Positive Detection of GPCR Antagonists Using a System for Inverted Expression of a Fluorescent Reporter Gene.

The yeast Saccharomyces cerevisiae is a useful eukaryotic host organism for studying GPCRs as monomolecular models. Fluorescent reporter gene assays for GPCRs provide a convenient assay for measuring receptor activity using fluorometric instruments. Generally, these assays detect receptor activation by agonistic ligands as the induction of fluorescent reporter expression, whereas antagonistic activities are detected by competition with agonistic ligands, resulting in decreases in fluorescence intensity. In the current study, we established a system for inverted expression of a fluorescent reporter by incorporating a PEST-tag and finding out a promoter inhibited by activation of the GPCR signaling pathway from yeast endogenous promoters. Because agonists prevent fluorescent reporter expression in this system, antagonists compete with agonists and yield increased fluorescence intensity. We used the yeast endogenous pheromone receptor as a model GPCR to demonstrate the feasibility of our system for positive detection targeted at antagonists. Compared to results when only agonists were added to yeast cells, more than 10-fold higher fluorescence intensity was observed when antagonists were added in combination with agonists. The approach described here has the potential to markedly accelerate the identification of GPCR antagonists by providing rapid and straightforward responses.