RAS-LIKE GTP-BINDING PROTEINS--REGULATION AND FUNCTION

RAS 样 GTP 结合蛋白——调控和功能

• 批准号: 3200701
• 负责人: IAN G MACARA
• 金额: $ 9.47万
• 依托单位: UNIVERSITY OF VERMONT & ST AGRIC COLLEGE
• 依托单位国家: 美国
• 财政年份: 1991
• 资助国家: 美国
• 起止时间: 1991-08-15 至 1996-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3200701
• 关键词: Escherichia coli; G protein; affinity chromatography; antibody; enzyme mechanism; gene expression; gene mutation; genetic regulatory element; guanine nucleotide binding protein; guanosine triphosphate; guanosinetriphosphatases; immunoprecipitation; intracellular transport; laboratory rabbit; molecular cloning; oncogenes; posttranslational modifications; protein purification; protein structure function; site directed mutagenesis; transcription factor; transfection; vesicle /vacuole

The goal of this project is to determine the mechanisms that regulate the activities of small, ras-like GTP binding proteins, and to investigate possible functions. A family exists of >20 such proteins, of unknown function, with 20-60% homology to the Ki-ras protein, that probably operate as "molecular switches'. Their medical importance is underlined by the discovery that one member, rap1 (or Krev), suppresses transformation by ras, and another, rho is a target for botulinum toxin. Multiple factors control the switch-states of these proteins, catalyzing GDP/GTP exchange or GTPase activity, or inhibiting GDP release. The ras proteins are also extensively modified, covalently. It is important to understand the functions and control of these GTP-binding proteins. The main focus will be on the rab subclass. The project is divided into 3 main sections: 1. Characterization. Specific antibodies are required against rab peptides and recombinant rab proteins expressed i E. coli. These reagents will be used to determine tissue distribution, subcellular localization and covalent modifications of the rab proteins. The hypothesis will be tested that different C-terminal consensus sequences control modification and localization. 2. Regulation. A ras-specific guanine nucleotide releasing factor has been discovered, that could function as an "on switch". The existence of similar factors specific to the rab proteins will be investigated. Other factors, that control rab GTPase activity (GAPS) will also be identified and purified, to determine control mechanisms. 3. Function. Three approaches will be used. (a) Affinity columns will be prepared from recombinant rab proteins and used to purify factors that bind specifically to the GTP-states of the proteins. Such factors are potential targets (b) The transforming potential, and transformation-suppressing activity of the rab proteins will be measured, to define the specificity of the properties of ras Krev. (c) Finally, the hypothesis will be tested, in collaboration with other laboratories, that the rab proteins normally function to control exocytosis and/or interorganelle transport, using in vitro assays.

本项目的目标是确定调节 小的ras样GTP结合蛋白的活性，并研究 可能的功能。 一个家族存在超过20种这样的蛋白质， 功能与Ki-ras蛋白具有20-60%的同源性，可能起作用 称为“分子开关”。他们的医学重要性是强调， 发现其中一个成员rap 1（或Krev）抑制转化， Ras和另一种Rho是肉毒杆菌毒素的靶标。 多重因素 控制这些蛋白质的开关状态，催化GDP/GTP交换或 GT3活性，或抑制GDP释放。 ras蛋白也是 被共价修饰过的重要的是要了解 这些GTP结合蛋白的功能和控制。 主要的重点将 属于RAB子类。 该项目分为3个主要部分： 1.特征描述。需要针对rab肽的特异性抗体 并在大肠杆菌中表达重组rab蛋白。杆菌这些试剂将 用于确定组织分布、亚细胞定位和 RAB蛋白的共价修饰。 假设将被检验 不同的C端共有序列控制着修饰， 本地化 2.调控Ras特异性鸟嘌呤核苷酸释放因子已被发现， 发现，它可以作为一个“开关”。 的存在 将研究rab蛋白特有的类似因素。其他 还将确定控制rab GT3活性（GAPS）的因素 并纯化，以确定控制机制。 3.功能 将采用三种方法。(a)亲和柱将是 由重组RAB蛋白制备并用于纯化结合 特别是蛋白质的GTP状态。 这些因素是潜在的 目标（B）转化潜力和转化抑制 将测量rab蛋白的活性，以确定 拉斯·克雷夫的财产(c)最后，假设将被测试，在 与其他实验室的合作，RAB蛋白通常 控制胞吐作用和/或细胞器间转运的功能，用于 体外测定。