A Colorimetric Assay for Measuring Activation of Gs- and Gq-Coupled Signaling Pathways

W. B. Chen, T. S. Shields, Philip Stork, R. D. Cone

Research output: Contribution to journalArticle

174 Citations (Scopus)

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

Fingerprint

Adenylyl Cyclases
Galactosidases
Assays
Chemical activation
Melanocortin Receptors
Bombesin Receptors
Melanocortins
Calcium
Cyclic AMP Response Element-Binding Protein
Lac Operon
Diglycerides
Enzyme Assays
Response Elements
Inositol
Phosphatidylinositols
Cyclic AMP
Radioimmunoassay
Cell Count
Pharmacology
Peptides

ASJC Scopus subject areas

  • Molecular Biology
  • Cell Biology
  • Biophysics
  • Biochemistry

Cite this

A Colorimetric Assay for Measuring Activation of Gs- and Gq-Coupled Signaling Pathways. / Chen, W. B.; Shields, T. S.; Stork, Philip; Cone, R. D.

In: Analytical Biochemistry, Vol. 226, No. 2, 04.1995, p. 349-354.

Research output: Contribution to journalArticle

Chen, W. B. ; Shields, T. S. ; Stork, Philip ; Cone, R. D. / A Colorimetric Assay for Measuring Activation of Gs- and Gq-Coupled Signaling Pathways. In: Analytical Biochemistry. 1995 ; Vol. 226, No. 2. pp. 349-354.
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