title: Development of an intact cell reporter gene beta-lactamase assay for G protein-coupled receptors for high-throughput screening.
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.
authors.affiliation: Department of Automated Biotechnology, Merck Research Laboratories, 502 Louise Lane, North Wales, PA 19454, USA. priya_kunapuli@merck.com
authors.forename: Priya
authors.initials: P
authors.lastname: Kunapuli
authors.forename: Richard
authors.initials: R
authors.lastname: Ransom
authors.forename: Kathy L
authors.initials: KL
authors.lastname: Murphy
authors.forename: Doug
authors.initials: D
authors.lastname: Pettibone
authors.forename: Julie
authors.initials: J
authors.lastname: Kerby
authors.forename: Sarah
authors.initials: S
authors.lastname: Grimwood
authors.forename: Paul
authors.initials: P
authors.lastname: Zuck
authors.forename: Peter
authors.initials: P
authors.lastname: Hodder
authors.forename: Raul
authors.initials: R
authors.lastname: Lacson
authors.forename: Ira
authors.initials: I
authors.lastname: Hoffman
authors.forename: James
authors.initials: J
authors.lastname: Inglese
authors.forename: Berta
authors.initials: B
authors.lastname: Strulovici
mesh_terms: D000818:Animals; D016466:CHO Cells; D002118:Calcium / Q000378:metabolism; D002452:Cell Count; D002460:Cell Line; D003001:Cloning, Molecular; D006224:Cricetinae; D004305:Dose-Response Relationship, Drug; D017930:Genes, Reporter / Q000235:genetics*; D020962:Heterotrimeric GTP-Binding Proteins / Q000378:metabolism*; D006801:Humans; D008024:Ligands; D015394:Molecular Structure; D043783:Receptor, Bradykinin B1; D018002:Receptors, Bradykinin / Q000819:agonists / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects; D001618:beta-Lactamases / Q000235:genetics / Q000378:metabolism*
chemical_list: D008024:Ligands; D043783:Receptor, Bradykinin B1; D018002:Receptors, Bradykinin; D001618:beta-Lactamases; D020962:Heterotrimeric GTP-Binding Proteins; D002118:Calcium