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...

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