Quantitative and Dynamic Analyses of G Protein-Coupled Receptor Signaling in Yeast Using Fus1, Enhanced Green Fluorescence Protein (EGFP), and His3 Fusion Protein

Abstract

The mechanism of G protein-coupled receptor (GPCR) signaling in yeasts is similar to that in mammalian cells. Therefore, yeasts can be used in GPCR assays, and several ligand detection systems using a pheromone signaling pathway in yeasts have been developed by employing yeasts with disrupted chromosomal genes that code for proteins producing specific effects. In this study, the construction of yeast strains that can detect ligand binding mediated by interactions between the G protein and GPCR using either fluorescence or auxotrophic selectivity is demonstrated. The strain was constructed by integrating the fusion gene of pheromone-responsive protein (FUS1), enhanced green fluorescence protein (EGFP), and auxotrophic marker protein (HIS3) into the FUS1 locus. Moreover, the influence of gene disruptions on the yeast signal transduction cascade is closely investigated with respect to both quantitative and dynamic aspects to further develop a high-throughput screening system for the GPCR assay using yeasts. Yeast strains with a disrupted SST2 gene, which is a member of the RGS (regulator of G protein signaling) family, and a disrupted FAR1 gene, which mediates cell cycle arrest in response to a pheromone, were monitored by measuring their fluorescence and growth rate. This method will be applicable to other comprehensive GPCR ligand screening methods.

Bright Fluorescence Monitoring System Utilizing Zoanthus sp. Green Fluorescent Protein (ZsGreen) for Human G-Protein-Coupled Receptor Signaling in Microbial Yeast Cells

“…The sst2 Δ allele is deficient in the yeast principal negative regulator of G-protein signaling (RGS). This deletion therefore results in hypersensitivity for the agonistic stimulus [5,32] and improves sensitivity towards low concentrations of ligand. The far1 Δ allele is deficient in yeast G1-cyclin-dependent kinase inhibitor; this inhibitor induces G1 cell cycle arrest in response to signaling [5,33].…”

Rapid, Facile Detection of Heterodimer Partners for Target Human G-Protein-Coupled Receptors Using a Modified Split-Ubiquitin Membrane Yeast Two-Hybrid System

“…An agar diffusion bioassay (halo assay) was performed to measure growth inhibition in response to signal-induced cell-cycle arrest [36]. Cells were grown in YPDA media overnight at 30°C.…”

Cell Wall Trapping of Autocrine Peptides for Human G-Protein-Coupled Receptors on the Yeast Cell Surface

“…The P FUS1 -FUS1-GFP or P FIG1 -GFP reporter gene was used to monitor signal transduction promoted by stimulating GPCRs in yeast (IMG-4, IMG-50 or IMFD-70 [5]). far1 Δ alleles were used to avoid G1 arrest and promote cell-cycle progression during signal activation [5], [28], [35] (IMG-4 and IMFD-70). sst2 Δ and ste2 Δ alleles were used to obtain hypersensitivity for ligand stimulation and to inhibit competitive expression of endogenous yeast GPCRs [5], [28] (IMG-50 and IMFD-70).…”