REGULATION OF CELLULAR LEVELS OF G PROTEIN SUBUNITS

G 蛋白亚基细胞水平的调节

• 批准号: 3305791
• 负责人: EVA J. NEER
• 金额: $ 16.64万
• 依托单位: BRIGHAM AND WOMEN'S HOSPITAL
• 依托单位国家: 美国
• 财政年份: 1991
• 资助国家: 美国
• 起止时间: 1991-09-01 至 1994-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3305791
• 关键词: G protein; brain cell; brain metabolism; cell growth regulation; cerebral cortex; clone cells; genetic translation; heart cell; heart metabolism; laboratory rat; liver cells; liver metabolism; messenger RNA; nuclear runoff assay; nucleic acid probes; polymerase chain reaction; protein biosynthesis; protein structure function

The G proteins are a family of proteins that couple a variety of membrane receptors to intracellular enzymes and ion channels. Individual cells contain many subtypes of G protein subunits in proportions which are characteristic of the different cell types. The overall goal of this project is to define the mechanisms which determine cellular levels of G protein subunits. At present, very little is known about how the level of G protein subunits are regulated: how rats of transcription, translation or protein degradation vary among G protein subunits, or which of these steps is most important in determining the level of a subunit. The problem can be divided into two parts: 1) How is the study-state level of mRNA related to the protein level? 2) What determines the study-state level of mRNA for a G protein subunit? We propose to begin with the first question, starting with the protein and, in future studies, working our way back to the gene. The specific aims are: 1. To quantitate the steady-state levels of protein mRNA for alpha(o), alpha(i-2) and beta1 in rat liver, heart, cerebral cortex and in three types of cultured cells (GH(4), PC12 and cardiocytes). 2. To determine the rates of synthesis and degradation of alpha(o) and alpha(i-2) in cell lines. 3. To determine the ratio of translationally active mRNA for alpha(o), alpha(i-2) and beta1 to translationally inactive messenger ribonucleoprotein. In selected tissues or cells, we will determine the polysome profile for these mRNAs and determine the elongation rate by a polysome run-off assay. 4. To use in vitro translation to analyze the translational properties of cellular mRNA and mRNA transcribed from modified cDNA. The translational properties of mRNA and mRNPs will be compared to protein-free mRNA. The translational properties of mRNA made from cDNAs modified in the 5' untranslated region will be measured to analyze the role of the 5' end of the mRNA in regulation of translation. The ability of cells to respond to hormones, neurotransmitters and mitogens depends not only on receptors for these agonists, but also on the type and amount of G proteins they express. These studies will provide the first systematic analysis of some of the mechanisms which set the levels of G protein subunits and lay the foundation for future studies to understand how these mechanisms function in development, differentiation and cell growth.

G 蛋白是耦合多种膜的蛋白质家族 细胞内酶和离子通道的受体。 单个细胞 含有多种 G 蛋白亚基亚型，其比例为 不同细胞类型的特征。 本次活动的总体目标 该项目旨在定义决定细胞 G 水平的机制 蛋白质亚基。 目前，人们对于其水平如何知之甚少。 G蛋白亚基被调控：大鼠如何转录、翻译 G 蛋白亚基之间的蛋白质降解或蛋白质降解有所不同，或者其中哪一个 步骤对于确定亚基的水平最重要。 问题 可以分为两部分：1）mRNA的研究状态水平如何 与蛋白质水平有关吗？ 2）什么决定了学习状态水平 G 蛋白亚基的 mRNA？ 我们建议从第一个问题开始， 从蛋白质开始，在未来的研究中，我们将回到 基因。 具体目标是： 1. 为了定量 alpha(o) 蛋白质 mRNA 的稳态水平， 大鼠肝脏、心脏、大脑皮层和三者中的 alpha(i-2) 和 beta1 培养细胞类型（GH(4)、PC12 和心肌细胞）。 2. 测定α(o)和α(o)的合成和降解速率 细胞系中的α(i-2)。 3. 确定 alpha(o) 翻译活性 mRNA 的比率， alpha(i-2) 和 beta1 为翻译不活跃的信使 核糖核蛋白。 在选定的组织或细胞中，我们将确定 这些 mRNA 的多核糖体图谱并通过 多核糖体流失测定。 4. 利用体外翻译分析翻译特性 细胞 mRNA 和从修饰的 cDNA 转录的 mRNA。 翻译的 mRNA 和 mRNA 的特性将与不含蛋白质的 mRNA 进行比较。 这 由 5' 端修饰的 cDNA 制成的 mRNA 的翻译特性 将测量非翻译区域以分析 5' 末端的作用 mRNA 的翻译调节。 细胞对激素、神经递质和有丝分裂原作出反应的能力 不仅取决于这些激动剂的受体，还取决于类型和 它们表达的 G 蛋白的量。 这些研究将提供第一个 对设定 G 水平的一些机制进行系统分析 蛋白质亚基并为未来的研究奠定基础 这些机制如何在发育、分化和细胞中发挥作用 生长。