Evidence for internalization of the recognition site of β-adrenergic receptors during receptor subsensitivity induced by (-)-isoproterenol

Abstract

In the supernatant (30,000 .times. g) of frog erythrocyte homogenates, protein that could bind [3H]dihydroalprenolol ([3H]DHA) with high affinity was detected by gel filtration. This binding was greatly enhanced when the erythrocytes were preincubated with (-)-isoproterenol. After various periods of incubation with (-)-isoproterenol, the extent of the increase in the density of [3H]DHA binding sites in the cytosol was paralleled by a proportional decrease in the number of [3H]DHA binding sites in the corresponding pellet; both events peaked after 2-3 h of incubation with (-)-isoproterenol. The Ka [association constant] of the (-)-isoproterenol-induced increase in [3H]DHA binding in cytosol and the decrease in this binding in the membrane ranged between 60-90 nM. The changes in the cytosol and particulate [3H]DHA binding sites were independent of RNA and protein synthesis. The increase in cytosol binding elicited by (-)-isoproterenol was blocked by exposure of the cells to (-)-alprenolol which failed to change the cytosol binding of [3H]DHA. Scatchard analysis revealed that the enhanced [3H]DHA binding to cytosol material was due to a 4-fold increase in the Bmax [binding maximum] with little or no change in Kd (.apprxeq. 9 nM). Binding displacement data show that the soluble [3H]DHA binding sites resemble the surface membrane recognition sites. The ability of various .beta.-adrenergic agents to increase [3H]DHA binding to cytosol after they were incubated with frog erythrocytes paralleled their affinity for membrane-bound .beta. receptors. Apparently the .beta.-adrenergic receptor desensitization caused by prolonged exposure to (-)-isoproterenol is due to an internalization of the recognition site of .beta.-adrenergic receptors.