The Effect of pH on β2 Adrenoceptor Function EVIDENCE FOR PROTONATION-DEPENDENT ACTIVATION

Abstract The transition of rhodopsin from the inactive to the active state is associated with proton uptake at Glu134 (1), and recent mutagenesis studies suggest that protonation of the homologous amino acid in the α1Badrenergic receptor (Asp142) may be involved in its mechanism of activation (2). To further explore the role of protonation in G protein-coupled receptor activation, we examined the effects of pH on the rate of ligand-induced conformational change and on receptor-mediated G protein activation for the β2adrenergic receptor (β2AR). The rate of agonist-induced change in the fluorescence of NBD-labeled, purified β2AR was 2-fold greater at pH 6.5 than at pH 8, even though agonist affinity was lower at pH 6.5. This biophysical analysis was corroborated by functional studies; basal (agonist-independent) activation of Gαs by the β2AR was greater at pH 6.5 compared with pH 8.0. Taken together, these results provide evidence that protonation increases basal activity by destabilizing the inactive state of the receptor. In addition, we found that the pH sensitivity of β2AR activation is not abrogated by mutation of Asp130, which is homologous to the highly conserved acidic amino acids that link protonation to activation of rhodopsin (Glu134) and the α1B adrenergic receptor (Asp142).