« Previous AbstractRapid inactivation of a moth pheromone    Next AbstractProtein-protein interactions and selection: yeast-based approaches that exploit guanine nucleotide-binding protein signaling »

Biotechnol Prog

Title:		"Quantitative and dynamic analyses of G protein-coupled receptor signaling in yeast using Fus1, enhanced green fluorescence protein (EGFP), and His3 fusion protein"
Author(s):		Ishii J; Matsumura S; Kimura S; Tatematsu K; Kuroda S; Fukuda H; Kondo A;
Address:		"Department of Molecular Science and Material Engineering, Graduate School of Science and Technology, Kobe University, Nada-ku, Japan"
Journal Title:		Biotechnol Prog
Year:		2006
Volume:		22
Issue:		4
Page Number:		954 - 960
DOI:		10.1021/bp0601387
ISSN/ISBN:		8756-7938 (Print) 1520-6033 (Linking)
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"
Keywords:		"Biological Assay Cell Membrane/chemistry/physiology Genes, Fungal/genetics Genes, Reporter/genetics Green Fluorescent Proteins/*genetics Histidine/genetics Mating Factor Membrane Proteins Organisms, Genetically Modified Peptides/pharmacology Receptors, G-;"
Notes:		"MedlineIshii, Jun Matsumura, Shizuka Kimura, Sakurako Tatematsu, Kenji Kuroda, Shun'ichi Fukuda, Hideki Kondo, Akihiko eng Research Support, Non-U.S. Gov't 2006/08/08 Biotechnol Prog. 2006 Jul-Aug; 22(4):954-60. doi: 10.1021/bp0601387"