A high affinity agonist . beta-adrenergic receptor complex is an intermediate for catecholamine stimulation of adenylate cyclase in turkey and frog erythrocyte membranes.

A high affinity nucleotide-sensitive complex of catecholamines with the /?-adrenergic receptor of turkey and frog erythrocyte membranes has been demonstrated by both adenylate cyclase and radioligandbinding experiments. This complex appears to serve as an intermediate which facilitates the activation of adenylate cyclase by regulatory guanine nucleotides. Turkey or frog erythrocyte membranes were incubated with the agonist (-)isoproterenol, then exposed to high concentrations of (+)propranolol, and extensively washed. Subsequent addition of guanyl-8-yl imidodiphosphate (Gpp(NH)p) in the presence of (?)propranolol produced, respectively, 100% and 200% greater activation of adenylate cyclase in the agonist pretreated than in control f rog or turkey erythrocyte membranes. This long lived agonist*receptor complex dissociates in the presence of GTP. Radioligand competition binding studies were performed using the antagonist [3H]dihydroalprenolol, and the data were analyzed by nonlinear least squares computer modeling procedures. In both turkey and f rog membranes the competition binding curve of the antagonist (+)propranolol was determined to best fit a oneaffinity state model and was unaffected by added guanine nucleotide. By contrast competition binding curves of the agonist (-)isoproterenol in both systems were best explained by two binding states with affinities differing by 64and 12-fold, respectively, in the frog and turkey erythrocyte membranes. In the presence of Gpp(NH)p agonist competition binding curves indicate a homogeneous lower binding affinity suggesting that nucleotide mediates a transition between the two affinity states associated with agonist occupancy of the receptors. These f indings demonstrate the existence of a long lived nucleotide-sensitive agonist receptor complex in both turkey and frog erythrocyte membranes and suggest that qualitatively similar mechanisms of receptor adenylate cyclase coupling may be operative in these two widely studied model systems.