Cytoskeletal constraint of the beta-adrenergic receptor in frog erythrocyte membranes.

Abstract

A fluorescence receptor binding assay, based on the high-affinity .beta.-adrenergic receptor antagonist propranolol, was utilized to probe the microenvironment of the antagonist-receptor complex in the frog (R. catesbeiana) erythrocyte membrane. The technique of steady-state fluorescence depolarization is applied to the propranolol-receptor complex, allowing quantitation of the rotational relaxation time of the complex. The complex is dynamically constrained at 20.degree. C. In the temperature range 6-10.degree. C, a sharp reversible release of constraint is observed. The addition of drugs that specifically disrupt the cytoskeleton (colchicine, vincristine and vinblastine) causes a similar but irreversible release of constraint at 20.degree. C. Cytochalasin B has a much smaller influence on the rotational mobility of the propranolol-receptor complex than do the other drugs that disrupt the cytoskeleton. Amphotericin B is without effect on the rotational constraint of the complex. Binding of the antagonist [3H]dihydroalprenolol is not influenced by colchicine. A model is proposed which postulates that cytoskeletal elements are linked to the antagonist-receptor complex. Antagonist binding does not result in cytoskeletal release, whereas agonist binding is postulated to lead to dissociation of the agonist-receptor complex from the cytoskeleton, thereby activating adenylate cyclase.