G Protein Coupled Receptor Activation Studied in Yeast

G蛋白偶联受体(GPCRs)在重要的生物学和医学信号转导中发挥着重要作用，但其激活细胞内G蛋白的分子机制尚不清楚。酵母信息素反应途径提供了一个GPCR信号的例子，这种信号可以通过在其他类型的细胞中不可能被遗传操纵的方式进行操作，但该途径的组件在功能上可以与哺乳动物系统的相应组件互换。该项目将使用只能在酵母中使用的遗传方法，通过异源表达的生长抑素2型受体和a交配信息素的内源酵母受体，逐步剖析GPCR信号传递的机制，这两种受体可以激活相同的G蛋白，但没有序列相似性。该项目的目的是：1.确定G蛋白与受体相互作用的部位。为了解决GPCR如何激活导致G蛋白进行核苷酸交换的问题，并绘制两种类型的受体和G蛋白之间的界面，将进行基因筛查，以确定G蛋白亚基中特定防止与受体相互作用的突变，以及特定补偿G蛋白亚基缺陷的受体突变。鉴定构成受体激活的构象变化。一种成对随机引入半胱氨酸残基的新技术将被用来筛选被二硫键锁定在激活状态的突变受体等位基因，作为识别在受体激活时经历分子内运动的位置的一种方式。识别介导对不同类型配体的反应的配体-受体相互作用。将进行筛选，以识别对某些配体反应改变的突变生长抑素受体，以及对信号影响改变的生长抑素配体改变。目的是确定受体激活和非激活状态所特有的配体-受体相互作用的特定部位。IV.受体齐聚对信号的影响的测定和受体-受体相互作用部位的确定。一种新的基于流式细胞术的分析将用于筛选随机突变的受体文库，以确定特定阻止受体寡聚的氨基酸替代。突变的受体将被用来确定寡聚对信号功能的影响。

G protein coupled receptors (GPCRs) play critical roles in biologically and medically important signaling; however, the molecular mechanisms of activation of intracellular G proteins by ligand-bound GPCRs are not understood. The yeast pheromone response pathway provides an example of GPCR signaling that can be genetically manipulated in ways that would be impossible in other types of cells, yet components of this pathway are functionally interchangeable with corresponding components of mammalian systems. This project will use genetic approaches only possible in yeast to pursue a step-by-step dissection of the mechanisms of GPCR signaling by heterologously expressed somatostatin type 2 receptor and the endogenous yeast receptor for the a-mating pheromone, two receptors that can activate the same G protein but share no sequence similarity. The aims of the project are: I. Identification of sites of G protein interaction with receptors. To address the question of how activation of a GPCR leads to nucleotide exchange by a G protein and to map the interfaces between the two types of receptors and G proteins, a genetic screen will be conducted to identify mutations in G protein subunits that specifically prevent the interaction with receptors and mutations in receptors that specifically compensate for defects in G protein subunits. II. Identification of the conformational changes that constitute receptor activation. A new technique for pair-wise random introduction of cysteine residues will be used to screen for mutant receptor alleles that are locked in the activated state by disulfide bonds as a way of identifying sites that undergo intramolecular motion upon activation of receptors. III. Identification of ligand-receptor interactions mediating responses to different types of ligands. Screens will be conducted to identify mutant somatostatin receptors with altered responses to certain ligands and altered somatostatin ligands with altered effects on signaling. The goal is to identify particular sites of ligand-receptor interaction that are specific for activated and un-activated states of the receptor. IV. Determination of effects of receptor oligomerization on signaling and identification of sites of receptor-receptor interaction. A new flow cytometry-based assay will be used to screen libraries of randomly mutagenized receptors to identify amino acid substitutions that specifically block receptor oligomerization. The mutant receptors will then be used to determine the effects of oligomerization on signaling function.