Said A. Goueli1,2, Meera Kumar1, Kevin Hsiao1, Jolanta Vidugiriene1, and Bob Bulleit1
1Cell Signaling Group, Cellular Analysis Platform, Dept. R&D, Promega Corporation, 2800 Woods Hollow Road, Madison WI 53711, USA
2Dept. of Pathology and Laboratory Medicine, University of Wisconsin School of Medicine and Public Health, Madison, WI
G Protein-Coupled Receptors (GPCRs) represent a large validated target for drug discovery research. They are classified into three main groups based on the G protein associated with the receptor. The Gs group is coupled to activation of adenylate cyclase, and the Gi group is coupled to inhibition of adenylate cyclase, while Gq is coupled to activation of phospholipase b. There are two main strategies to monitor the activation of GPCRs, reporter-based assays and cAMP accumulation-based assays. We report here on a new assay for monitoring modulation of GPCRs that are linked to activation or inhibition of adenylate cyclase (Gs or Gi). The assay can be used to monitor cAMP accumulation or depletion in the cell upon treatment with agonists or antagonists of Gs- or Gi-coupled receptors. The assay is homogenous and amenable to high-throughput screening of modulators of GPCRs. A Z′ value higher than 0.7 attests to the robustness of the assay and is carried out in 96- and 384-well plates and potentially adaptable to 1536-well format. The assay is based on luminescence, and thus it does not suffer from fluorescence interference by library compounds. We have successfully generated EC50 values for agonists and IC50 values for antagonists of Gs-coupled receptors that are similar to those reported in the literature. The assay is easy to use, can be carried out in less than 60 minutes and does not require antibodies or expensive instrumentation for signal detection. The signal output is relatively stable for several hours and thus can be used for screening large numbers of plates. This luminescent assay is fast, homogenous, and reliable, as indicated by high Z′ values, making it an attractive screening tool for identifying agonist or antagonists that modulate Gs- and Gi-coupled receptors.