NEUROTRANSMITTER RECEPTOR PURIFICATION AND STRUCTURE

神经递质受体的纯化和结构

• 批准号: 4696972
• 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/4696972
• 关键词: affinity chromatography; conformation; electrophysiology; gel electrophoresis; guanosine triphosphate; high performance liquid chromatography; human tissue; ion exchange chromatography; liposomes; monoclonal antibody; neural conduction; proteolysis

Neurotransmitter receptors; adrenergic (beta1, beta2, alpha1 and alpha2), cholinergic (muscarinic and nicotinic), dopaminergic, and serotonergic 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. Our data have demonstrated that structural similarities exist amongst 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. 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 are now permitting simultaneous detailed structural comparisons of all adrenergic and cholinergic receptor proteins. Functional receptor proteins isolated by affinity chromatography and HPLC are being reconstituted into phospholipid vesicles together with purified GTP-regulatory proteins (Gi and Go) to study molecular events involved in the control of cellular events by 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），多巴胺能和5-羟色胺能 受体被隔离和纯化，以了解 受体功能和神经元通信的分子基础。 具体的 正在进行项目，以提供有关每一个的精确结构信息 上述受体蛋白。 获得的结构数据包括 主要序列数据，蛋白水解消化图，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，膜结构域和效应子偶联蛋白 识别域。 我们的数据证明了结构性 非药理学与神经递质之间存在相似之处 受体（毒蕈碱，胆碱能和α肾上腺素能），这些 神经递质受体通过蛋白质介导细胞调节 神经递质与结合结合引发的构象变化 位于细胞外蛋白质结构域中的位置。 受体耦合是由 受体的细胞质“尾巴”似乎是效应器 受体的蛋白质（GTP调节蛋白）识别部分。 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。 分离的功能受体蛋白 通过亲和力色谱和HPLC，正在重构 磷脂囊泡与纯化的GTP调节蛋白（GI）一起 然后去研究与细胞控制有关的分子事件 受体蛋白的事件。