Molecular & Pharmacological Studies Of Dopamine Receptor

分子

• 批准号: 6841904
• 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/6841904
• 关键词: AMPA receptors; G protein; biological signal transduction; cell surface receptors; chimeric proteins; dopamine agonists; dopamine antagonists; dopamine receptor; gene mutation; genetic library; genetically modified animals; glioma; laboratory mouse; laboratory rat; molecular cloning; neural information processing; neuropharmacology; phosphorylation; protein isoforms; protein structure function; receptor binding; receptor coupling; receptor expression; receptor sensitivity

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. Homologous desensitization of G protein-coupled receptors (GPCRs) is mediated by G protein receptor kinases (GRKs). Upon agonist binding and activation, the GPCR becomes a substrate for GRK phosphorylation at serine and/or threonine residues. In, FY2003 we discovered evidence that the GRK4 isoform exhibits constitutive or agonist-independent phosphorylation of the D1 dopamine receptor. Co-expression of GRK4 and the rat D1 receptor in HEK293T cells results in increased basal phosphorylation of the receptor that is similar to the level of phosphorylation induced by dopamine stimulation in the absence of GRK4. The addition of dopamine to GRK4 co-transfected cells produces only a modest increase in phosphorylation over basal levels. Radioligand binding assays and confocal fluorescence microscopy using a D1 receptor-GFP chimera reveals that GRK4 co-expression induces a shift in the number of receptors from the plasma membrane into intracellular locations independent of agonist stimulation. GRK4 co-expression also results in a ~50% decline in the accumulation of cAMP in response to a maximally-effective concentration of dopamine. However, the population of receptors that remain at the plasma membrane in the presence of GRK4 desensitize and internalize similarly as D1 receptors co-expressed without GRK4. Mutation or truncation of serine and threonine residues in the carboxyl terminus of the D1 receptor reduces the amount of phosphorylation when the receptor is co-expressed with GRK4, but the reduction in phosphorylation is independent of agonist stimulation. These data suggest that the D1 dopamine receptor can be constitutively phosphorylated by a GRK4 in an agonist-independent manner and that this phosphorylation results in constitutive desensitization and internalization of the receptor. GRK4 might thus play a critical role in regulating the D1 receptor within cells or tissues that co-express these two proteins. Previously, D2 dopamine receptors (D2DARs) have been shown to undergo G protein-coupled receptor kinase (GRK) phosphorylation in an agonist-specific fashion. In FY2003 have investigated the ability of the second-messenger activated protein kinases, protein kinase A (PKA) and protein kinase C (PKC) to mediate phosphorylation and desensitization of the D2DAR. HEK293T cells were transiently transfected with the rat D2DAR and then treated with various intracellular activators and inhibitors of PKA or PKC. Treatment with agents that increase cAMP, and activate PKA, had no effect on the phosphorylation state of the D2DAR suggesting that PKA does not phosphorylate the D2DAR, at least in HEK293T cells. In contrast, cellular treatment with 1 uM phorbol 12-myristate 13-acetate (PMA), a PKC activator, resulted in ~3-fold increase in D2DAR phosphorylation within 10 min of treatment. The phosphorylation was specific for PKC as the PMA effect was mimicked by PDBu, but not by 4-alpha-PDD, active and inactive, phorbol diesters, respectively. PMA treatment produced only a small change in the cell surface expression of the D2DAR (5% decrease). The PMA-mediated D2DAR phosphorylation was completely blocked by co-treatment with the PKC inhibitor, bisindolylmaleimide II. In contrast, PKC inhibition had no effect on agonist-promoted phosphorylation suggesting that PKC is not involved in this response. To assess D2DAR desensitization, we examined the ability of the receptor to attenuate cAMP accumulation. PMA pretreatment of the cells resulted in a ~30-40% desensitization in the maximum response for dopamine inhibition of forskolin-stimulated cAMP accumulation. We also examined agonist-stimulated [35S]-GTP-gamma-S binding. PMA treatment diminished dopamine-stimulated [35S]-GTP-gamma-S binding by ~ 50% and also produced >5 fold shift (to the lower affinity) in the EC50. These results suggest that PKC mediates phosphoryation of the D2DAR and may be involved in its heterologous desensitization. We have previously shown that D3 dopamine receptors (D3DARs) and AMPA receptor subunits can be co-immunoprecipitated from HEK293T cells. These effects were specific and suggested that D3 and AMPA receptors may form hetero-oligomers when expressed in the same cell. In FY2003, we have investigated the functional consequences of these receptor-receptor interactions using intact cells. Using radioligand binding assays, we found that co-expression of the GluR2 or GluR3 subunits resulted in a 25-50% decrement in D3DAR expression levels. To examine D3DAR function, it was necessary to co-transfect adenylyl cyclase type V in the cells and then examine D3DAR-mediated inhibition of forskolin-stimulated cAMP accumulation. Both dopamine and quinpirole inhibited the adenylyl cyclase activity in a dose-dependent fashion. Co-expression of GluR2 with the D3DAR resulted in a 4-6 fold rightward shift in the agonist dose-response curves (increase in EC50) without a major effect on the maximum response. Interestingly, the addition of 30 uM AMPA to the cAMP assay abolished the shift in agonist potency such that the GluR2 subunit expression was without effect. These results suggest that D3DAR and GluR subunits may constitutively hetero-oligomerize in HEK293T cells and that this attenuates D3DAR-G protein coupling. Further, the state of D3DAR/GluR hetero-oligomerization may be dependent on agonist occupancy/activation of the GluR receptor. In terms of AMPA receptor function, co-expression of D3DARs with GluR1+2 in HEK293T cells did not alter the functional expression of GluR-mediated responses as measured by the density of current activated by 100 uM AMPA + 100 uM cyclothiazide. The expression of GluR2 also did not appear to be altered, as current/voltage relationships were linear. We are currently further investigating the effects of D3DAR expression on GluR activity. The D2 dopamine receptor exists in two protein isoforms that are generated by alternative mRNA splicing. In order to identify proteinsthat may differentially interact with these isofomrs, we have performed yeast two-hybrid screens of rat brain cDNA libraries using the 3rd cytoplasmic loops of the receptors as baits. Our strategy was to first identify positive interacting clones using the D2S and D2L 3rd loops and then test for cross-interaction using the opposite 3rd loop. Using the D2S 3rd loop, we identified 282 positive clones whereas 193 clones were isolated using the D2L 3rd loop. Following sib elimination, insert identification and sizing, as well as DNA sequencing, there remained 55 and 81 unique clones positive for D2S and D2L, respectively. Cross transformations revealed that, of the 55 clones identified with the D2S isoform, 16 were found to be highly positive with the D2L receptor whereas 21 of the 81 clones originally identified with the D2L isoform, where found to be highly positive with the D2S receptor. The identification and characterization of the unique as well as the cross-reacting clones are currently 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。 G蛋白偶联受体（GPCR）的同源脱敏是由G蛋白受体激酶（GRKS）介导的。激动剂结合和激活后，GPCR成为丝氨酸和/或苏氨酸残基上GRK磷酸化的底物。在2003财年，我们发现了GRK4同工型表现出D1多巴胺受体的构成或激动剂非依赖性磷酸化的证据。 HEK293T细胞中GRK4和大鼠D1受体的共表达导致受体的基底磷酸化增加，这与在没有GRK4的情况下与多巴胺刺激诱导的磷酸化水平相似。在GRK4共转染的细胞中添加多巴胺只会在基础水平上产生适度的磷酸化增加。使用D1受体-GFP嵌合体的放射性结合测定和共聚焦荧光显微镜表明，GRK4共表达诱导受体数量从质膜到独立于激动剂刺激的细胞内位置的变化。 GRK4的共表达也导致cAMP积累的响应最大浓度的多巴胺的积累下降了约50％。然而，在GRK4存在下脱敏和内在化与D1受体相似的情况下，在没有GRK4的情况下，质膜的受体群体仍保留在质膜上。当受体与GRK4共表达时，D1受体羧基末端中丝氨酸和苏氨酸残基的突变或截断会减少磷酸化的量，但是磷酸化的降低与激动剂刺激无关。这些数据表明，D1多巴胺受体可以通过grk4的组成型磷酸化，而这种磷酸化可以导致受体的组成型脱敏和内在化。因此，GRK4可能在调节这两种蛋白质的细胞或组织中调节D1受体中起关键作用。 以前，D2多巴胺受体（D2DAR）已显示出以激动剂特异性的方式进行G蛋白偶联受体激酶（GRK）磷酸化。在2003财年中，已经研究了第二届元激活蛋白激酶，蛋白激酶A（PKA）和蛋白激酶C（PKC）介导D2DAR磷酸化和脱敏的能力。 HEK293T细胞用大鼠D2DAR瞬时转染，然后用PKA或PKC的各种细胞内激活剂和抑制剂处理。用增加cAMP并激活PKA的药物的处理对D2DAR的磷酸化状态没有影响，这表明PKA至少在HEK293T细胞中不会磷酸化D2DAR。相比之下，PKC激活剂1 UM佛波尔13-乙酸盐（PMA）的细胞处理，在治疗后的10分钟内，D2DAR磷酸化增加了约3倍。磷酸化对PKC是特异性的，因为PDBU模仿了PMA效应，但分别由4-Alpha-PDD模仿，活性和不活跃的Phorbol Diesters。 PMA处理仅产生了D2DAR的细胞表面表达的微小变化（降低了5％）。 PMA介导的D2DAR磷酸化完全通过与PKC抑制剂Bisindolylylmaleimide II完全阻断。相反，PKC抑制对激动剂促进的磷酸化没有影响，这表明PKC与这种反应无关。为了评估D2DAR脱敏，我们检查了受体减轻营地积累的能力。细胞的PMA预处理导致大约30-40％的脱敏，以最大程度地响应多巴胺抑制福斯科林刺激的cAMP积累。我们还检查了激动剂刺激的[35S] -GTP-GAMMA-S结合。 PMA治疗减少了多巴胺刺激的[35S] -GTP-GAMMA-S结合约50％，并且在EC50中也产生> 5倍的移位（至较低亲和力）。这些结果表明，PKC介导D2DAR的磷酸化，并可能参与其异源脱敏。 我们先前已经表明，D3多巴胺受体（D3DAR）和AMPA受体亚基可以从HEK293T细胞中共免疫沉淀。这些作用是特定的，并表明在同一细胞中表达时D3和AMPA受体可能形成异弱者。在2003财年，我们使用完整的细胞研究了这些受体受体相互作用的功能后果。使用放射性结合测定法，我们发现GLUR2或GLUR3亚基的共表达导致D3DAR表达水平降低了25-50％。为了检查D3DAR功能，有必要在细胞中共接触腺苷酸环化酶V型V，然后检查D3DAR介导的Forskolin刺激的CAMP积累的抑制作用。多巴胺和喹硫吡疑都以剂量依赖性的方式抑制腺苷酸环化酶活性。与D3DAR的GLUR2共表达导致激动剂剂量反应曲线的4-6倍移动（EC50的增加），对最大响应没有重大影响。有趣的是，在CAMP分析中添加30 UM AMPA消除了激动剂效力的转移，因此GlUR2亚基表达无效。这些结果表明，D3DAR和GLUR亚基可能在HEK293T细胞中组成性异构物，并且这会减弱D3DAR-G蛋白偶联。此外，D3DAR/GLUR杂波的状态可能取决于Glur受体的激动剂占用/激活。在AMPA受体功能方面，HEK293T细胞中D3DAR与GLUR1 + 2的共表达不会改变Glur介导的响应的功能表达，如通过由100 UM AMPA + 100 UM Cyclothiazide激活的电流密度所测量的。由于电流/电压关系是线性的，因此Glur2的表达似乎也没有改变。我们目前正在进一步研究D3DAR表达对Glur活性的影响。 D2多巴胺受体存在于两个通过替代mRNA剪接产生的蛋白质同工型中。为了鉴定蛋白质可能与这些异f子差异相互作用，我们使用受体的第三个细胞质环作为诱饵进行了大鼠脑cDNA库的酵母两杂交筛选。我们的策略是首先使用D2S和D2L 3循环识别正相互作用的克隆，然后使用相反的第三环测试交叉交流。使用D2S 3循环，我们确定了282个正克隆，而使用D2L 3循环分离了193个克隆。消除SIB消除，插入识别和大小以及DNA测序后，D2S和D2L呈阳性55和81个独特的克隆。交叉转化表明，在用D2S同工型鉴定的55个克隆中，发现16个具有D2L受体的高度阳性，而最初用D2L同工型鉴定的81个克隆中有21个，其中发现与D2S受体高度阳性。目前正在进行唯一和交叉反应克隆的识别和表征。