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; 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蛋白是一个蛋白质家族，伴随各种膜 细胞内酶和离子通道的受体。 单个细胞 在比例中包含许多G蛋白亚基的亚型 不同细胞类型的特征。 总体目标 项目是定义确定G细胞水平的机制 蛋白质亚基。 目前，关于如何水平知之甚少 G蛋白亚基受到调节：转录大鼠如何翻译 或蛋白质降解在G蛋白亚基之间有所不同，或其中哪种 步骤对于确定亚基的水平是最重要的。 问题 可以分为两个部分：1）mRNA的研究状态水平如何 与蛋白质水平有关？ 2）什么决定了研究状态的水平 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，在以后的研究中，我们回到 基因。 具体目的是： 1。量化α（O）的蛋白mRNA的稳态水平， 大鼠肝脏，心脏，脑皮质中的α（I-2）和β1 培养细胞的类型（GH（4），PC12和心脏细胞）。 2。确定α（O）和降解的速率（O）和 细胞系中的α（I-2）。 3。确定α（O）的翻译活性mRNA的比率 alpha（i-2）和beta1到翻译不活跃的使者 核糖核蛋白。 在选定的组织或细胞中，我们将确定 这些mRNA的多元组曲线，并确定A的伸长率 多层径流测定法。 4。使用体外翻译分析的翻译特性 从修饰的cDNA转录的细胞mRNA和mRNA。 翻译 mRNA和mRNP的特性将与无蛋白质mRNA进行比较。 这 在5'中修改的cDNA制成的mRNA的翻译特性 将测量未翻译的区域以分析5'末端的作用 翻译调节中的mRNA。 细胞对激素，神经递质和有丝分子的反应能力 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蛋白量。 这些研究将提供第一个 对设定G水平的某些机制的系统分析 蛋白质亚基并为未来的研究奠定基础 这些机制如何在开发，分化和细胞中起作用 生长。