Molecular And Pharmacological Studies Of Dopamine Recept

多巴胺受体的分子和药理学研究

• 批准号: 6659609
• 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/6659609
• 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 the neurotransmitter dopamine. To characterize these receptors at the biochemical and molecular levels and study their regulation, two interrelated lines of research are underway: 1) investigation of the cell biology, function and regulation of the receptors at the protein level; and 2) the molecular cloning of receptor-interacting cDNAs/genes and investigation of receptor structure, pharmacology and regulation in cultured cell lines and transgenic mice. In FY-2002, the mechanisms of agonist-induced regulation of D1 receptors were further investigated. D1 dopamine receptor (DAR) is stoichiometrically phosphorylated in C6 glioma cells and this phosphorylation is increased 2-3 fold upon agonist activation and desensitization. Potential G protein-coupled receptor kinase (GRK) phosphorylation sites exist in both the COOH terminus and the 3rd intracellular loop (3rdICL) of the receptor. To investigate the role of these two regions in desensitization, we created mutant receptors where either the cytoplasmic tail is mostly deleted (T347) or where three serines in the 3rdICL were mutated (3rdICL-234). The T347 mutant desensitized normally, while the 3rdICL-234 mutant exhibited impaired desensitization. Since potential GRK phosphorylation sites have been mutated or deleted in these two mutants, we investigated how over-expression of different GRKs would affect their desensitization. Desensitization was documented as a decrease in the maximal cAMP response as well as an EC50 shift in the dopamine dose-response curve. When co-transfected with the 3rdICL-234 receptor, GRK2, GRK3, and GRK6 each corrected the impairment of desensitization seen previously with this mutant. When co-transfected with the T347 receptor, GRK3 and GRK6 had no effect on desensitization whereas GRK2 induced a larger EC50 shift. Transfection of GRK5 to either the 3rdICL-234 or T347 receptors did not enhance their desensitization; however, GRK5 expression induced a larger agonist-induced EC50 shift with the wild-type receptor. These results suggest that GRK2/3/5/6 phosphorylation sites exist in the receptor's COOH terminus whereas only GRK2 phosphorylates residues in the receptor's 3rdICL. We have begun to investigate the role that protein phosphorylation plays in D2 receptor regulation. In the present year, we used HEK293T cells to investigate D2 dopamine receptor phosphorylation and sequestration. For receptor sequestration, we used [3H]sulpiride, a hydrophilic ligand that is cell surface-restricted and intact cell radioligand binding assays. Treatment with dopamine for 1 hr resulted in a 25% loss of cell surface receptor binding. This effect was not blocked by pertussis toxin indicating that G-protein coupling was not required. Agonist-induced receptor sequestration was blocked by co-expression with a dominant-negative mutant of dynamin which itself resulted in increased receptor expression at the cell surface. Co-expression of GRKs 2&3 decreased cell surface D2 receptor expression and enhanced agonist-induced sequestration, whereas GRKs 5&6 were without effect. Expression of either arrestin2 or arrestin3 increased agonist-induced receptor sequestration and this effect was enhanced by GRK2. These results suggest that the D2 receptor exhibits constitutive sequestration that is blocked by the dynamin mutant and enhanced by GRK2/3 phosphorylation. The D2 receptor is also phosphorylated under basal conditions and phosphorylation is increased 2-3 fold upon exposure to dopamine. GRKs 2&3 enhanced both basal and dopamine-stimulated receptor phosphorylation whereas GRKs 5&6 had no effect. Simultaneous mutation of serines 285,286,288 and threonines 287,293 within the 3rd cytoplasmic loop attenuated, but did not completely block, agonist- and GRK2- enhanced receptor phosphorylation and sequestration. These results suggest that GRK2/3 phosphorylation of Ser/Thr residues in the 3rd loop modulates intracellular trafficking of the D2 receptor. D2/D3 dopamine receptors directly interact with GRIP, a PDZ domain-containing protein that also interacts with GluR2/3 AMPA receptor subunits. GRIP functions as a scaffolding protein linking AMPA receptors and other signaling proteins into macromolecular complexes within postsynaptic membranes. Given this, and the fact that D2-like and AMPA receptors show cellular co-localization in the CNS we wondered if D2 or D3 receptors might form heterodimers with AMPA receptors, perhaps in a GRIP-facilitated fashion. As an initial test of this hypothesis, we co-expressed a FLAG-tagged D2 receptor or a c-myc-tagged D3 receptor with either GluR1, GluR2 or GluR4 AMPA receptor subunits in HEK293T cells. Radioligand binding assays confirmed the expression of the dopamine receptors while immunoblots confirmed the expression of the AMPA receptors. When the cells were detergent-solubilized and the D2 or D3 receptors were immunoprecipitated with either anti-FLAG or anti-c-myc antisera, respectively, D2 and D3 receptors were co-immunoprecipitated with AMPA receptor subunits GluR1, GluR2 and GluR4. However, D3 receptors were only co-immunoprecipitated with the GluR2 subunit when co-expressed with GRIP. These results suggest that GRIP facilitates interactions between D3 and GluR2. GluR3 subunit interactions are currently under investigation. In contrast, no immunoprecipitation of any AMPA receptor subunit was observed from cells expressing the GluR subunits alone or with the GluR subunit co-expressed with either the FLAG or c-myc peptides only. Further, co-expression with AMPA receptors altered D2 or D3 radioligand binding activity in HEK293T cells. Thus, D2 and D3 dopamine receptors directly interact with AMPA receptors in a heterologous cellular expression system.

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在神经元膜之间的调节。正在研究的主要受体系统是神经递质多巴胺的受体系统。为了在生化和分子水平上表征这些受体并研究其调节，正在进行两种相互关联的研究线：1）研究受体在蛋白质水平上的细胞生物学，功能和调节； 2）受体相互作用cDNA/基因的分子克隆以及对培养的细胞系和转基因小鼠的受体结构，药理学和调节的研究。 在2002财年，进一步研究了激动剂诱导的D1受体调节的机制。 D1多巴胺受体（DAR）在C6神经胶质瘤细胞中被石化磷酸化，并且在激活和脱敏后，这种磷酸化增加了2-3倍。在COOH末端和受体的第三个细胞内环（3rdicl）中，潜在的G蛋白偶联受体激酶（GRK）磷酸化位点均存在。为了研究这两个区域在脱敏中的作用，我们创建了突变受体，其中细胞质尾巴大部分被删除（T347）或3rdicl中的三个丝氨酸被突变（3RDICL-234）。 T347突变体脱敏，而3RDICL-234突变体表现出脱敏受损。由于潜在的GRK磷酸化位点已在这两个突变体中突变或删除，因此我们研究了不同GRK的过表达如何影响其脱敏。脱敏被记录为最大cAMP反应的减少以及多巴胺剂量反应曲线的EC50偏移。当与3RDICL-234受体GRK2，GRK3和GRK6共转染时，各自纠正了先前使用该突变体看到的脱敏的损害。当与T347受体共转染时，GRK3和GRK6对脱敏没有影响，而GRK2诱导了较大的EC50偏移。将GRK5转染至3RDICL-234或T347受体不会增强其脱敏。然而，GRK5表达诱导了野生型受体较大的激动剂诱导的EC50移位。这些结果表明，GRK2/3/5/6磷酸化位点存在于受体的COOH末端中，而GRK2仅磷酸化受体3rdicl中的残基。 我们已经开始研究蛋白磷酸化在D2受体调节中起作用的作用。今年，我们使用HEK293T细胞研究了D2多巴胺受体磷酸化和隔离。对于受体隔离，我们使用了[3H]硫磺，这是一种亲水性配体，是细胞表面限制和完整的细胞放射性结合测定法。多巴胺治疗1小时，导致细胞表面受体结合损失25％。百日咳毒素不会阻止这种作用，表明不需要G蛋白偶联。激动剂诱导的受体隔离通过与元素的显性阴性突变体共表达阻塞，这本身在细胞表面导致受体表达增加。 GRK 2和3的共表达降低了细胞表面D2受体表达和增强的激动剂诱导的隔离，而GRKS 5和6则无效。抑制素2或抑制素3的表达增加了激动剂诱导的受体隔离，而GRK2增强了这种作用。这些结果表明，D2受体表现出由Dynamin突变体阻止并通过GRK2/3磷酸化增强的组成式隔离。在基础条件下，D2受体也被磷酸化，暴露于多巴胺后磷酸化增加了2-3倍。 GRK 2和3增强了基底和多巴胺刺激的受体磷酸化，而Grks 5和6均无效。在第三个细胞质环中，丝氨酸的同时突变285,286,288和苏氨酸287,293被减弱，但没有完全阻断，激动剂和GRK2增强的受体磷酸化和螯合。这些结果表明，在第三环中，Ser/THR残基的GRK2/3磷酸化调节了D2受体的细胞内运输。 D2/D3多巴胺受体直接与Grip相互作用，Grip是一种含PDZ结构域的蛋白质，它也与GLUR2/3 AMPA受体亚基相互作用。握力充当脚手架蛋白，将AMPA受体和其他信号蛋白连接到突触后膜内的大分子复合物中。鉴于此，以及D2样和AMPA受体在中枢神经系统中显示细胞共定位的事实，我们想知道D2或D3受体是否可能以握把的方式形成具有AMPA受体的异二聚体。作为该假设的初步检验，我们在HEK293T细胞中与GlUR1，GlUR2或GlUR4 AMPA受体亚基共同表达了标记为标记的D2受体或C-MYC标记的D3受体。放射性结合测定法证实了多巴胺受体的表达，而免疫印迹证实了AMPA受体的表达。当细胞进行洗涤剂 - 溶解并分别用抗FLAG或抗C-MYC抗血清免疫沉淀D2或D3受体时，将D2和D3受体与AMPA受体亚基Glur1，Glur1，GluR2和Glur4共对D2和D3受体进行了共免疫沉淀。然而，当与握把共表达时，D3受体仅与Glur2亚基共免疫沉淀。这些结果表明，抓地力促进了D3和Glur2之间的相互作用。 GLUR3亚基相互作用目前正在研究中。相比之下，从单独表达Glur亚基的细胞或与仅与FLAG或C-MYC肽共表达的GLUR亚基，没有观察到任何AMPA受体亚基的免疫沉淀。此外，与AMPA受体共表达改变了HEK293T细胞中D2或D3放射性结合活性。因此，D2和D3多巴胺受体在异源细胞表达系统中直接与AMPA受体相互作用。