INHIBITION OF G PROTEIN BETA-SUBUNIT SIGNALING IN YEAST

酵母中 G 蛋白 β 亚基信号传导的抑制

• 批准号: 6628918
• 负责人: Jeanne P. Hirsch
• 金额: $ 21.27万
• 依托单位: ICAHN SCHOOL OF MEDICINE AT MOUNT SINAI
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-02-01 至 2005-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6628918
• 关键词: G protein; Saccharomyces cerevisiae; biological signal transduction; cell cell interaction; cell fusion; chimeric proteins; fungal genetics; fungal proteins; hormone receptor; immunoprecipitation; molecular genetics; molecular site; pheromone; posttranslational modifications; protein localization; protein protein interaction; protein structure function; receptor binding; receptor expression; site directed mutagenesis; yeast two hybrid system

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.

通过异三聚体G蛋白起作用的信号转导途径介导了许多不同生物体中对各种细胞外信号的反应。在酵母中，信息素反应途径通过细胞外信息素与G蛋白偶联受体的结合激活，该受体特异于MATA或MATALPHA交配类型的细胞。 G蛋白α-亚基的激活导致β-Gamma-subunits释放，从而将信号传递到下游激酶。该提案中描述的研究将研究ASTE3P（A因子受体）的新功能。这种新型功能（称为受体抑制作用）通过独立于α-亚基的方式抑制β-亚基的活性，从而导致信号传播的障碍。受体抑制只有在细胞含有A-特异性和α特异性蛋白质时才发生，并且在两个配合细胞融合后可能会立即起作用，以确保信号传导途径迅速关闭。此过程需要新鉴定的称为ASG7的A特异性基因的乘积。因此，当同一细胞同时表达ASG7P（A a特异性蛋白ASG7P）和α特异性蛋白的Ste3p时，就开始了直接作用于β-亚基并抑制其活性的过程。当A因子受体和新型调节剂ASG7P存在于同一细胞中时，该项目将如何抑制β-亚基信号传导。一个具体的目的是确定该过程如何阻止β亚基的活性。 ASG7P丰度，定位和与β-亚基的结合的影响将被测试，以测试其对β-亚基活性的影响。还将进行其他受体抑制所需基因的屏幕。第二个特定目的是确定如何通过Ste3p受体的存在激活ASG7P。将确定膜定位，翻译后修饰和Ste3p结合在AS97P激活中的潜在作用。最终的具体目的是研究Ste3P在其受体抑制功能方面的结构/功能关系。通过在Ste3中和构建嵌合受体中分离特异性突变，将确定参与受体抑制的Ste3p区域。在阐明信号转导机制方面取得了重大进展，在生物体中可以轻松地进行复杂的遗传操作。在酵母中阐明受体抑制过程可能会提供有关其他系统中G蛋白β-亚基的调节的信息，在其他系统中，β-亚基在信号传输中起着积极作用。