FUNCTION OF MODIFIED BRAIN SIGNALING PROTEINS

这个项目的主要目标是确定一个新的G蛋白α/o亚单位在大脑信号机制中的作用。G蛋白介导细胞外信号对细胞内信号通路的影响。它们在大脑中尤其重要，因为许多神经递质的作用是由G蛋白偶联受体介导的。G/O是脑内含量最丰富的异三聚体G蛋白，约占总颗粒蛋白的0.2%-1%。α/o蛋白只有一个哺乳动物基因，它可以产生多个mRNAs，编码至少两种不同的GoAlpha蛋白。然而，至少有三种主要的GoAlpha蛋白已经从大脑中纯化出来。本文报道的初步结果表明，第三种主要亚型(α/oc)是两种蛋白质的混合物，这些蛋白质来源于主要的α/omRNA(α/o1)表达的蛋白，但在两个特定的天冬酰胺残基中的一个或另一个修饰。这两个天冬酰胺残基毗邻在蛋白质的C末端附近，参与上游受体和下游效应器的相互作用。这项拟议的工作将在对纯化和重组蛋白进行生化研究的基础上，表征这些修饰蛋白的功能、分布和来源。具体目标是：(1)确定GoAlpha亚型之间的功能差异。(2)确定Goapha蛋白在神经组织、细胞系和亚细胞组分中的定位和分布。(3)确定α/oc亚型起源的机制。这些特定的目的是为了检验以下假设：α/oc是α/oc蛋白的前体，并且α/oc的产生速度取决于G蛋白信号系统的活性，从而导致具有新性质的信号通路的改变。这种影响主要大脑信号蛋白的分子机制很可能是记忆、神经可塑性或其他相关神经适应过程的重要组成部分。

The primary goal of this project is to determine the role of a novel G protein alpha/o subunit in signaling mechanisms in the brain. G proteins mediate the effects of extracellular signals on intracellular signaling pathways. They are particularly important in brain where the effects of many neurotransmitters are mediated by G protein-coupled receptors. G/o is the most abundant heterotrimeric G protein in brain, comprising about 0.2%-1% of total particulate protein. There is a single mammalian gene for the alpha/o protein, which gives rise to multiple mRNAs encoding at least two different Goalpha proteins. However, at least three major Goalpha proteins have been purified from brain. Preliminary Results reported here show that the third major isoform (alpha/oc) is a mixture of two proteins derived from the protein expressed from the major alpha/o mRNA (alpha/o1), but modified at one or the other of two specific asparagine residues. These two asparagine residues are next to each other and near the C-terminus in a region of the protein involved in upstream receptor and downstream effector interactions. The proposed work will characterize the function, distribution and origin of these modified proteins based upon biochemical studies of purified and recombinant proteins. The Specific Aims are: (1) To determine the functional differences between the Goalpha isoforms. (2) To determine the localization and distribution of the Goapha proteins in neural tissues, cell lines and subcellular fractions. (3) To determine the mechanism underlying the origin of the alpha/oc isoforms. These Specific Aims are designed to test the hypotheses that alpha/oa is a precursor of the alpha/oc proteins, and that the rate of production of alpha/oc is dependent upon the activity of the G protein signaling system, resulting in an altered signaling pathway with new properties as a result of the production of alpha/oc. Such a molecular mechanism affecting a major brain signaling protein would likely be an important component of memory, neural plasticity or other related neuroadaptive processes.