NEUROTRANSMITTER RECEPTOR PURIFICATION AND STRUCTURE

神经递质受体；肾上腺素能(β1、β2、α1和α2)， 胆碱能(毒鼠碱和烟碱)和苯二氮卓类受体 被分离和提纯以了解其分子基础 感受器功能和神经元通讯。具体项目有 正在进行中，以提供上述每一项的准确结构信息 受体蛋白。正在获得的结构数据包括初级序列 数据、蛋白水解图、拓扑信息和结构功能 数据，例如，神经递质结合部位定位，糖定位， 膜结构域和效应器偶联蛋白识别结构域。这个 阿尔茨海默病和亨廷顿病患者受体的结构 正在接受免疫学技术的检查。 我们的数据表明，在结构上存在相似性 非药物相关的神经递质受体(毒鼠碱， 胆碱能和α肾上腺素能)，这些神经递质受体 通过蛋白质构象变化介导细胞调节 神经递质与胞外蛋白结合部位的结合 域。受体偶联是由细胞质“尾巴”介导的 似乎是效应蛋白(GTP调节蛋白)的受体 受体的识别部分。电子显微镜，性能 大小排斥层析纯化的受体表明 α2和β2受体以同源二聚体的形式存在，而毒扁豆碱 受体是单体。 已经建立了蛋白质制备程序，其中包括各种 高效液相色谱步骤、配基亲和层析、单抗亲和 层析、制备性十二烷基硫酸钠凝胶电泳法、凝集素亲和力 层析、离子交换层析和柱等电层析 集中注意力。这些纯化方案的建立现在是 允许同时进行所有肾上腺素能的详细结构比较 和胆碱能受体蛋白。

Neurotransmitter receptors; adrenergic (beta1, beta2, alpha1 and alpha2), cholinergic (muscarinic and nicotinic), and benzodiazepine receptors are being isolated and purified in order to understand the molecular basis of receptor function and neuronal communication. Specific projects are underway to provide precise structural information on each of the above receptor proteins. Structural data being obtained include primary sequence data, proteolytic digest maps, topology information and structure-function data, e.g., neurotransmitter binding site localization, sugar localization, membrane domain and effector coupling protein recognition domains. The structure of receptors from Alzheimer's and Huntington's disease patients are being examined by immunological techniques. Our data have demonstrated that structural similarities exist among non-pharmacologically related neurotransmitter receptors (muscarinic, cholinergic and alpha adrenergic) and that these neurotransmitter receptors mediate cellular modulation via protein conformational changes initiated by neurotransmitter binding to the binding site in the extracellular protein domain. Receptor coupling is mediated by the cytoplasmic "tail" of the receptors which appears to be the effector protein (GTP-regulatory protein) recognition portion of the receptor. Electron microscopy, performance size-exclusion chromatography of purified receptors indicate that the alpha2 and beta2 receptors exist as homodimers while the muscarinic receptor is monomeric. Protein preparative procedures have been established which include various HPLC steps, ligand affinity chromatography, monoclonal antibody affinity chromatography, preparative SDS-gel electrophoresis, lectin affinity chromatography, ion exchange chromatography and column isoelectric focusing. The establishment of these purification protocols is now permitting simultaneous detailed structural comparisons of all adrenergic and cholinergic receptor proteins.