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Development of an intact cell reporter gene beta-lactamase assay for g protein-coupled receptors for high-throughput screening

Academic Article
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Overview

authors

  • Kunapuli, P.
  • Ransom, R.
  • Murphy, K. L.
  • Pettibone, D.
  • Kerby, J.
  • Grimwood, S.
  • Zuck, P.
  • Hodder, Peter
  • Lacson, R.
  • Hoffman, I.
  • Inglese, J.
  • Strulovici, B.

publication date

  • March 2003

journal

  • Analytical Biochemistry  Journal

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.

subject areas

  • Animals
  • CHO Cells
  • Calcium
  • Cell Count
  • Cell Line
  • Cloning, Molecular
  • Cricetinae
  • Dose-Response Relationship, Drug
  • Genes, Reporter
  • Heterotrimeric GTP-Binding Proteins
  • Humans
  • Ligands
  • Molecular Structure
  • Receptor, Bradykinin B1
  • Receptors, Bradykinin
  • Signal Transduction
  • beta-Lactamases
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Research

keywords

  • GPCR
  • beta-lactamase reporter
  • bradykinin B1 receptor
  • high-throughput screening
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Identity

International Standard Serial Number (ISSN)

  • 0003-2697

Digital Object Identifier (DOI)

  • 10.1016/s0003-2697(02)00587-0

PubMed ID

  • 12633598
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Additional Document Info

start page

  • 16

end page

  • 29

volume

  • 314

issue

  • 1

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