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蛋白起作用的信号转导途径介导对多种细胞外信号的应答。在酵母中，通过细胞外信息素与G蛋白偶联受体的结合激活信息素应答途径，所述G蛋白偶联受体对MATa或MATalpha交配型细胞具有特异性。G蛋白α-亚基的活化导致β-γ-亚基的释放，其将信号传递到下游激酶。本提案中描述的研究将调查a因子受体Step 3 p的新功能。这种新的功能称为受体抑制，通过以独立于α亚基的方式抑制β亚基的活性来阻断信号传递。受体抑制仅在细胞含有α-特异性和α-特异性蛋白质时发生，并且可能在两个交配细胞融合后立即起作用，以确保信号传导途径迅速关闭。这个过程需要一个新发现的称为ASG 7的特异性基因的产物。因此，当同一细胞同时表达Asg 7 p（一种α-特异性蛋白）和Ste 3 p（一种α-特异性蛋白）时，就会启动一个直接作用于β-亚基并抑制其活性的过程。该项目将研究当a因子受体和新型调节剂Asg 7 p存在于同一细胞中时，β亚基信号传导如何被抑制。一个具体的目的是确定β亚基的活性如何被这一过程阻断。将测试Asg 7 p丰度、定位和与β亚基结合的变化对β亚基活性的影响。还将对受体抑制所需的其他基因进行筛选。第二个具体目标是确定Asg 7 p如何被Ste 3 p受体的存在激活。膜定位，翻译后修饰，和Ste 3 p结合在激活As 97 p的潜在作用将被确定。最后一个具体的目的是调查的结构/功能的关系，Ste 3 p相对于其受体抑制功能。通过分离STE 3中的特定突变并通过构建嵌合受体，将鉴定参与受体抑制的Ste 3 p区域。在阐明生物体中的信号转导机制方面已经取得了重大进展，在这些生物体中可以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提供关于G蛋白β亚基在其他系统中的调节的信息，其中β亚基在信号传递中起着积极的作用。

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.