| Title: | Positive Detection of GPCR Antagonists Using a System for Inverted Expression of a Fluorescent Reporter Gene |
| Author(s): | Fukuda N; Kaishima M; Ishii J; Honda S; |
| Address: | "Biomedical Research Institute, National Institute of Advanced Industrial Science and Technology (AIST) , Higashi 1-1-1, Tsukuba, Ibaraki 305-8566, Japan. Department of Chemical Science and Engineering, Graduate School of Engineering, Kobe University , 1-1 Rokkodai, Nada, Kobe 657-0013, Japan. Graduate School of Science, Technology and Innovation, Kobe University , 1-1 Rokkodai, Nada, Kobe 657-0013, Japan" |
| DOI: | 10.1021/acssynbio.7b00056 |
| ISSN/ISBN: | 2161-5063 (Electronic) 2161-5063 (Linking) |
| Abstract: | "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" |
| Keywords: | "Gene Expression Regulation, Fungal/genetics Genes, Reporter/*genetics Green Fluorescent Proteins/*genetics Molecular Imaging Promoter Regions, Genetic/genetics Protein Engineering/*methods Receptors, G-Protein-Coupled/*antagonists & inhibitors/*genetics/m;" |
| Notes: | "MedlineFukuda, Nobuo Kaishima, Misato Ishii, Jun Honda, Shinya eng 2017/05/14 ACS Synth Biol. 2017 Aug 18; 6(8):1554-1562. doi: 10.1021/acssynbio.7b00056. Epub 2017 May 18" |
