Our goal is to identify molecular mechanisms involved in the regulation of the beta-adrenergic receptor (BAR)-coupled adenylyl cyclase. Although there are three subtypes, B1AR, B2AR and B3AR, the physiological relevance of each remains to be elucidated. One possibility is that they may be regulated differently by agonists and other modulators. At least three major mechanisms of receptor regulation have been identified: desensitization, internalization, and down-regulation. We and others have shown that for the human subtypes, the ability to undergo regulation is B2AR <B1AR << B3AR. We have been investigating the differences in agonist-mediated internalization of human B1AR and B2AR stably expressed at different levels in baby hamster kidney (BHK) cells. For each subtype, the rate and extent of internalization varied inversely with receptor density; however, at similar expression levels, hB2AR always exhibited more internalization than hB1AR. Thus at 1 pmol/mg protein, 37% of hB2AR underwent internalization in 30 min vs. 17% of hB1AR. Beta-arrestin has been implicated in hB2AR internalization where it is believed to act as an adapter protein between the receptor and clathrin. Therefore, we over expressed bovine beta-arrestin in the BHK cells and determined its effects on hBAR internalization. For both subtypes, agonist-mediated internalization increased to ~50%. Thus, over expression of beta-arrestin appears to enhance the internalization of both subtypes and to minimize their differences. One interpretation is that hB1AR has a lower affinity for beta-arrestin than hB2AR and that at the low endogenous levels of beta-arrestin, fewer hB1AR complexes are formed and recruited into the clathrin-mediated endocytic pathway. We also are using a comprehensive approach to understand the mechanism of hB1AR down-regulation. When Chinese hamster fibroblasts (CHW) expressing hB1AR were exposed to agonist, there was a time-dependent loss of binding activity. Cells exposed to permeable cAMP derivatives displayed less extensive and rapid down-regulation. For both effectors, cells expressing high hB1AR levels exhibited less down-regulation than cells expressing low levels. Using a ribonuclease protection assay to quantify hB1AR mRNA, we found that both agonist and cAMP treatments resulted in a similar time-dependent reduction in steady-state mRNA levels that appeared to be due to increased destabilization. To follow the fate of the receptors during down-regulation, we carried out western blotting on cell lysates using antibodies to the C-terminus of hB1AR. In cells exposed to agonist, loss of hB1AR immunoreactivity paralleled the down-regulation of binding activity whereas in cells exposed to cAMP, there was no reduction in immunoreactivity despite the loss of binding activity. Thus, down-regulation of hB1AR appears to be complex and to involve both cAMP-mediated destabilization of receptor mRNA and inactivation of receptor protein, and agonist-mediated receptor degradation.