Quantitative and dynamic analyses of G protein-coupled receptor signaling in yeast using FUS1, enhanced green fluorescence protein (EGFP), and HIS3 fusion protein
Quantitative and dynamic analyses of G protein-coupled receptor signaling in yeast using FUS1, enhanced green fluorescence protein (EGFP), and HIS3 fusion protein
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DOI：
10.1021/bp0601387
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发表时间：
2006-08-01
影响因子：
2.9
通讯作者：
Kondo, Akihiko
中科院分区：
文献类型：
--
作者：
Ishii, Jun;Matsumura, Shizuka;Kondo, Akihiko
酵母中G蛋白偶联受体（GPCR）信号转导机制与哺乳动物细胞中的类似。因此，酵母可用于GPCR测定，并且已经通过使用具有破坏的染色体基因的酵母开发了在酵母中使用信息素信号传导途径的几种配体检测系统，所述染色体基因编码产生特定效应的蛋白质。在这项研究中，酵母菌株的建设，可以检测G蛋白和GPCR之间的相互作用，使用荧光或营养缺陷型选择性介导的配体结合证明。通过将信息素应答蛋白（FUS 1）、增强型绿色荧光蛋白（EGFP）和营养缺陷型标记蛋白（HIS 3）的融合基因整合到FUS 1基因座中，构建了该菌株。此外，基因中断对酵母信号转导级联的影响进行了密切的调查，在定量和动态方面，以进一步开发一个高通量的筛选系统，使用酵母的GPCR检测。通过测量它们的荧光和生长速率来监测具有破坏的SST 2基因（其是RGS（G蛋白信号传导调节器）家族的成员）和破坏的FAR1基因（其响应于信息素而介导细胞周期停滞）的酵母菌株。该方法也适用于其他GPCR配体的综合筛选方法。
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.