Human β1-Adrenergic Receptor Is Subject to Constitutive and Regulated N-terminal Cleavage

• Published in: The Journal of biological chemistry Vol. 285; no. 37; pp. 28850 - 28861
• Main Authors: Hakalahti, Anna E., Vierimaa, Miia M., Lilja, Minna K., Kumpula, Esa-Pekka, Tuusa, Jussi T., Petäjä-Repo, Ulla E.
• Format: Journal Article
• Language: English
• Published: 9650 Rockville Pike, Bethesda, MD 20814, U.S.A Elsevier Inc 10.09.2010; American Society for Biochemistry and Molecular Biology
• Subjects: Adrenergic Receptor; Cell Biology; G Protein-coupled Receptors (GPCR); Glycosylation; Metalloprotease; Post-translational Modification; Protein Processing; Receptor Regulation; Signal Transduction; Receptor Regulation; G Protein-coupled Receptors (GPCR); Post-translational Modification; Adrenergic Receptor; Metalloprotease; Glycosylation; Protein Processing
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M110.149989

The β1-adrenergic receptor (β1AR) is the predominant βAR 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 β1AR and reveal novel features relevant to its regulation using an inducible heterologous expression system in HEK293i 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 mucin-type O-glycosylation in addition to one N-glycan attached to Asn15. 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 Arg31 and Leu32 and Pro52 and Leu53, 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 βAR 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 Arg31–Leu32 cleavage site stabilized the mature receptor. We hypothesize that the N-terminal cleavage represents a novel regulatory mechanism of cell surface β1ARs.