Journal
ANALYTICAL BIOCHEMISTRY
Volume 314, Issue 1, Pages 16-29Publisher
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/S0003-2697(02)00587-0
Keywords
GPCR; bradykinin B1 receptor; beta-lactamase reporter; high-throughput screening
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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. (C) 2003 Elsevier Science (USA). All rights reserved.
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