MECHANISMS OF ACETYLCHOLINE RECEPTOR CLUSTERING

脊椎动物的神经肌肉接头是一种典型的突触 用于将信息从运动神经快速局部化传输到肌肉 通过高度专业化的突触前和突触后结构。突触后 膜上含有高密度的烟碱型乙酰胆碱 受体(AChR)恰好与脑干的分支相对 突触前神经末梢。这些AChR簇对突触至关重要 功能。这个项目的目标是阐明分子机制。 突触后AChR簇的基本形成和维持 神经肌肉连接处的膜。具体地说，这项研究将 细胞内43kD和87kD突触后蛋白的作用 (43k和87k蛋白)在AChR聚集性中。当用异源基因表达时 细胞，43K蛋白在细胞表面形成簇状，并可诱导 当两者都表达时，AChRs的共同聚集。此外，43k蛋白 可以单独对每个AChR亚单位(阿尔法、贝塔、伽马和增量)进行聚类 在细胞表面。使用最近开发的一种高效的鹌鹑 成纤维细胞(Qt-6)瞬时转染系统，我们将研究 蛋白质-蛋白质相互作用对AChR集群很重要。 首先，阿尔法亚单位的区域负责其 与43K蛋白的相互作用将通过突变特定的位点来确定 在α亚基内，然后将突变体与43k蛋白在 免疫荧光法检测QT-6细胞的聚集情况 显微镜。这一突变分析还将得到测试的补充 在嵌合体中，阿尔法亚单位的一部分已插入到 确定非聚集跨膜蛋白是否为这个阿尔法 亚基区含有43k蛋白相互作用位点。一旦这个网站 已在阿尔法亚基中发现，在另一个亚基中发现同源位点 亚基将发生突变以显示同源功能。第二， 聚集所需的最少43k个蛋白质相互作用位点数 完全组装的五聚体AChR将通过共表达43k来确定 具有完全组装的受体的蛋白质，其中一个或多个43k 蛋白质相互作用位点已被定点失活 诱变。突变的AChRs在转基因细胞中的聚集将 如上所述地确定。最后，我们将研究87k的作用 分离和鉴定AChR基因克隆中的蛋白质 小鼠87K蛋白及其特异性多克隆的制备 用于表达研究的抗体。87K蛋白将得到共表达 用43k蛋白或用43k蛋白和AChR测定其是否 与AChR/43k蛋白簇共定位或改变 这些星团。突变分析将在87k进行 蛋白质来确定哪些结构域负责其活性。这些 实验将有助于阐明潜在的分子机制。 地球表面高密度AChR星团的形成和维持 神经肌肉接头。此外，这项研究可能有助于定义 某些先天性肌无力综合征的分子基础 以突触后膜AChRs缺乏为特征的。

The vertebrate neuromuscular junction is a prototypical synapse designed for rapid localized transmission of information from motor nerve to muscle via highly specialized pre- and postsynaptic structures. The postsynaptic membrane contains high density clusters of nicotinic acetylcholine receptor (AChR) positioned precisely opposite the branches of the presynaptic nerve terminal. These AChR clusters are critical to synapse function. The goal of this project is to clarify the molecular mechanisms underlying formation and maintenance of AChR clusters in the postsynaptic membrane of the neuromuscular junction. Specifically, this study will address the roles of the cytosolic 43kD and 87kD postsynaptic proteins (43k and 87k protein) in AChR clustering. When expressed in heterologous cells, 43k protein forms clusters at the cell surface, and can induce coclustering of AChRs when both are expressed. In addition, 43k protein can cluster each AChR subunit (alpha, beta, gamma, and delta) individually at the cell surface. Using a recently developed, highly efficient quail fibroblast (QT-6) transient transfection system, we will investigate the protein-protein interactions that are important for AChR clustering. First, the region of the alpha subunit that is responsible for its interaction with 43k protein will be identified by mutating specific sites within the alpha subunit, then coexpressing the mutant with 43k protein in QT-6 cells to test for clustering as assessed by immunofluorescence microscopy. This mutational analysis will be complemented by also testing chimeras in which a portion of the alpha subunit has been inserted into a non-clustering transmembrane protein to determine whether this alpha subunit region contains the 43k protein interaction site. Once this site has been identified in alpha subunit, homologous sites in the other subunit will be mutated to demonstrate homologous function. Second, the minimum number of 43k protein interaction sites required for clustering of a fully assembled pentameric AChR will be determined by coexpressing 43k protein with fully assembled receptors in which one or more of the 43k protein interaction sites has been inactivated by site-directed mutagenesis. Clustering of the mutant AChRs in the transfected cells will be determined as above. Finally, we will investigate the role of 87k protein in AChR clustering by isolating and characterizing a cDNA clone for mouse 87k protein and preparing 87k protein-specific polyclonal antibodies for use in expression studies. 87k protein will be coexpressed with 43k protein or with 43k protein and AChR to determine whether it colocalizes with AChR/43k protein clusters or alters the appearance of these clusters. Mutational analysis will then be carried out on 87k protein to determine which domains are responsible for its activity. These experiments will help to elucidate the molecular mechanisms underlying the formation and maintenance of high density AChR clusters at the neuromuscular junction. In addition, this study may help to define the molecular basis of some congenital myasthenic syndromes which are characterized by a deficiency in AChRs in the postsynaptic membrane.