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: 6290619
• 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/6290619
• 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. In FY99, the role of protein phosphorylation in regulating D1 receptor function was examined using metabolic labeling techniques. We created an epitope-tagged D1 receptor construct by adding the FLAG peptide sequence to the NH2 terminus of the rat receptor. This receptor construct was stably expressed to high levels in C6 glioma cells. Prior investigations by this laboratory, and others, have shown the D1 receptor to undergo rapid (t1/2 = 10-15 min) agonist-induced desensitization in C6 cells which is characterized by a reduction in agonist potency and maximum cAMP response. In our present studies, we used metabolic labeling to perform whole cell phosphorylation assays in which the receptor is immunopurified using antisera directed against the FLAG epitope. We find that the D1 receptor is phosphorylated under basal conditions and that its phosphorylation state is increased by about 3-fold upon pre-exposure of the cells to dopamine. The rate of agonist-induced phosphorylation is rapid, t1/2 < 1 min, and is much faster than the rate of agonist- induced desensitization. This suggests that receptor phosphorylation is not the rate limiting step for receptor desensitization. The dopamine- induced receptor phosphorylation is blocked by D1-selective antagonists and is mimicked by D1-selective agonists. The recovery of receptor phosphorylation to basal levels upon agonist removal is also rapid (t1/2 = 6-7 min) and is not blocked by agents (i.e., Con A) which inhibit receptor internalization. This suggests that receptor internalization may not be required for receptor de-phosphorylation. - dopamine, receptor, desensitization, mutation, transgenic

该项目的长期目标是表征神经递质受体介导的信息转导及其跨神经元膜的调节。正在研究的主要受体系统是多巴胺受体系统。为了在生化和分子水平上表征这些受体并研究它们的调节，正在进行两个相互关联的研究：1）在蛋白质水平上研究受体的细胞生物学、功能和调节； 2）受体cDNA/基因的分子克隆以及培养细胞系和转基因小鼠中受体结构、药理学和调控的研究。 2099 财年，利用代谢标记技术检查了蛋白质磷酸化在调节 D1 受体功能中的作用。我们通过将 FLAG 肽序列添加到大鼠受体的 NH2 末端，创建了表位标记的 D1 受体构建体。该受体构建体在 C6 神经胶质瘤细胞中稳定高水平表达。该实验室和其他实验室之前的研究表明，D1 受体在 C6 细胞中经历快速（t1/2 = 10-15 分钟）激动剂诱导的脱敏，其特征是激动剂效力和最大 cAMP 反应降低。在我们目前的研究中，我们使用代谢标记进行全细胞磷酸化测定，其中使用针对 FLAG 表位的抗血清对受体进行免疫纯化。我们发现 D1 受体在基础条件下被磷酸化，并且在细胞预暴露于多巴胺时其磷酸化状态增加了约 3 倍。激动剂诱导的磷酸化速率很快，t1/2<1分钟，并且比激动剂诱导的脱敏速率快得多。这表明受体磷酸化不是受体脱敏的限速步骤。多巴胺诱导的受体磷酸化被 D1 选择性拮抗剂阻断，并被 D1 选择性激动剂模拟。去除激动剂后，受体磷酸化恢复至基础水平的速度也很快（t1/2 = 6-7 分钟），并且不会被抑制受体内化的药物（即 Con A）所阻断。这表明受体内化可能不是受体去磷酸化所必需的。 - 多巴胺、受体、脱敏、突变、转基因