MECHANISM AND REGULATION OF RECEPTOR-G PROTEIN SIGNALLIN

许多激素，autacoids，神经调节剂，和感官刺激的作用， 通过细胞表面受体与细胞内鸟嘌呤结合 核苷酸结合蛋白（G蛋白）。 该家族的受体介导 人类的各种生理过程，包括血液调节 压力、炎症、心律、突触反应和神经元 可塑性，光和嗅觉的感觉，以及细胞的调节 增殖 受体间信号转导的机制和调控，G 蛋白质及其细胞内效应物是尚未解决的关键问题， 从开发药理学试剂的观点来看， 针对特定的受体亚型，并了解（也许 控制）某些细胞癌基因的转化活性。 虽然受体和G蛋白的结构特征， 跨膜信号开始出现， 受体、G蛋白和效应物的激活和调节较差 明白 这项研究计划的长期目标是建立基本的 G蛋白偶联信号系统的分子原理 监管. 出于这些目的，一个提供独特实验的系统 优势将被使用：α因子交配信息素信号 酵母Saccharomycescerevisiae的转导途径。 基因编码 α-因子受体及其α、β和γ亚基 已经鉴定了同源G蛋白。 药理学和生物化学 受体/G蛋白功能的测定现在已经发展得很好。 这些 生物化学技术将与传统的酵母技术相结合 遗传学、分子遗传学和酵母交配生理学来选择和 表征改变信号的突变受体和G蛋白亚基 在新的和启发性的方式转导过程。 此外， 将探索细胞内效应物的性质。 具体目标是：1. 识别阿尔法的结构元素- 因子受体参与跨膜信号转导，通过选择 受体变体：i）不再激活G蛋白; ii） 组成型激活G蛋白而没有结合激动剂;和iii）具有 获得了对α因子拮抗剂的反应能力。 2. 建立 受体突变是否影响G蛋白的相互作用或激活， 使用：i）等位基因特异性抑制技术以获得G蛋白 定义特定蛋白质-蛋白质相互作用的变体; ii）动力学， 平衡和竞争结合实验，以监测受体G 体外蛋白偶联; iii）激动剂刺激的GTP酶或GDP/GTP 交换以监测G蛋白激活。 3. 挖掘潜力 细胞内第二信使系统介导对α- 通过确定是否：i）α-因子受体及其 在非洲爪蟾卵母细胞中表达的同源酵母G蛋白亚基调节 调节离子通道活性的效应物;和ii）酵母细胞刺激 与α-因子通过水解产生推定的第二信使 膜磷脂。

Many hormones, autacoids, neuroregulatory agents, and sensory stimuli act through cell-surface receptors that are coupled to intracellular guanine nucleotide-binding proteins (G proteins). Receptors of this family mediate diverse physiological processes in man, including regulation of blood pressure, inflammation, cardiac rhythm, synaptic response and neuronal plasticity, sensation of light and olfactants, and regulation of cellular proliferation. The mechanisms and regulation of signal transduction between receptors, G proteins and their intracellular effectors are unresolved issues of key importance from the standpoint of developing pharmacological agents targeted to specific receptor subtypes, and understanding (and perhaps controlling) the transforming activity of certain cellular oncogenes. Although the structural features of receptors and G proteins that mediate transmembrane signaling are beginning to emerge, the mechanisms by which receptors, G proteins and effectors are activated and regulated are poorly understood. The long-term goal of this research program is to establish the fundamental molecular principles by which G protein-coupled signaling systems are regulated. for these purposes a system offering unique experimental advantages will be used: the alpha-factor mating pheromone signal transduction pathway of the yeast Saccharomyces cerevisiae. Genes encoding the alpha-factor receptor and the alpha, beta and gamma subunits of its cognate G protein have been identified. Pharmacological and biochemical assays for receptor/G protein function are now well-developed. These biochemical techniques will be combined with those of classical yeast genetics, molecular genetics and yeast mating physiology to select and characterize mutant receptors and G protein subunits that alter the signal transduction process in novel and illuminating ways. Also, the potential nature of intracellular effectors will be explored. Specific objectives are to: 1. Identify structural elements of the alpha- factor receptor involved in transmembrane signaling by selecting for receptor variants that: i) no longer activate G protein; ii) constitutively activate G protein without binding agonist; and iii) have gained the ability to respond to alpha-factor antagonists. 2. Establish whether receptor mutations affect G protein interaction or activation by using: i) techniques of allele-specific suppression to obtain G protein variants that define specific protein-protein interactions; ii) kinetic, equilibrium, and competition binding experiments to monitor receptor-G protein coupling in vitro; iii) of agonist-stimulated GTPase or GDP/GTP exchange to monitor G protein activation. 3. Explore the potential intracellular second messenger system(s) that mediate response to alpha- factor by determining whether: i) the alpha-factor receptor and its cognate yeast G protein subunits expressed in Xenopus oocytes modulate effectors that regulate ion channel activity; and ii) yeast cell stimulated with alpha-factor produce putative second messengers through the hydrolysis of membrane phospholipids.