MODEL SCREENING SYSTEM FOR MEMBRANE RECEPTOR LIGANDS IN COMBINATION WITH GPCR FLUORESCENCE ASSAY USING A SINGLE CELL, HROMYCESSACCA CEREVISIAE
For the basis of ligand screening system using combinatorial peptide libraries displayed on yeast cellular membrane, a factor, which is an endogenous peptide ligand of Ste2p, the G protein-coupled receptor (GPCR) for yeast pheromone response pathway, was displayed on cellular membrane using glycosylphosphatidylinositol (GPI)-anchored plasma membrane protein YPS1 with several different lengths of linkers. Compared to the conventional cell surface display system, the peptide ligand is displayed not on cell wall but on cellular membrane, which is considered to be optimal in terms of interaction with membrane receptors. As a result, displayed α factor functionally activated pheromone response pathway in Saccharomyces cerevisiae, which was confirmed by fluorescent reporter gene assay, fluorescent microscope, FACS analysis, and Western blot. Interestingly, lengths of linkers have crucial influence in activating the pathway. This membrane ligand screening system in combination with yeast GPCR assay will be a robust tool for identification of novel and unknown peptide ligands for exogenous mammalian GPCRs.
Yeast GPCR assay ligand screening high-throughput assay membrane ligand display system fluorescent reporter gene assay raft research
Keisuke HARA Kouichi Kuroda Mitsuyoshi UEDA
Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Kitashirakawa-oiwake-cho, Sakyo-ku, Kyoto 606-8502, Japan
国际会议
The 6th International Forum on Post-genome Technologies(6IFPT)(第六届国际后基因组生命科学技术学术论坛)
北京
英文
375-376
2009-09-17（万方平台首次上网日期，不代表论文的发表时间）