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The β1-adrenergic receptor (BIAR) is the predominant BAR 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 BAR 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. [ABSTRACT FROM AUTHOR]

Abstract: High-sensitivity, high-throughput, and user-friendly lanthanide-based assays for receptor–ligand interactions provide an attractive alternative to the traditional radioligand displacement assays. In this study, three small-molecule pindolol ligand derivatives were synthesized and their binding properties were tested in a radioligand displacement assay. The ligand derivatives were further labeled with fluorescent europium(III) chelate for β2-adrenergic receptor–ligand binding assay. The europium-labeled pindolol ligands having no spacer (C0) or a 12-carbon spacer (C12) arm bound to the human β2-adrenergic receptors overexpressed in human embryonic kidney HEK293i cells. Europium ligand with a 6-carbon spacer arm (C6) showed no binding. Competitive binding assays were developed with the functional labeled ligands. The IC50 values for β2-adrenergic antagonist propranolol were 60 and 37nM, the Z′ values were 0.51 and 0.77, and the signal-to-background ratios were 5.5 and 16.0 for C0 and C12, respectively. This study shows that functional time-resolved fluorescent assays can be constructed using fluorescent lanthanide chelates conjugated to small-molecule ligands. [Copyright &y& Elsevier]