Abstract

Constitutive activation of G protein-coupled receptors (GPCRs) is now well recognized and many classical GPCR antagonists have been found to be inverse agonists. For the α_2A-adrenergic receptor (AR) we determine the relative inverse efficacies of a series of antagonists and utilize the extended ternary complex model to estimate the fraction of constitutively active mutant (CAM) receptors in the active state. Stable Chinese hamster ovary cell lines expressing the porcine α_2A-AR in its wild-type (WT) and constitutively activated (CAM-T373K) form were isolated. Activation of both G_i and G_s was enhanced for CAM receptors. cAMP production was suppressed in cells with the CAM α_2A-AR and this suppression was reversed by α₂-adrenergic antagonists with an order of inverse efficacy of rauwolscine > yohimbine > RX821002 > MK912, whereas phentolamine and idazoxan were essentially neutral antagonists. This striking difference in inverse efficacy between idazoxan and RX821002 may account for in vivo pharmacological differences between these two α₂-adrenergic antagonists. Agonist binding affinity to the non-G protein-coupled CAM receptor was 3- to 9-fold higher than to WT, whereas binding of the most efficacious inverse agonists, yohimbine and rauwolscine, was 1.7- and 2.1-fold weaker. Analysis of this difference by the extended ternary complex model indicates that approximately 50% of the CAM α_2A-AR is in the active (R*) state although there is no detectable constitutive activity of the WT receptor in the absence of agonist.