Development of an intact cell reporter gene β-lactamase assay for G protein-coupled receptors for high-throughput screening
G protein-coupled receptors (GPCRs) are involved in a large variety of physiological disorders, and are thus important pharmaceutical drug targets. Here, we describe the development and characterization of a β-lactamase reporter gene assay as a functional readout for the ligand-induced activation of...
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Veröffentlicht in: | Analytical biochemistry 2003-03, Vol.314 (1), p.16-29 |
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Format: | Artikel |
Sprache: | eng |
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Zusammenfassung: | G protein-coupled receptors (GPCRs) are involved in a large variety of physiological disorders, and are thus important pharmaceutical drug targets. Here, we describe the development and characterization of a β-lactamase reporter gene assay as a functional readout for the ligand-induced activation of the human bradykinin B1 receptor, expressed recombinantly in CHO cells. The β-lactamase reporter gene assay provides high sensitivity due to the absence of endogenous β-lactamase activity in mammalian cells. The cell-permeable fluorogenic substrate allows single-cell cloning of cells expressing functional BK1 receptors. Pharmacological characterization reveals comparable sensitivity and potency of known BK1 receptor agonists and antagonists between the β-lactamase assay, competition-binding assay, and other direct measurements of second messengers. The β-lactamase assay has been optimized for cell density, time of agonist stimulation, and DMSO sensitivity. This CHO-hBK1-β-lactamase assay is well suited to automation and miniaturization required for high-throughput screening. |
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ISSN: | 0003-2697 1096-0309 |
DOI: | 10.1016/S0003-2697(02)00587-0 |