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 毒扁豆碱酸乙酰胆碱的生化、发育及细胞调控 感受器。这些受体介导了很大比例的中枢胆碱能 信号转导，在许多行为和临床上都有牵连 显著的现象，并显示出广泛的换能器和 监管回应。目前看来，分离最合适的方法是 并开始鉴定受体系统蛋白质并产生受体 M细胞和发育研究中的特异性抗体 生物学。因此，我们选择了以下三个目标： 目的#1--构建M受体特异性单抗文库 抗体。部分纯化(250倍)的牛脑受体将被 用于使用已经测试的多种方法的组合来诱导抗体 因为他们的有效性。结合分析和免疫印迹将用于 检测抗体的特异性。 目的#2--提纯并开始鉴定受体分子。 免疫亲和层析将添加到测试的分离中，基于 亚细胞分级、疏水性、电荷、相对分子质量和 特定的糖基化。二维凝胶、沉淀平衡和N-末端 分析将用于检测纯度。配基的特异性、数量 亚基、结合位点数和总的氨基酸组成 将对纯化的分子进行评估。 目标#3--定位受体作为突触发育的功能， 翻译后修饰和胆碱能刺激。这个 以上获得的单抗将用于定位受体。 亚细胞和超微结构水平的鸟类视网膜神经元。一个 一种新的分离分化神经元和HVEM整体的方法 将使用装载分析。

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.