The ␤ 1 -adrenergic receptor (␤ 1 AR) is the predominant ␤AR in the heart, mediating the catecholamine-stimulated increase in cardiac rate and force of contraction. Regulation of this important G protein-coupled receptor is nevertheless poorly understood. We describe here the biosynthetic profile of the human ␤ 1 AR and reveal novel features relevant to its regulation using an inducible heterologous expression system in HEK293 i cells. Metabolic pulse-chase labeling and cell surface biotinylation assays showed that the synthesized receptors are efficiently and rapidly transported to the cell surface. The N terminus of the mature receptor is extensively modified by sialylated mucintype O-glycosylation in addition to one N-glycan attached to Asn 15 . Furthermore, the N terminus was found to be subject to limited proteolysis, resulting in two membrane-bound C-terminal fragments. N-terminal sequencing of the fragments identified two cleavage sites between Arg 31 and Leu 32 and Pro 52 and Leu 53 , which were confirmed by cleavage site and truncation mutants. Metalloproteinase inhibitors were able to inhibit the cleavage, suggesting that it is mediated by a matrix metalloproteinase or a disintegrin and metalloproteinase (ADAM) family member. Most importantly, the N-terminal cleavage was found to occur not only in vitro but also in vivo. Receptor activation mediated by the ␤AR agonist isoproterenol enhanced the cleavage in a concentration-and time-dependent manner, and it was also enhanced by direct stimulation of protein kinase C and adenylyl cyclase. Mutation of the Arg 31 -Leu 32 cleavage site stabilized the mature receptor. We hypothesize that the N-terminal cleavage represents a novel regulatory mechanism of cell surface ␤ 1 ARs.The ␤ 1 -adrenergic receptor (␤ 1 AR) 3 is one of the three ␤AR subtypes that are activated by the endogenous catecholamines adrenaline and noradrenaline (1). These receptors belong to the G protein-coupled receptor (GPCR) family, one of the largest membrane protein families involved in cellular signaling (2, 3).The ␤ 1 AR is the predominant ␤AR subtype in the heart, mediating the increase in cardiac rate and force of contraction (4, 5). This makes it the most important target receptor for the ␤-blockers that are used to treat common cardiac diseases such as chronic heart failure, coronary artery disease, hypertension, and arrhythmias. The mechanisms that regulate human ␤ 1 AR (h␤ 1 AR) are therefore of considerable interest.The h␤ 2 AR is one of the most extensively studied GPCRs, but much less is known about h␤ 1 AR. The suggestion that it may be more resistant to agonist-mediated desensitization (6), internalization (7-12), and down-regulation (7, 10, 13, 14) could indicate that the two receptors are regulated by distinct mechanisms. Their ligand-binding sites are well conserved, but the overall homology of the two ␤ARs is only 54% (15). The most diverse regions are the intervening loops that connect the transmembrane domains, the extracellular N terminus and the intracellular C terminu...

The b 1 -adrenergic receptor (b 1 AR) is the predominant bAR in the heart and is the main target for b-adrenergic antagonists, widely used in the treatment of cardiovascular diseases. Previously, we have shown that the human (h) b 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 b 1 AR regulation, focusing on the effects of long-term treatment with b-adrenergic ligands on receptor processing in stably transfected human embryonic kidney 293 i cells. The key findings were verified using the transiently transfected hb 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 b 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 b-adrenergic ligands and extend this property from receptor activation and signaling to the regulation of b 1 AR levels. This phenomenon may contribute to the exceptional resistance of b 1 ARs to downregulation and tendency toward upregulation following long-term ligand treatments.

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