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
Hauptverfasser: 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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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.
ISSN:0003-2697
1096-0309
DOI:10.1016/S0003-2697(02)00587-0