NEUROTRANSMITTER RECEPTOR PURIFICATION AND STRUCTURE

神经递质受体的纯化和结构

• 批准号: 3969050
• 负责人: J C VENTER
• 金额: --
• 依托单位: NATIONAL INSTITUTE OF NEUROLOGICAL DISORDERS AND STROKE
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/3969050
• 关键词: Alzheimer's disease; Huntington's disease; affinity chromatography; benzodiazepines; conformation; electrofocusing; electron microscopy; electrophysiology; gel electrophoresis; guanosine triphosphate; high performance liquid chromatography; human tissue; immunochemistry; ion exchange chromatography; liposomes; monoclonal antibody; neural conduction; proteolysis; Alzheimer's disease; Huntington's disease; affinity chromatography; benzodiazepines; conformation; electrofocusing; electron microscopy; electrophysiology; gel electrophoresis; guanosine triphosphate; high performance liquid chromatography; human tissue; immunochemistry; ion exchange chromatography; liposomes; monoclonal antibody; neural conduction; proteolysis

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.

神经递质受体；肾上腺素能（beta1，beta2，alpha1和alpha2）， 胆碱能（毒蕈碱和烟碱）和苯二氮卓受体是 被隔离和纯化，以了解 受体功能和神经元通信。 特定项目是 正在进行中，以提供上述每一个的精确结构信息 受体蛋白。 获得的结构数据包括主要序列 数据，蛋白水解摘要图，拓扑信息和结构功能 数据，例如，神经递质结合位点定位，糖定位， 膜结构域和效应子耦合蛋白识别域。 这 阿尔茨海默氏症和亨廷顿氏病的受体结构 正在通过免疫学技术进行检查。 我们的数据表明，结构性相似 非药理学相关的神经递质受体（毒蕈碱，，， 胆碱能和α肾上腺素能）以及这些神经递质受体 通过蛋白质构象变化引发的介导细胞调节 神经递质与细胞外蛋白的结合位点结合 领域。 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 纯化受体的尺寸排斥色谱法表明 α2和β2受体作为同二聚体存在，而毒蕈碱则存在 受体是单体。 已经建立了蛋白质制备程序，其中包括各种 HPLC步骤，配体亲和色谱，单克隆抗体亲和力 色谱，制备SDS-凝胶电泳，凝集素亲和力 色谱，离子交换色谱和色谱柱等电 专注。 这些纯化协议的建立现在已经 允许同时对所有肾上腺素的详细结构比较 和胆碱能受体蛋白。