Molecular And Pharmacological Studies Of Dopamine Recept

多巴胺受体的分子和药理学研究

• 批准号: 6501275
• 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/6501275
• 关键词: AMPA receptors; biological signal transduction; chimeric proteins; clone cells; dopamine agonists; dopamine antagonists; dopamine receptor; gene mutation; genetic library; genetically modified animals; glioma; laboratory mouse; laboratory rat; molecular cloning; mutant; neural information processing; neuropharmacology; phosphorylation; protein isoforms; protein structure function; receptor binding; receptor coupling; receptor expression; receptor sensitivity; transfection; yeast two hybrid system

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 mice. In FY-2001, the mechanisms of agonist-induced regulation of D1 receptors were further investigated. We previously showed that the rat D1 dopamine receptor (DAR) is stoichiometrically phosphorylated in C6 glioma cells and that this phosphorylation is increased 2-3 fold upon agonist activation and desensitization. In the current year, we attempted to identify the sites of regulatory phosphorylation within the D1 DAR using mutagenesis techniques and transient expression in HEK-293 cells. We created two 3rd cytoplasmic loop mutants, one in which all Ser and Thr residues were mutated to Ala or Val residues (3rdTOT) and another in which Ser residues 256, 258 & 259 were mutated to alanines (3rd234). We also prepared a series of mutants where the D1 DAR was truncated subsequent to the indicated positions in the COOH terminus: T347, T369, T394 & T404. Expression of these mutant and wild-type constructs resulted in similar levels of receptor binding activity (~1 pmol/mg) with the exception of the T347 mutant which was about 50% lower. Dopamine (DA) stimulated cAMP accumulation in a similar fashion for all constructs except for the 3rdTOT mutant which showed a 10-fold reduction in DA potency. DA-induced desensitization was investigated for the 3rdTOT and T347 mutants. Both mutants exhibited desensitization, however, desensitization of the 3rdTOT and T347 mutants were impaired and enhanced, respectively, compared to wild-type. DA-induced phosphorylation of all constructs was investigated. Relative to wild-type, the 3rdTOT mutant exhibited about a 50% decrease which was mimicked by the 3rd234 mutant. This suggests that Ser residues 256, 258 & 259 account for the majority of 3rd loop phosphorylation. Mutant T404 also exhibited a 50% decrease in phosphorylation with further reductions observed for each of the truncation mutants. Surprisingly, phosphorylation of T347 mutant was barely detectable. These results suggest that phosphorylation of COOH terminal residues is necessary for phosphorylation of 3rd loop to take place. The mechanism of desensitization in the T347 mutant is currently under investigation. In order to identify potential proteins that interact with the rat D3 dopamine receptor (DAR), we have started to use the DupLex-A yeast two-hybrid system i FY-2001. The entire third cytoplasmic loop (residues 210-375) of the rat D3 DAR was used as a bait to identify clones encoding interacting proteins from a rat whole brain cDNA library. One of the most highly positive clones appeared to represent a partial-length cDNA of GRIP-1, (Glutamate Receptor-Interacting Protein-1) a scaffolding protein known to interact with AMPA receptors. Our partial-length cDNA contains the fourth and fifth PDZ domains of the GRIP-1 protein, which had been shown previously to be required for AMPA receptor interactions. The specificity of the interaction of the D3 DAR and the partial-length GRIP-1 clone was verified using a corresponding yeast expression vector that lacks inserts and showed no growth on -leu plates and no color change on X-gal plates. The partial-length GRIP-1 clone was further shown not to interact with the third cytoplasmic loops of the rat D1, D2L, D4, or D5 DARs. Co-expression of the partial-length GRIP-1 clone with the D3 DAR in HEK-293T cells resulted in ~50% reduction of receptor binding activity. Co-expression of the GRIP-1 clone had no effect the receptor expression or binding activities of the D1, D4 or D5 DARs, however, a decrease in receptor binding was surprisingly observed with the D2L DAR. These findings represent the first report of GRIP-1 interactions with a G-protein coupled receptor. Further experiments confirming and characterizing the interactions between GRIP-1 and the D3 DAR are in progress.

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-2001中，进一步研究了激动剂诱导的D1受体调节机制。我们之前表明，大鼠 D1 多巴胺受体 (DAR) 在 C6 神经胶质瘤细胞中按化学计量磷酸化，并且这种磷酸化在激动剂激活和脱敏后增加 2-3 倍。今年，我们尝试使用诱变技术和 HEK-293 细胞中的瞬时表达来鉴定 D1 DAR 内的调节性磷酸化位点。我们创建了两种第三个细胞质环突变体，一种中所有 Ser 和 Thr 残基突变为 Ala 或 Val 残基 (3rdTOT)，另一种中 Ser 残基 256、258 和 259 突变为丙氨酸 (3rd234)。我们还制备了一系列突变体，其中 D1 DAR 在 COOH 末端指定位置之后被截短：T347、T369、T394 和 T404。这些突变体和野生型构建体的表达导致了相似水平的受体结合活性（~1 pmol/mg），但 T347 突变体除外，其降低了约 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，该突变体的 DA 效力降低了 10 倍。研究了 3rdTOT 和 T347 突变体的 DA 诱导脱敏作用。两种突变体都表现出脱敏作用，然而，与野生型相比，3rdTOT 和 T347 突变体的脱敏作用分别受损和增强。研究了 DA 诱导的所有构建体的磷酸化。相对于野生型，3rdTOT 突变体表现出约 50% 的减少，这与 3rd234 突变体相似。这表明 Ser 残基 256、258 和 259 占第三环磷酸化的大部分。突变体 T404 的磷酸化也表现出 50% 的降低，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，几乎检测不到T347突变体的磷酸化。这些结果表明 COOH 末端残基的磷酸化对于第三环的磷酸化是必要的。 T347突变体的脱敏机制目前正在研究中。 为了鉴定与大鼠 D3 多巴胺受体 (DAR) 相互作用的潜在蛋白质，我们已开始使用 DupLex-A 酵母双杂交系统 i FY-2001。使用大鼠 D3 DAR 的整个第三细胞质环（残基 210-375）作为诱饵，从大鼠全脑 cDNA 文库中鉴定编码相互作用蛋白的克隆。阳性率最高的克隆之一似乎代表了 GRIP-1（谷氨酸受体相互作用蛋白-1）的部分长度 cDNA，GRIP-1 是一种已知与 AMPA 受体相互作用的支架蛋白。我们的部分长度 cDNA 包含 GRIP-1 蛋白的第四和第五个 PDZ 结构域，之前已证明这些结构域是 AMPA 受体相互作用所必需的。使用相应的酵母表达载体验证了 D3 DAR 和部分长度 GRIP-1 克隆相互作用的特异性，该酵母表达载体缺乏插入片段，并且在 -leu 平板上没有显示生长，在 X-gal 平板上没有颜色变化。部分长度的 GRIP-1 克隆进一步显示不与大鼠 D1、D2L、D4 或 D5 DAR 的第三个细胞质环相互作用。在 HEK-293T 细胞中，部分长度 GRIP-1 克隆与 D3 DAR 共表达导致受体结合活性降低约 50%。 GRIP-1克隆的共表达对D1、D4或D5 DAR的受体表达或结合活性没有影响，然而，令人惊奇地观察到D2L DAR的受体结合减少。这些发现代表了 GRIP-1 与 G 蛋白偶联受体相互作用的第一份报告。确认和表征 GRIP-1 与 D3 DAR 之间相互作用的进一步实验正在进行中。