G Protein Coupled Receptor Activation Studied in Yeast

G蛋白偶联受体（gpcr）在生物学和医学上重要的信号传导中发挥关键作用；然而，配体结合的gpcr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信号传导的例子，可以通过其他类型细胞不可能的方式进行遗传操纵，然而该途径的组成部分在功能上与哺乳动物系统的相应组成部分可互换。该项目将使用遗传方法，只可能在酵母中通过异源表达的生长抑素2型受体和内源性酵母a-交配信息素受体来逐步解剖GPCR信号传导机制，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 . G蛋白与受体相互作用位点的鉴定。为了解决GPCR的激活如何导致G蛋白进行核苷酸交换的问题，并绘制两种受体和G蛋白之间的界面，将进行遗传筛选，以确定G蛋白亚基中特异性阻止与受体相互作用的突变，以及特异性补偿G蛋白亚基缺陷的受体突变。2。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用于筛选被二硫键锁定在激活状态的突变受体等位基因，作为识别受体激活后经历分子内运动的位点的一种方法。3。鉴定配体-受体相互作用介导对不同类型配体的反应。将进行筛选，以确定突变的生长抑素受体对某些配体的反应改变，以及改变的生长抑素配体对信号传导的影响改变。目的是确定配体与受体相互作用的特定位点，这些位点对受体的激活和非激活状态具有特异性。受体寡聚化对信号传导的影响以及受体-受体相互作用位点的鉴定。一种新的基于流式细胞术的检测将用于筛选随机诱变的受体文库，以鉴定特异性阻断受体寡聚化的氨基酸取代。突变受体将被用来确定寡聚化对信号功能的影响。

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.