FUNCTION OF MODIFIED BRAIN SIGNALING PROTEINS

该项目的主要目标是确定一种新的G蛋白α/o亚基在大脑信号传导机制中的作用。G蛋白介导细胞外信号对细胞内信号通路的影响。它们在脑中特别重要，其中许多神经递质的作用由G蛋白偶联受体介导。 G/o是脑中最丰富的异源三聚体G蛋白，约占总颗粒蛋白的0.2%-1%。α/o蛋白有一个单一的哺乳动物基因，它产生编码至少两种不同Goalpha蛋白的多个mRNA。然而，已经从脑中纯化了至少三种主要的Goalpha蛋白。 本文报道的初步结果表明，第三种主要同种型（α/α）是两种蛋白质的混合物，这两种蛋白质来源于主要α/o mRNA（α/o 1）表达的蛋白质，但在两个特定天冬酰胺残基中的一个或另一个处进行了修饰。这两个天冬酰胺残基彼此相邻，并且靠近参与上游受体和下游效应物相互作用的蛋白质区域中的C-末端。拟开展的工作将基于纯化和重组蛋白的生化研究来表征这些修饰蛋白的功能、分布和起源。具体目的是：（1）确定Goalpha异构体之间的功能差异。(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/α亚型起源的潜在机制。这些特定目的旨在检验以下假设：α/oa是α/oc蛋白的前体，α/oc的产生速率取决于G蛋白信号传导系统的活性，导致信号传导途径改变，并因α/oc的产生而具有新特性。这种影响主要脑信号蛋白的分子机制可能是记忆、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.