A COLORIMETRIC ASSAY FOR MEASURING ACTIVATION OF G(S)-COUPLED AND G(Q)-COUPLED SIGNALING PATHWAYS

Current assays for functional activation of G(s)-coupled receptors usu ally involve quantitation of adenylyl cyclase or measurement of cAMP c oncentration by radioimmunoassay. The activation of G(q)-coupled recep tors is commonly assayed by measurement of the production of inositol triphosphate or diacylglycerol from phosphatidylinositol 4,5-bisphosph ate or of changes in intracellular calcium. These assays generally req uire large numbers of cells (10(5)-10(6)) and/or the use of radioactiv e materials. We have developed a rapid nonradioactive colorimetric ass ay 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 cAM P 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 ass ay 1000 samples a day. Using this assay, the fold increase in beta-gal actosidase activity was similar in magnitude to increases in cAMP or a denylyl cyclase activity and was approximately linear from 0.01 to 0.2 7 fmol/cell of intracellular cAMP. Furthermore, pharmacological charac terization of one of the melanocortin receptors, mMC5-R, using this as say resulted in a similar order of potency for several melanocortin pe ptides to that obtained with a commonly used adenylyl cyclase enzyme a ssay. This assay is also useful for the characterization of G(q)-coupl ed 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 G (s)- and G(q)-coupled receptors. (C) 1995 Academic Press, Inc.