β-Adrenergic Agonists Mediate Enhancement of β1-Adrenergic Receptor N-terminal Cleavage and Stabilization In Vivo and In Vitro

The beta(1)-adrenergic receptor (beta(1)AR) is the predominant beta AR in the heart and is the main target for beta-adrenergic antagonists, widely used in the treatment of cardiovascular diseases. Previously, we have shown that the human (h) beta(1)AR is cleaved in its N terminus by a metalloproteinase, both constitutively and in a receptor activation-dependent manner. In this study, we investigated the specific events involved in beta(1)AR regulation, focusing on the effects of long-term treatment with beta-adrenergic ligands on receptor processing in stably transfected human embryonic kidney 293(i) cells. The key findings were verified using the transiently transfected h beta(1)AR and the endogenously expressed receptor in neonatal rat cardiomyocytes. By using flow cytometry and Western blotting, we demonstrated that isoproterenol, S-propranolol, CGP-12177 [4-[3-[(1,1-dimethylethyl)amino]2-hydroxypropoxy]-1,3-dihydro-2H-benzimidazol-2-one], pindolol, and timolol, which displayed agonistic properties toward the beta(1)AR in either the adenylyl cyclase or the mitogen-activated protein kinase signaling pathways, induced cleavage of the mature cell-surface receptor. In contrast, metoprolol, bisoprolol, and CGP-20712 [1-[2-((3-carbamoyl-4-hydroxy)phenoxy)ethylamino]-3-[4-(1-methyl-4-trifluoromethyl-2-imidazolyl) phenoxy]-2-propanol], which showed no agonistic activity, had only a marginal or no effect. Importantly, the agonists also stabilized intracellular receptor precursors, possibly via their pharmacological chaperone action, and they stabilized the receptor in vitro. The opposing effects on the two receptor forms thus led to an increase in the amount of cleaved receptor fragments at the plasma membrane. The results underscore the pluridimensionality of beta-adrenergic ligands and extend this property from receptor activation and signaling to the regulation of beta(1)AR levels. This phenomenon may contribute to the exceptional resistance of beta(1)ARs to downregulation and tendency toward upregulation following long-term ligand treatments.