BIOCHEMICAL AND PHARMACOLOGICAL STUDIES OF DOPAMINE RECEPTORS

多巴胺受体的生物化学和药理学研究

• 批准号: 6111829
• 负责人: DAVID R. SIBLEY
• 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/6111829
• 关键词: biological signal transduction; chimeric proteins; clone cells; dopamine agonists; dopamine antagonists; dopamine receptor; genetically modified animals; laboratory mouse; molecular cloning; neural information processing; neuropharmacology; protein isoforms; protein structure function; receptor coupling; receptor expression; receptor sensitivity; serotonin receptor; transfection

The long term goal of this project is to characterize neurotransmitter receptor-mediated information transduction, and its regulation, across neuronal membranes. The primary receptor systems under investigation are those for dopamine. In order to characterize these receptors at the biochemical and molecular levels, and study their regulation, there are two interrelated lines of research being performed: 1) investigation of the cell biology, function and regulation of the receptors at the protein level; and 2) the molecular cloning of the receptor cDNAs/genes and investigation of receptor structure, pharmacology and regulation in cultured cell lines and transgenic mice. The role of protein phosphorylation in regulating D-1 receptor function was examined using mutagenesis and metabolic labeling techniques. We had previously found that when four potential protein kinase A (PKA) phosphorylation sites in the receptor were eliminated, the mutant receptor exhibited slower kinetics of desensitization subsequent to dopamine exposure. Further analyses of single mutated receptors, in which only one of the four sites is modified, reveals that Thr-268 in the 3rd cytoplasmic loop of the receptor is primarily responsible for the effects of PKA. We have also mutated potential G protein-coupled receptor kinase (GRK) phosphorylation sites within the 3rd cytoplasmic loop of the receptor. Several of these mutant receptors demonstrate delayed/reduced agonist-induced desensitization. Using C6 cells stably transfected with an epitope tagged D-1 receptor, we have been able to directly demonstrate agonist-induced phosphorylation of the receptor protein. The phosphorylation status of mutated D-1 receptors is presently under investigation. The epitope-tagged D-1 receptor has also been visualized in transfected cells using both fluorescence and confocal microscopy and we are investigating "internalization" motifs in the receptor protein using mutagenesis approaches. Similar regulatory work has also begun on the D-2S and D-2L receptor isoforms. Work also continued on cloning a third "D-1 like" receptor which stimulates phosphatidylinositol (PI) turnover and calcium mobilization. Homozygous D-5 receptor knock-out mice were generated and determined to be viable, fertile and neurologically intact. Preliminary behavioral experimentation suggests that the animals exhibit greater exploratory activity in an open field test which is not due to reduced anxiety. The animals also exhibit superior motor learning skills as assessed with the rotarod test. These animals are currently undergoing additional physiological/behavioral characterization.

该项目的长期目标是表征 神经递质受体介导的信息转导，以及 它的调节是跨神经元膜的。主要受体 正在研究的系统是多巴胺系统。为了 在生化和分子方面表征这些受体 水平，并研究其调节，有两条相互关联的线路 正在进行的研究：1）细胞生物学研究， 受体在蛋白质水平上的功能和调节；和 2) 受体 cDNA/基因的分子克隆以及 受体结构、药理学和调控的研究 培养的细胞系和转基因小鼠。蛋白质的作用 检查了调节 D-1 受体功能的磷酸化 使用诱变和代谢标记技术。我们有 先前发现当四种潜在的蛋白激酶A（PKA） 受体中的磷酸化位点被消除，突变体 受体表现出较慢的脱敏动力学 多巴胺暴露。对单个突变受体的进一步分析， 仅修改了四个位点之一，揭示了 Thr-268 受体的第三个细胞质环主要负责 PKA 的影响。我们还突变了潜在的G 蛋白偶联受体激酶 (GRK) 磷酸化位点 位于受体的第三个细胞质环内。其中几个 突变受体表现出激动剂诱导的延迟/减少 脱敏。使用稳定转染表位的 C6 细胞 标记的 D-1 受体，我们已经能够直接证明 激动剂诱导的受体蛋白磷酸化。这 突变的 D-1 受体的磷酸化状态目前处于 调查。表位标记的 D-1 受体也已被 使用荧光和共聚焦在转染细胞中可视化 显微镜下我们正在研究“内化”基序 使用诱变方法的受体蛋白。类似监管 D-2S 和 D-2L 受体亚型的研究工作也已开始。 克隆第三个“D-1 样”受体的工作也在继续进行。 刺激磷脂酰肌醇 (PI) 周转和钙 动员。纯合 D-5 受体敲除小鼠 生成并确定为可存活、可生育且具有神经功能 完好无损的。初步的行为实验表明 动物在旷场测试中表现出更大的探索活动 这并不是因为焦虑减少。动物们也展示了 通过旋转测试评估出卓越的运动学习技能。 这些动物目前正在接受额外的治疗 生理/行为特征。