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Inactive conformations of α1B-AR and β2-AR are structurally similar but exhibit distinct pharmacological properties. Pharmacological heterogeneity in GPCRs: G protein–coupled receptors (GPCRs) share a common overall structure that switches between inactive and active conformations, with the latter being stabilized by ligand binding. Ragnarsson et al. investigated the role of a switch formed by hydrogen bonds between the NPxxY motif in transmembrane helix 7 (TMH7) and a tyrosine residue in TMH5 in activation of the α1B- and β2-adrenoceptors (ARs). Mutating the switch stabilized the inactive receptor conformations, resulting in reduced signaling. Although the mutations reduced the agonist affinity of the β2-AR without affecting signaling efficacy, they enhanced the agonist affinity and reduced the signaling efficacy of the α1B-AR. These findings show that inactive conformations of individual GPCRs have different pharmacological properties, which may help with designing new drugs or improving the efficacy and reducing the side effects of existing drugs. 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 α1B-adrenoceptor (α1B-AR) and β2-AR activation. Disrupting this bond by mutagenesis stabilized the α1B-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 α1B-AR and β2-AR inactive-state mutants. However, the inactive-state β2-ARs exhibited decreased agonist affinity, whereas the inactive-state α1B-ARs had enhanced agonist affinity. Conversely, antagonist affinity was unchanged for inactive-state conformations of both α1B-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 α1B-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. [ABSTRACT FROM AUTHOR]