Changes in immunohistochemical properties of beta-adrenergic receptors in frog erythrocytes by isoproterenol-induced desensitization

Immunohistochemical properties of beta-adrenergic receptor (BAR) in frog erythrocytes have been studied by using antiserum raised against purified guinea pig BAR. Immunoblotting of frog erythrocyte membranes with the anti-BAR serum revealed prominent staining of a protein with Mr of 65,000-67,000. BARs present in intact frog erythrocytes were made visible by incubation with the anti-BAR serum and then goat-anti rabbit IgG conjugated with colloidal gold. About 50-60% of the cells showed small, punctate dots by staining with the anti-BAR serum. After 4 hr exposure of the cells to isoproterenol, the density of the staining was markedly increased. Labeling of BAR after permeabilization of erythrocytes with saponin was markedly enhanced in isoproterenol-desensitized, but not in control cells. The BARs present in cytoslic fraction of desensitized cells migrated in the void volume of Sepharose-4B and were effectively labeled by a lipophilic BAR ligand capable of penetrating the cell membranes, but not by a hydrophilic ligand. Thus, isoproterenol-induced desensitization is associated with alteration of the immunoreactivity of BAR. Moreover, our immunochemical and biochemical data provide further evidence for the internalization of BAR in desensitized frog erythrocytes.