MECHANISM AND REGULATION OF RECEPTOR-G PROTEIN SIGNALING

受体-G 蛋白信号传导的机制和调控

• 批准号: 2182603
• 负责人: Kendall J Blumer
• 金额: $ 20.74万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1990
• 资助国家: 美国
• 起止时间: 1990-07-01 至 1995-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2182603
• 关键词: G protein; Saccharomyces cerevisiae; Xenopus oocyte; alleles; antibody formation; crosslink; hormone receptor; hormone regulation /control mechanism; immunoprecipitation; laboratory rabbit; membrane lipids; mutant; phase contrast microscopy; pheromone; phospholipids; protein structure function; receptor coupling; scintillation spectrometry; second messengers; site directed mutagenesis; ultracentrifugation

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.

许多激素，自闭症，神经调节剂和感觉刺激作用 通过与细胞内鸟嘌呤耦合的细胞表面受体 核苷酸结合蛋白（G蛋白）。 这个家庭的受体调解 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，光和嗅觉剂的感觉以及细胞的调节 增殖。 受体之间信号转导的机制和调节 蛋白质及其细胞内效应子是尚未解决的关键问题 从开发药理学剂的角度来看 针对特定受体亚型和理解（也许 控制）某些细胞癌基因的转化活性。 虽然受体和G蛋白的结构特征介导 跨膜信号开始出现，该机制的机制 受体，G蛋白和效应子被激活，调节很差 理解。 该研究计划的长期目标是建立基本 G蛋白偶联信号系统是分子原理 受监管。 出于这些目的，提供独特的实验的系统 将使用优点：alpha因子交配信息素信号 酿酒酵母的酵母糖疗法的转导途径。 基因编码 其α因子受体以及其α，β和伽玛亚基 认知G蛋白已被鉴定出来。 药理和生化 现在，对受体/G蛋白功能的测定已发达。 这些 生化技术将与经典酵母菌相结合 选择和 表征突变体受体和G蛋白亚基，以改变信号 新颖和照明方式的转导过程。 另外，潜力 将探索细胞内效应子的性质。 特定目标是：1。确定α-的结构元素 通过选择参与跨膜信号传导的因子受体 受体变体：i）不再激活G蛋白； ii） 组成性激活G蛋白而无需结合激动剂；和iii） 获得了对α因素拮抗剂反应的能力。 2。建立 受体突变是影响G蛋白相互作用还是通过 使用：i）等位基因特异性抑制以获得G蛋白 定义特定蛋白质蛋白相互作用的变体； 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；激动剂刺激的GTPase或GDP/GTP的iii） 交换以监测G蛋白激活。 3。探索潜力 细胞内二信使系统介导对α-的反应 通过确定是否：i）α因子受体及其ITS来确定是否 在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子； ii）刺激的酵母细胞 用α因子通过水解产生推定的第二使者 膜磷脂。