MECHANISM AND REGULATION OF RECEPTOR-G PROTEIN SIGNALLIN

许多激素、耳石、神经调节剂和感官刺激物起作用 通过与细胞内鸟嘌呤偶联的细胞表面受体 核苷酸结合蛋白(G蛋白)。这个家族的受体在 人类不同的生理过程，包括血液调节 压力、炎症、心律、突触反应和神经元 可塑性、光和嗅觉与细胞调节 扩散。 受体间信号转导的机制和调控 蛋白质及其细胞内效应器是尚未解决的关键问题 从发展药理药物的角度看其重要性 以特定受体亚型为目标，并了解(可能 控制)某些细胞癌基因的转化活性。 尽管受体和G蛋白的结构特征 跨膜信号开始出现，其机制是 受体、G蛋白和效应器被激活和调节较差 明白了。 这项研究计划的长期目标是建立 G蛋白偶联信号系统的分子原理 受监管的。出于这些目的，该系统提供了独特的实验性 优点将被使用：阿尔法因子交配信息素信号 酿酒酵母的转导途径。编码基因 α-因子受体及其α、β、γ亚基 同源G蛋白已被鉴定。药理和生化 受体/G蛋白功能的分析现在已经发展得很好。这些 生化技术将与经典酵母的技术相结合 遗传学、分子遗传学和酵母菌交配生理学 表征突变的受体和改变信号的G蛋白亚基 以新颖而富有启发性的方式进行转导过程。此外，潜在的 我们将探索细胞内效应器的性质。 具体目标是：1.确定阿尔法的结构要素- 因子受体通过选择Fas参与跨膜信号转导 受体变异体：i)不再激活G蛋白；ii) 在没有结合激动剂的情况下结构性地激活G蛋白；以及iii)具有 获得了对阿尔法因子拮抗剂做出反应的能力。2.建立 受体突变是否会影响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， 监测G受体的平衡实验和竞争结合实验 体外蛋白质偶联；III)激动剂刺激的GTP酶或GDP/GTP Exchange用于监测G蛋白的激活情况。3.发掘潜力 细胞内第二信使系统(S)介导对α-氨基丁酸的反应 通过确定是否：i)α因子受体和其 在非洲爪哇卵母细胞中表达的同源酵母G蛋白亚基的调节作用 调节离子通道活性的效应器；以及ii)刺激的酵母细胞 与α-因子通过水解产生可能的第二信使 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.