INHIBITION OF G PROTEIN BETA-SUBUNIT SIGNALING IN YEAST

在许多不同的生物体中，通过异源三聚体G蛋白作用的信号转导通路介导了对多种胞外信号的反应。在酵母中，信息素反应途径是通过细胞外信息素与G蛋白偶联受体的结合来激活的，G蛋白偶联受体是MATA或MATAlpha交配型细胞的特异性受体。G蛋白α亚基的激活导致β-伽马亚基的释放，后者将信号传递到下游的激酶。这项提案中描述的研究将探索a因子受体Ste3p的一种新功能。这种被称为受体抑制的新功能，通过以一种独立于阿尔法亚基的方式抑制β亚基的活性，导致信号传递受阻。只有当细胞同时含有α-特异性和α-特异性蛋白时，受体抑制才会发生，而且可能在两个交配细胞融合后立即发挥作用，以确保信号通路迅速关闭。这一过程需要一种新发现的称为ASG7的a-特异性基因的产物。因此，当同一细胞同时表达a-特异性蛋白Asg7p和α-特异性蛋白Ste3p时，就会启动一个直接作用于β亚基并抑制其活性的过程。这个项目将研究当a因子受体和新的调节因子Asg7p存在于同一细胞中时，β亚基信号是如何被抑制的。一个具体的目标是确定这个过程是如何阻止β亚基的活动的。Asg7p丰度、定位和与β亚基结合的变化对β亚基活性的影响将被测试。还将对受体抑制所需的其他基因进行筛查。第二个特定的目的是确定Asg7p是如何通过Ste3p受体的存在而激活的。膜定位、翻译后修饰和Ste3p结合在As97p激活中的潜在作用将被确定。最后一个特定的目标是研究Ste3p与其受体抑制功能之间的结构/功能关系。通过分离STE3中的特定突变和构建嵌合受体，将确定参与受体抑制的Ste3p区域。在阐明信号转导机制方面已经取得了重大进展，在生物体中可以很容易地进行复杂的遗传操作。阐明酵母中受体抑制的过程可能会提供有关G蛋白β亚基在其他系统中的调节信息，在这些系统中，β亚基在信号传递中发挥积极作用。

Signal transduction pathways that act through heterotrimeric G proteins mediate the response to a wide variety of extracellular signals in many different organisms. In yeast, the pheromone response pathway is activated by the binding of extracellular pheromones to G protein- coupled receptors that are specific to cells of either the MATa or MATalpha mating type. Activation of the G protein alpha-subunit results in release of the beta-gamma-subunits, which transmit the signal to downstream kinases. Studies described in this proposal will investigate a novel function of Ste3p, the a-factor receptor. This novel function, called receptor inhibition, causes a block in signal transmission by inhibiting the activity of the beta-subunit in a manner that is independent of the alpha-subunit. Receptor inhibition only occurs when cells contain both a- specific and alpha-specific proteins, and probably functions immediately after the fusion of two mating cells to ensure that the signaling pathway is rapidly turned off. This process requires the product of a newly identified a-specific gene called ASG7. Thus, when the same cell expresses both Asg7p, an a-specific protein, and Ste3p, an alpha-specific protein, a process is initiated that acts directly on the beta-subunit and inhibits its activity. This project will investigate how beta-subunit signaling is inhibited when the a-factor receptor and the novel regulator Asg7p are present in the same cell. One specific aim is to determine how the activity of the beta- subunit is blocked by this process. The effect of changes in Asg7p abundance, localization, and binding to the beta-subunit will be tested for their effects on beta-subunit activity. A screen for other genes required for receptor inhibition will also be carried out. A second specific aim is to determine how Asg7p is activated by the presence of the Ste3p receptor. The potential roles of membrane localization, posttranslational modification, and Ste3p binding in the activation of As97p will be determined. A final specific aim is to investigate the structure/function relationships of Ste3p with respect to its receptor inhibition function. By isolating specific mutations in STE3 and by constructing chimeric receptors, the region of Ste3p involved in receptor inhibition will be identified. Significant progress in elucidating signal transduction mechanisms has been made in organisms in which sophisticated genetic manipulations can easily be performed. Elucidation of the process of receptor inhibition in yeast is likely to provide information about the regulation of G protein beta-subunits in other systems in which the beta-subunit plays an active role in signal transmission.