A colorimetric assay for measuring activation of Gs- and Gq-coupled signaling pathways

Wen Biao Chen, Tammy S. Shields, Philip J.S. Stork, Roger D. Cone

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Abstract

Current assays for functional activation of Gs-coupled receptors usually involve quantitation of adenylyl cyclase or measurement of cAMP concentration by radioimmunoassay. The activation of Gq-coupled receptors is commonly assayed by measurement of the production of inositol triphosphate or diacylglycerol from phosphatidylinositol 4,5-bisphosphate or of changes in intracellular calcium. These assays generally require large numbers of cells (105-106) and/or the use of radioactive materials. We have developed a rapid nonradioactive colorimetric assay that utilizes a β-galactosidase (lacZ) gene fused to five copies of the cyclic AMP response element (CRE) to detect the activation of CRE-binding protein that results from an increase in intracellular cAMP or calcium. This assay can be performed using as few as 30,000 cells in a 96-well format with the end products measured simultaneously in a microplate reader. Consequently, a single individual can readily assay 1000 samples a day. Using this assay, the fold increase in β-galactosidase activity was similar in magnitude to increases in cAMP or adenylyl cyclase activity and was approximately linear from 0.01 to 0.27 fmol/cell of intracellular cAMP. Furthermore, pharmacological characterization of one of the melanocortin receptors, mMC5-R, using this assay resulted in a similar order of potency for several melanocortin peptides to that obtained with a commonly used adenylyl cyclase enzyme assay. This assay is also useful for the characterization of Gq-coupled receptors as is demonstrated here using cells transfected with the mouse bombesin receptor. The large-scale capacity of this assay makes it an excellent method for screening molecules of interest acting on Gs- and Gq-coupled receptors.

Original languageEnglish (US)
Pages (from-to)349-354
Number of pages6
JournalAnalytical Biochemistry
Volume226
Issue number2
DOIs
StatePublished - Apr 1995

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ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Molecular Biology
  • Cell Biology

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