MOLECULAR CHARACTERIZATION OF MUSCARINIC ACH RECEPTORS

这项工作的长期目标是了解信号转导是如何 在中枢神经系统胆碱能突触建立和维持;其重点是 毒蕈碱乙酰胆碱生物化学、发育和细胞调节 受体。 这些受体介导大部分中枢神经系统胆碱能 信号，已经牵连到许多行为和临床 并显示出广泛的转导和 监管回应。 目前看来最合适的办法是 并开始表征受体系统蛋白， 用于研究毒蕈碱细胞和发育的特异性抗体 生物学 因此，选定了以下三个目标： 目的#1--构建毒蕈碱受体特异性单克隆抗体文库 抗体的 将部分纯化的（250倍）牛脑受体 使用已测试的方法组合用于抗体诱导 因为他们的有效性。 结合试验和蛋白质印迹将用于 检测抗体特异性。 目的#2--纯化并开始表征受体分子。 免疫亲和色谱将根据以下条件添加到检测分离中： 亚细胞分级、疏水性、电荷、分子量和 特异性糖基化。 二维凝胶、沉降平衡和N-末端 分析将用于测试纯度。 配体特异性， 亚基，结合位点的数量，和总的氨基酸组成， 将评估纯化的分子。 目的#3--将受体定位为突触发育的功能， 翻译后修饰和胆碱能刺激。 的 上面获得的单克隆抗体将用于定位受体， 鸟类视网膜神经元的亚细胞和超微结构水平。 一 采用分离分化的神经元和HVEM整体的新方法 将使用安装分析。

The long-range goal of this work is to understand how signal transduction is established and maintained at CNS cholinergic synapses; its focus is on the biochemistry, development and cellular regulation of muscarinic ACh receptors. These receptors mediate a large proportion of CNS cholinergic signaling, have been implicated in many behaviorally and clinically significant phenomena and show a broad spectrum of transductional and regulatory responses. At present, it appears most appropriate to isolate and begin characterizing receptor system proteins and to generate receptor specific antibodies for studies of muscarinic cell and developmental biology. The following three aims have therefore been chosen: AIM #1--To generate a library of muscarinic receptor-specific monoclonal antibodies. Partially purified (250-fold) bovine brain receptors will be used for antibody induction using a combination of methods already tested for their effectiveness. Binding assays and western blots will be used in testing antibody specificity. AIM #2--To purify and begin characterization of receptor molecules. Immunoaffinity chromatography will be added to tested separations based on subcellular fractionation, hydrophobicity, charge, molecular weight and specific glycosylation. 2D-Gel, sedimentation equilibrium and N-terminal analyses will be used in testing purity. The ligand specificity, number of subunits, number of binding sites, and overall amino acid composition of the purified molecules will be assessed. AIM #3--To localize receptors as a function of synaptic development, post-translational modification, and cholinergic stimulation. The monoclonal antibodies obtained above will be used to localize receptors on avian retina neurons at the subcellular and ultrastructural levels. A novel approach employing isolation of differentiated neurons and HVEM whole mount analysis will be used.