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

Anal Biochem. 1995 Apr 10;226(2):349-54. doi: 10.1006/abio.1995.1235.


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 (10(5)-10(6)) and/or the use of radioactive materials. We have developed a rapid nonradioactive colorimetric assay that utilizes a beta-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 beta-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.

Publication types

  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Base Sequence
  • Bombesin / metabolism
  • Cells, Cultured
  • Colforsin / pharmacology
  • Colorimetry
  • Cyclic AMP / metabolism
  • Cyclic AMP Response Element-Binding Protein / metabolism*
  • Enzyme Activation
  • GTP-Binding Proteins / metabolism*
  • Humans
  • Kidney / chemistry
  • Kidney / metabolism
  • Kinetics
  • Molecular Sequence Data
  • Receptors, Bombesin / metabolism
  • Receptors, Cell Surface / metabolism
  • Receptors, Corticotropin / metabolism
  • Receptors, Melanocortin
  • Signal Transduction*
  • Transfection
  • alpha-MSH / metabolism
  • beta-Galactosidase / genetics
  • beta-Galactosidase / metabolism


  • Cyclic AMP Response Element-Binding Protein
  • Receptors, Bombesin
  • Receptors, Cell Surface
  • Receptors, Corticotropin
  • Receptors, Melanocortin
  • Colforsin
  • alpha-MSH
  • Cyclic AMP
  • beta-Galactosidase
  • GTP-Binding Proteins
  • Bombesin