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: 6432884
• 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/6432884
• 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-related cDNAs/genes and investigation of receptor structure, pharmacology and regulation in cultured cell lines and transgenic miceIn FY-2000, the mechanisms of agonist-induced regulation of D1 and D2 receptors were further investigated. We attempted to identify the sites of regulatory phosphorylation (PHOS) in the D1 dopamine receptor (DAR) using mutagenesis techniques and expression in HEK-293 cells. We created two 3rd cytoplasmic loop mutants, one in which all serine and threonine residues were mutated (3rdTOT) and another in which just serines 256, 258 & 259 were mutated (3rd234). We also prepared a series of mutants where the D1 DAR was truncated subsequent to the indicated positions in the carboxyl terminus: T347, T369, T394 & T404. Expression of the mutant and wild-type (WT) constructs revealed similar levels of receptor binding except for the T347 mutant which was 50% lower. Dopamine (DA) stimulated cAMP accumulation in a similar fashion for all constructs except for the 3rdTOT mutant which showed a lower potency for DA. DA-induced desensitization (DES) was investigated for the 3rdTOT and T347 mutants. Interestingly, DES of the 3rdTOT and T347 mutants was impaired and enhanced, respectively, compared to WT. DA-induced PHOS of all constructs was investigated. Relative to WT, the 3rdTOT mutant exhibited a 50% decrease which was mimicked by the 3rd234 mutant. This suggests that serines 256, 258 & 259 account for the majority of 3rd loop PHOS. Mutant T404 also exhibited a 50% decrease in PHOS with further reductions observed for each of the truncation mutants. Surprisingly, PHOS of the T347 mutant was barely detectable. These results suggest that PHOS of carboxyl terminal residues is necessary for PHOS of 3rd loop residues. The mechanism of DES in the T347 mutant is currently under investigation. To investigate the intracellular trafficking of the D2 receptor in response to DA treatment, we constructed D2-YFP chimeras in which YFP was fused to the carboxyl termini of the human D2L and D2S receptors. Expression of these constructs in HEK293 cells revealed receptor binding characteristics identical to those of wild-type receptors. Fluorescent visualization of the expressed D2-YFP constructs was performed via confocal microscopy and a live-cell, real-time format. Under basal conditions, the D2L-YFP receptor is expressed in the plasma membrane (PM), however, significant levels of cytoplasmic (CP) fluorescence (FL) is also observed. After DA treatment, the PM FL is decreased and more diffuse whereas the CP FL is increased and more punctate. Similar results are observed with the D2S-YFP construct. We also co-expressed the D2L-YFP receptor with GRKs 2, 3, 5 or 6, or arrestin 2 or 3. Co-expression with any of the GRKs, or either of the arrestins, results in a decrease in PM FL and an increase in CP FL such that the D2L-YFP receptor is mostly intracellular. In GRK-2 and/or arrestin-2 co-transfected cells, DA exposure results in a slight further decrease in PM FL and increase in CP FL. In other experiments, we found that DA treatment results in a doubling of the D2L receptor binding activity. These results suggest that the DA-induced D2 receptor up-regulation is not due to recruitment of CP receptors to the PM.

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）受体相关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在FY-2000中，进一步研究了激动剂诱导的D1和D2受体的调节机制。我们尝试使用诱变技术和 HEK-293 细胞中的表达来鉴定 D1 多巴胺受体 (DAR) 中的调节磷酸化 (PHOS) 位点。我们创建了两种第三个细胞质环突变体，一种中所有丝氨酸和苏氨酸残基均发生突变（3rdTOT），另一种中仅丝氨酸 256、258 和 259 发生突变（3rd234）。我们还制备了一系列突变体，其中 D1 DAR 在羧基末端指定位置之后被截短：T347、T369、T394 和 T404。突变体和野生型 (WT) 构建体的表达显示出相似的受体结合水平，但 T347 突变体低 50%。对于所有构建体，多巴胺 (DA) 以类似的方式刺激 cAMP 积累，但 3rdTOT 突变体除外，该突变体对 DA 的效力较低。研究了 3rdTOT 和 T347 突变体的 DA 诱导脱敏 (DES)。有趣的是，与 WT 相比，3rdTOT 和 T347 突变体的 DES 分别受损和增强。研究了所有构建体的 DA 诱导的 PHOS。相对于 WT，3rdTOT 突变体表现出 50% 的降低，这与 3rd234 突变体相似。这表明丝氨酸 256、258 和 259 占第三环 PHOS 的大部分。突变体 T404 还表现出 PHOS 降低 50%，并且对于每个截短突变体观察到进一步降低。令人惊讶的是，T347 突变体的 PHOS 几乎检测不到。这些结果表明羧基末端残基的 PHOS 对于第三环残基的 PHOS 是必需的。 T347突变体中DES的机制目前正在研究中。为了研究 D2 受体响应 DA 处理的细胞内运输，我们构建了 D2-YFP 嵌合体，其中 YFP 融合到人 D2L 和 D2S 受体的羧基末端。这些构建体在 HEK293 细胞中的表达揭示了与野生型受体相同的受体结合特征。通过共聚焦显微镜和活细胞实时格式对表达的 D2-YFP 构建体进行荧光可视化。在基础条件下，D2L-YFP 受体在质膜 (PM) 中表达，但也观察到显着水平的细胞质 (CP) 荧光 (FL)。 DA 处理后，PM FL 降低且更加弥散，而 CP FL 增加且更加点状。使用 D2S-YFP 构建体观察到类似的结果。我们还将 D2L-YFP 受体与 GRK 2、3、5 或 6，或抑制蛋白 2 或 3 共表达。与任何 GRK 或任一抑制蛋白共表达，都会导致 PM FL 降低和CP FL 增加，使得 D2L-YFP 受体主要位于细胞内。在GRK-2和/或arrestin-2共转染细胞中，DA暴露导致PM FL进一步轻微降低和CP FL增加。在其他实验中，我们发现 DA 处理导致 D2L 受体结合活性加倍。这些结果表明，DA 诱导的 D2 受体上调并非由于 CP 受体募集到 PM 所致。