Development Of Novel Assays For Detection Of GPCR Mediated Second Messenger-cAMP And Ca²⁺

G-protein-coupled receptors (GPCRs) are involved in a wide range of physiological processes and diseases, and represents significant and attractive targets for drug discovery. GPCRs interact with heterotrimeric G proteins to regulate a range of second messenger pathways to enable communication from the cell surface to the nucleus. Ca2+ and cAMP are important second messengers that regulate multiple cellular processes. Based on cAMP response element (CRE) and nuclear factor of activated T-cells (NFAT), we have developed a novel assay system with dual-reporter gene to detect both cellular cAMP and Ca2+ signals, providing a G-protein subtype-independent method to determine agonist-stimulated activation of receptors. This assay system has been further validated with six functionally divergent GPCRs:the insect adipokinetic hormone receptor (AKHR), the human cannabinoid receptor 1 and 2 (CB1 and CB2), the human Histamine receptor 1 and 3 (H1R and H3R), the human nicotinic acid receptor (HM74a) and the human Glucagon-like peptide 1 (GLP-1). The EC50 data obtained for known agonists are in close agreement with the results of previous reports. Our data derived from this assay were found to be comparable to the results obtained with the classical cAMP and Ca2+ assay methods, indicating that this assay system has sufficient sensitivity for detection of GPCR-mediated second messenger signal. In the current study, we used this assay system to analyze the crosstalk between cAMP and Ca2+signal pathways, and also crosstalk between different GPCRs. This cell-based assay has been demonstrated to be easily manipulated, and to be cost-effective, with no requirements for sophisticated instruments and expensive reagents as a primary assay for GPCR. Therefore, this rapid and quantitative assay could be used as a universal and cell-based function assay for GPCR high-throughput screening for drug discovery.