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

ACS Synthetic Biology
Nobuo FukudaShinya Honda

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 app...Continue Reading

References

Mar 25, 1992·Nucleic Acids Research·D GietzR H Schiestl
Sep 5, 2002·Nature Reviews. Drug Discovery·Andrew L Hopkins, Colin R Groom
Oct 7, 2004·Biochemical and Biophysical Research Communications·Mark H PauschSeung Kwak
Jun 11, 2009·Methods in Molecular Biology·Richard M Eglen, Terry Reisine
Jul 6, 2011·Analytical Biochemistry·Nobuo FukudaAkihiko Kondo
Sep 13, 2014·Current Pharmaceutical Biotechnology·Milt Teitler, Katharine Herrick-Davis
Jul 28, 2015·International Journal of Molecular Sciences·Andre GrossPeter Koltay
Feb 28, 2016·Biochemical Pharmacology·Rongfang LiuAdriaan P IJzerman
Aug 26, 2016·Nature Communications·Alvaro BanderasVictor Sourjik
Feb 10, 2017·Nature Communications·Chun-Hua LiuJin-Peng Sun

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Citations

Apr 12, 2018·ACS Synthetic Biology·Nobuo Fukuda, Shinya Honda
Oct 30, 2018·Chemical Reviews·Oh Seok KwonJyongsik Jang

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