G PROTEIN STRUCTURE/FUNCTION

描述（研究者摘要）： 信号，如光，激素和神经递质开始由一个特定的 配体或刺激物与受体蛋白的相互作用。 最终 细胞特异性反应是通过特异性的受体激活产生的。 GTP结合蛋白。 虽然这一过程的功能方面是 定义明确，G蛋白功能的结构基础， 受体、G蛋白和效应子之间的相互作用， 过程只被部分理解。 在最后一个赠款期间， 用于产生大量野生型和突变型G 蛋白质被排除在外。 此外，三维结构的 α和β/γ亚基以及异源三聚体α/β/γ复合物 是在保罗·西格勒的实验室的合作下解决的。 这种结构性 信息，结合功能研究，建议详细 假设的机制，保持G蛋白不活跃的情况下， 激活受体，导致G蛋白的连续激活， 在信号转导过程中通过激活受体的效应物， 通过控制GTP水解来确定活性状态的时间 rates. 这些假设将在本提案中得到检验， G蛋白α的定点突变和异源表达 β/γ亚单位。coli和杆状病毒感染的SF 9细胞。 的 模型系统包括多种受体、G蛋白和效应物， 无论是天然的还是过表达的，在生物化学测定中测试， 重组膜。 关键的氨基酸残基参与这些 过程将被确定。 与西格勒实验室的合作 将继续结晶和解析 受体-G蛋白复合物和G蛋白α和β/γ复合物 亚基与许多下游效应子。 结合结构和 功能信息将有助于我们了解基本的 通过各种信号激活细胞的机制，也将 提供对影响G蛋白功能的疾病的深入了解。

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.