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跨神经元膜的调节。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它们的调节，两个相互关联的研究正在进行中：1）在蛋白质水平上研究受体的细胞生物学、功能和调节； 2) 受体相互作用 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。 FY-2002进一步研究了激动剂诱导的D1受体调节机制。 D1 多巴胺受体 (DAR) 在 C6 神经胶质瘤细胞中按化学计量被磷酸化，并且这种磷酸化在激动剂激活和脱敏后增加 2-3 倍。潜在的 G 蛋白偶联受体激酶 (GRK) 磷酸化位点存在于受体的 COOH 末端和第三胞内环 (3rdICL) 中。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，其中细胞质尾部大部分被删除（T347）或 3rdICL 中的三个丝氨酸发生突变（3rdICL-234）。 T347突变体正常脱敏，而3rdICL-234突变体表现出脱敏受损。由于这两个突变体中潜在的 GRK 磷酸化位点已发生突变或删除，我们研究了不同 GRK 的过度表达将如何影响其脱敏。脱敏被记录为最大 cAMP 反应的降低以及多巴胺剂量反应曲线中 EC50 的变化。当与 3rdICL-234 受体共转染时，GRK2、GRK3 和 GRK6 各自纠正了之前在该突变体中观察到的脱敏损伤。当与 T347 受体共转染时，GRK3 和 GRK6 对脱敏没有影响，而 GRK2 则诱导较大的 EC50 变化。将 GRK5 转染至 3rdICL-234 或 T347 受体并不会增强其脱敏作用；然而，GRK5 表达诱导了野生型受体更大的激动剂诱导的 EC50 变化。这些结果表明GRK2/3/5/6磷酸化位点存在于受体的COOH末端，而只有GRK2磷酸化受体的3rdICL中的残基。 我们已经开始研究蛋白质磷酸化在 D2 受体调节中的作用。今年，我们使用 HEK293T 细胞研究 D2 多巴胺受体磷酸化和隔离。对于受体隔离，我们使用[3H]舒必利，这是一种亲水性配体，是细胞表面限制的完整细胞放射性配体结合测定。用多巴胺处理 1 小时导致细胞表面受体结合丧失 25%。这种效应没有被百日咳毒素阻断，表明不需要 G 蛋白偶联。激动剂诱导的受体隔离被与动力蛋白显性失活突变体共表达所阻断，该突变体本身导致细胞表面受体表达增加。 GRKs 2和3的共表达降低了细胞表面D2受体的表达并增强了激动剂诱导的隔离，而GRKs 5和6则没有效果。抑制蛋白2或抑制蛋白3的表达增加了激动剂诱导的受体隔离，并且GRK2增强了这种作用。这些结果表明，D2 受体表现出组成型隔离，该隔离被动力突变体阻断并被 GRK2/3 磷酸化增强。 D2 受体在基础条件下也会被磷酸化，并且在接触多巴胺后磷酸化会增加 2-3 倍。 GRKs 2和3增强基础和多巴胺刺激的受体磷酸化，而GRKs 5和6则没有效果。第三细胞质环内丝氨酸285,286,288和苏氨酸287,293的同时突变减弱，但没有完全阻断激动剂和GRK2增强的受体磷酸化和隔离。这些结果表明，GRK2/3 第三环中 Ser/Thr 残基的磷酸化调节 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 是一种包含 PDZ 结构域的蛋白质，也与 GluR2/3 AMPA 受体亚基相互作用。 GRIP 充当支架蛋白，将 AMPA 受体和其他信号蛋白连接成突触后膜内的大分子复合物。鉴于此，以及 D2 样受体和 AMPA 受体在中枢神经系统中表现出细胞共定位的事实，我们想知道 D2 或 D3 受体是否可能与 AMPA 受体形成异二聚体，也许以 GRIP 促进的方式。作为对该假设的初步测试，我们在 HEK293T 细胞中共表达 FLAG 标记的 D2 受体或 c-myc 标记的 D3 受体与 GluR1、GluR2 或 GluR4 AMPA 受体亚基。放射性配体结合测定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。当细胞被去污剂溶解并且D2或D3受体分别用抗FLAG或抗c-myc抗血清免疫沉淀时，D2和D3受体与AMPA受体亚基GluR1、GluR2和GluR4共免疫沉淀。然而，当与 GRIP 共表达时，D3 受体仅与 GluR2 亚基发生共免疫沉淀。这些结果表明 GRIP 促进 D3 和 GluR2 之间的相互作用。 GluR3 亚基相互作用目前正在研究中。相反，从单独表达GluR亚基或仅与FLAG或c-myc肽共表达的GluR亚基的细胞中没有观察到任何AMPA受体亚基的免疫沉淀。此外，与 AMPA 受体共表达改变了 HEK293T 细胞中的 D2 或 D3 放射性配体结合活性。因此，D2和D3多巴胺受体直接与异源细胞表达系统中的AMPA受体相互作用。