In frog erythrocyte, agonist induced down regulation of Beta-adrenergic receptors (BAR) is associated with internalization of BAR binding sites; these internalized BAR sites are present in the 30,000 x g supernatant of erythrocyte homogenate. We found that these sites are associated with vesicular structures of more than 20 million daltons. These BAR sites can be labeled by lipophilic ligands (dihydroalprenolol, cyanopindolol and hydroxybenzylpindolol) but not by hydrophilic ligands (CGP-12177 and isoproterenol), suggesting that these internalized BAR sites are present in inside-out endocytotic vesicles. In an attempt to elucidate the molecular events involved in BAR internalization, monoclonal antibodies against erythrocyte membrane components that react with BAR have been produced. Moreover we have investigated whether BAR internalization occurs in the CNS. Since clathrin-coated vesicles (CVs) have been implicated in both endocytotic and intracellular transport of a variety of receptors, we have isolated CVs from bovine brain and examined them for the presence of BAR and adenylate cyclase activities. Purified CVs were found to contain BAR binding sites assessed with 125I-cyanopindolol as ligand in Sepharose column assays (Bmax=32plus/minus3 fmol/mg protein). The addition of GppNHp does not affect displacement of cyanopindolol binding to CVs by (-)isoproterenol, suggesting that these sites are uncoupled to the nucleotide regulatory protein. Moreover, these sites in CVs appear to be cryptic in nature, because 3H-CGP 12177 fails to label BAR in CVs. In addition, adenylate cyclase activity can be detected in the pure preparation of CVx (24+0.5 pmol cAMP/mg//min). These activities are unaffected by GTP or isoproterenol + GTP. These data suggest that BAR and adenylate cyclase detected in brain CVs may be molecular entities undergoing endocytosis or intracellular transport.
