G PROTEIN COUPLED RECEPTOR ACTIVATION STUDIED IN YEAST

G蛋白偶联受体(GPCRs)激活胞质三聚体G蛋白，在多种细胞信号通路中发挥重要作用。然而，配体与受体结合影响G蛋白变化的机制尚不清楚。这项建议将重点放在酵母STE2基因编码的阿尔法因子受体上，作为研究G蛋白激活的模型。这种受体在酵母交配中的作用导致了对受体功能的敏感筛选和选择，这些筛选和选择允许从随机突变文库中分离出功能改变的受体。由于STE2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该具有广泛的适用性。该项目有四个主要目标：1.识别相互作用的表面和跨膜片段的相对运动。GPCRs预测的七个跨膜螺旋的三维排列尚不清楚。通过分离功能缺失和第二位基因内抑制突变与分子内二硫键交联，将鉴定跨膜片段之间的相互作用。这些相互作用将被用作阿尔法因子受体结构建模的约束条件。此外，分子内交联对受体功能的影响将被用来检测跨膜片段的激活依赖运动。II.配体结合部位的定位。这将通过筛选干扰配体结合的受体突变和能够补偿配体结构变化的受体突变来实现。受体间相互作用机制的确定。我们实验室已经分离出了干扰共表达的正常受体功能的显性负性突变受体。此外，超敏和结构性活性突变受体的信号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于受体在信号传递过程中的寡聚化或通过非活性受体隔离G蛋白而导致的。这些可能性将通过假设的寡聚体的交联、通过检查G蛋白过度表达的影响以及通过将G蛋白α亚单位与各种受体等位基因共价融合来区分。IV.确定受体-G蛋白相互作用的部位。为了确定受体和G蛋白接触部位的特定氨基酸残基，将进行基因筛查，寻找专门阻止与受体相互作用的G蛋白突变，以及抑制G蛋白突变的受体突变。

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.