title: Human beta1-adrenergic receptor is subject to constitutive and regulated N-terminal cleavage.
abstract: The beta(1)-adrenergic receptor (beta(1)AR) is the predominant betaAR 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 beta(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 mucin-type 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 betaAR 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 beta(1)ARs.
authors.affiliation: Department of Anatomy and Cell Biology, Institute of Biomedicine, University of Oulu, FI-90014 Oulu, Finland.
authors.forename: Anna E
authors.initials: AE
authors.lastname: Hakalahti
authors.forename: Miia M
authors.initials: MM
authors.lastname: Vierimaa
authors.forename: Minna K
authors.initials: MK
authors.lastname: Lilja
authors.forename: Esa-Pekka
authors.initials: EP
authors.lastname: Kumpula
authors.forename: Jussi T
authors.initials: JT
authors.lastname: Tuusa
authors.forename: Ulla E
authors.initials: UE
authors.lastname: Petäjä-Repo
mesh_terms: D000262:Adenylyl Cyclases / Q000235:genetics / Q000378:metabolism; D000318:Adrenergic beta-Agonists / Q000494:pharmacology; D002460:Cell Line; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation / Q000187:drug effects / Q000502:physiology; D006031:Glycosylation / Q000187:drug effects; D006801:Humans; D007545:Isoproterenol / Q000494:pharmacology; D009154:Mutation; D010447:Peptide Hydrolases / Q000235:genetics / Q000378:metabolism*; D011493:Protein Kinase C / Q000235:genetics / Q000378:metabolism; D017434:Protein Structure, Tertiary; D021381:Protein Transport / Q000187:drug effects / Q000502:physiology; D018342:Receptors, Adrenergic, beta-1 / Q000235:genetics / Q000378:metabolism*
chemical_list: D000318:Adrenergic beta-Agonists; D018342:Receptors, Adrenergic, beta-1; D011493:Protein Kinase C; D010447:Peptide Hydrolases; D000262:Adenylyl Cyclases; D007545:Isoproterenol