Development of an intact cell reporter gene beta-lactamase assay for G protein-coupled receptors for high-throughput screening |
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Authors: | Kunapuli Priya Ransom Richard Murphy Kathy L Pettibone Doug Kerby Julie Grimwood Sarah Zuck Paul Hodder Peter Lacson Raul Hoffman Ira Inglese James Strulovici Berta |
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Affiliation: | Department of Automated Biotechnology, Merck Research Laboratories, 502 Louise Lane, North Wales, PA 19454, USA. priya_kunapuli@merck.com |
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Abstract: | 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 beta-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 beta-lactamase reporter gene assay provides high sensitivity due to the absence of endogenous beta-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 beta-lactamase assay, competition-binding assay, and other direct measurements of second messengers. The beta-lactamase assay has been optimized for cell density, time of agonist stimulation, and DMSO sensitivity. This CHO-hBK1-beta-lactamase assay is well suited to automation and miniaturization required for high-throughput screening. |
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Keywords: | GPCR Bradykinin B1 receptor β-Lactamase reporter High-throughput screening |
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