G PROTEIN STRUCTURE/FUNCTION

描述(研究人员摘要)：生物的转导 光、激素和神经递质等信号由特定的 配体或刺激物与受体蛋白的相互作用。终极的 细胞特异性反应是通过特异性受体激活产生的。 GTP结合蛋白。虽然这个过程的功能方面是 定义明确，G蛋白功能的结构基础，调控 受体、G蛋白和效应器的相互作用及其激活 人们对流程的理解只有一部分。在上一次拨款期间， 大量产生野生型和突变型G的表达系统 蛋白质被研究出来了。此外，它的三维结构 α和β/伽马亚基，以及异三聚体α/β/伽马复合体 都是与保罗·西格勒的实验室合作解决的。这一结构 与功能研究相结合的信息表明， 在缺乏G蛋白的情况下保持G蛋白不活跃的机制假说 激活的受体，导致G蛋白的一系列激活和 在信号转导过程中由激活的受体激活效应器，并且 通过控制GTP水解度来确定激活状态的时间 费率。这些假设将在本提案中得到验证，使用 G蛋白α和G蛋白的定点突变及异源表达 大肠杆菌和杆状病毒感染的SF9细胞中的β/γ亚基。这个 模型系统包括各种受体、G蛋白和效应器， 无论是天然的还是过度表达的，在生化分析中都进行了测试 重组膜。它们所涉及的关键氨基酸残基 进程将被确定。与西格勒实验室的合作 将继续进行结晶和结构解析 受体-G蛋白复合体和G蛋白α、β/γ复合体 具有多个下游效应器的子单元。组合的结构和 功能信息将有助于我们理解基本的 通过各种信号激活细胞的机制，还将 洞察影响G蛋白功能的疾病。

DESCRIPTION (Investigator's Abstract): The transduction of biological signals such as light, hormones and neurotransmitters starts by a specific interaction of the ligand or stimulus with a receptor protein. The ultimate cell-specific responses are produced via receptor activation of specific GTP-binding proteins. While the functional aspects of this process are well-defined, the structural basis of G protein function, the regulated interactions between receptor, G protein and effector, and the activation processes are understood only partially. In the last grant period, expression systems for generation of large amounts of wild type and mutant G proteins were worked out. In addition, the three-dimensional structures of alpha and beta/gamma subunits, and heterotrimeric alpha/beta/gamma complex were solved in collaboration with Paul Sigler's laboratory. This structural information, in conjunction with functional studies, suggests detailed hypotheses for the mechanisms that keep G proteins inactive in the absence of activated receptors, that lead to serial activation of G proteins and effectors by activated receptors during signal transduction, and that determine the timing of the active state by controlling GTP hydrolysis rates. These hypotheses will be tested in this proposal, using site-directed mutagenesis and heterologous expression of G protein alpha and beta/gamma subunits in E. coli and Baculovirus-infected SF9 cells. The model systems include a variety of receptors, G proteins and effectors, either native or overexpressed, tested in biochemical assays in reconstituted membranes. The critical amino acid residues involved in these processes will be determined. The collaboration with Sigler's laboratory will continue with crystallization and resolution of structures of receptor-G protein complexes and complexes of G protein alpha and beta/gamma subunits with a number of downstream effectors. The combined structural and functional information will contribute to our understanding of basic mechanisms of cellular activation by a variety of signals, and will also provide insight into diseases that affect G protein function.