AR and the β₂-AR in inactive-state conformations, which displayed decreased agonist potency for stimulating downstream IP₁ and cAMP signaling, respectively. Compared to that for wild-type receptors, agonist-mediated β-arrestin recruitment was substantially reduced or abolished for all α_1B-AR and β₂-AR inactive-state mutants. However, the inactive-state β₂-ARs exhibited decreased agonist affinity, whereas the inactive-state α_1B-ARs had enhanced agonist affinity. Conversely, antagonist affinity was unchanged for inactive-state conformations of both α_1B-AR and β₂-AR. Removing the influence of agonist affinity on agonist potency gave a measure of signaling efficacy, which was markedly decreased for the α_1B-AR mutants but little altered for the β₂-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.