Development of an intact cell reporter gene beta-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 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.
Kunapuli
Priya
P
Department of Automated Biotechnology, Merck Research Laboratories, 502 Louise Lane, North Wales, PA 19454, USA. priya_kunapuli@merck.com
Ransom
Richard
R
Murphy
Kathy L
KL
Pettibone
Doug
D
Kerby
Julie
J
Grimwood
Sarah
S
Zuck
Paul
P
Hodder
Peter
P
Lacson
Raul
R
Hoffman
Ira
I
Inglese
James
J
Strulovici
Berta
B
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*
D008024:Ligands; D043783:Receptor, Bradykinin B1; D018002:Receptors, Bradykinin; D001618:beta-Lactamases; D020962:Heterotrimeric GTP-Binding Proteins; D002118:Calcium