G PROTEIN COUPLED RECEPTOR ACTIVATION STUDIED IN YEAST

G蛋白偶联受体（GPCR）激活细胞质三聚体G蛋白在不同的细胞信号转导途径中发挥关键作用。 然而，配体与受体结合影响G蛋白变化的机制尚不清楚。 该提案的重点是α因子受体，由酵母STE 2基因编码作为研究G蛋白活化的模型。 这种受体在酵母交配中的作用导致了对受体功能和对受体功能的敏感筛选和选择的发展，这些筛选和选择允许从随机突变文库中分离具有改变的功能的受体。 由于STE 2基因产物与其他GPCR相似，并且在功能上可互换，因此所使用的结果和技术应广泛适用。该项目有四个主要目标：一。跨膜段相互作用表面和相对运动的识别。 GPCR的七个预测的跨膜螺旋的三维排列尚不清楚。 通过将功能丧失和第二位点基因内抑制突变的分离与分子内二硫键交联相结合，将鉴定跨膜片段之间的相互作用。这些相互作用将被用作α因子受体的结构建模中的约束。 此外，分子内交联对受体功能的影响将用于检测跨膜片段的激活依赖性运动。二.配体结合位点的定位。 这将通过筛选干扰配体结合的受体突变和可以补偿配体结构改变的受体突变来实现。 三.确定受体之间的相互作用机制。我们的实验室已经分离出显性负性突变受体，其干扰共表达的正常受体的功能。 此外，超敏和组成型活性突变受体的信号传导可以被共表达的正常受体抑制。 这种受体间的相互作用可能是由信号传导过程中受体的寡聚化或失活受体对G蛋白的螯合引起的。 这些可能性将通过假定的寡聚体的交联，通过检查G蛋白过表达的影响，以及通过将G蛋白α亚基共价融合到各种受体等位基因来区分。 四.识别受体-G蛋白相互作用的位点。 为了鉴定受体和G蛋白之间接触位点的特定氨基酸残基，将对特异性阻断与受体相互作用的G蛋白突变和抑制G蛋白突变的受体突变进行遗传筛选。

G protein coupled receptors (GPCRs) that activate cytoplasmic trimeric G proteins play critical roles in diverse cellular signaling pathways. However, the mechanisms by which ligand binding to receptors effect changes in the G proteins are not understood. This proposal focuses on the alpha-factor receptor, encoded by the yeast STE2 gene as a model for studying G protein activation. The role of this receptor in yeast mating has led to development of sensitive screens and selections both for and against receptor function that allow isolation of receptors with altered function from random mutational libraries. Since the STE2 gene product is similar to, and functionally interchangeable with, other GPCRs, the results and techniques used should be widely applicable. The project has four major aims: I. Identification of interacting surfaces and relative motions of transmembrane segments. The three-dimensional arrangement of the seven predicted transmembrane helices of GPCRs is not known. By combining the isolation of loss-of function and second-site intragenic suppressor mutations with intramolecular disulfide crosslinking, interactions between transmembrane segments will be identified. These interactions will be used as constraints in structural modeling of the alpha-factor receptor. In addition, effects of intramolecular crosslinking on receptor function will be used to detect activation-dependent motions of transmembrane segments. II. Localization of the site of ligand binding. This will be accomplished by screening for receptor mutations that interfere with ligand binding and for receptor mutations that can compensate for alterations in the structure of the ligand. III. Determination of the mechanism of interactions between receptors. Our laboratory has isolated dominant negative mutant receptors that interfere with the function of co-expressed normal receptors. In addition, signaling by hypersensitive and constitutively active mutant receptors can be suppressed by co-expressed normal receptors. Such inter-receptor interactions could result from oligomerization of receptors during signaling or sequestration of G protein by inactive receptors. These possibilities will be distinguished by crosslinking of putative oligomers, by examining the effects of G protein overexpression, and by covalently fusing the G protein alpha subunit to various receptor alleles. IV. Identification of sites of receptor-G protein interactions. To identify particular amino acid residues at sites of contact between the receptor and the G protein, genetic screens will be conducted for G protein mutations that specifically block interactions with the receptor and for receptor mutations that suppress G protein mutations.