Molecular Analysis of Dopamine 2 Like Receptor Function

多巴胺 2 样受体功能的分子分析

• 批准号: 7126922
• 负责人: ALAN S KOPIN
• 金额: $ 15.92万
• 依托单位: TUFTS MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-09-28 至 2010-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7126922
• 关键词: Drosophilidae; RNA interference; RNA splicing; arthropod genetics; biochemical evolution; biological models; biological signal transduction; cell line; chimeric proteins; confocal scanning microscopy; dopamine agonists; dopamine receptor; gene expression profiling; gene mutation; human genetic material tag; molecular biology; neural transmission; neurogenetics; neuropharmacology; protein isoforms; psychomotor function; receptor expression; site directed mutagenesis; species difference; transposon /insertion element

Dopamine receptors in the central nervous system regulate locomotor function and as such are important therapeutic targets for neuropathologic conditions including Parkinson's disease. To better understand the processes that determine receptor-mediated function in mammals we will utilize Drosophila as a model organism. Flies are amenable to a unique spectrum of genetic approaches for which advanced research tools (e.g. microarrays, repository of mutant fly lines) are readily available. It is of note that there is a high degree of structural and functional conservation between the Drosophila and mammalian proteins that modulate dopaminergic neurotransmission. Our laboratory has recently extended these known parallels with the cloning and pharmacologic characterization of a Drosophila dopamine 2 like receptor (DD2R), the homolog of a well-established mammalian protein that is a key regulator of locomotor function. In vitro pharmacologic comparison between the fly and human D2 receptors reveals a similar profile of agonist (endogenous amines and selected synthetic ligands) induced signaling. At the same time, a subset of anti-Parkinsonian D2R drugs preferentially binds and activates the human (vs. the fly) receptor homolog. In Aim 1, receptor mutants will be generated in which divergent residues are exchanged between the human D2R and the DD2R. These constructs will be utilized to identify human amino acids that confer functional activity to clinically important agonists. In Aim 2, we propose to define the tissue specific expression and the pharmacologic profile of multiple DD2R splice variants that we have identified. As a rationale for these studies, it is well established that mammalian D2 receptor isoforms have distinct cellular distributions and modulate different functions in vivo. The assessment of DD2R splice variants will rely on a combination of approaches including quantitative PCR to assess relative transcript abundance, confocal microscopy with DD2R antibodies to assess expression profiles, and in vitro cell based assays to assess pharmacologic properties. To examine in vivo function, we have generated trangenic RNA interference (RNAi) flies with reduced expression of the DD2R. Characterization of these flies has revealed a highly significant decrease in locomotor function. In Aim 3, we propose to identify novel genes that are linked to dopaminergic signaling pathways, utilizing two complementary approaches (i) transcriptome analysis of the DD2R RNAi (vs. control) flies, and (ii) screening an existing collection of insertion bearing fly lines for alterations in locomotor activity/dopaminergic signaling. Once identified, these genes will be functionally assessed to determine their potential role in dopaminergic signaling.

中枢神经系统中的多巴胺受体调节运动功能，因此是包括帕金森病在内的神经病理疾病的重要治疗靶点。为了更好地了解哺乳动物中决定受体介导功能的过程，我们将以果蝇作为模式生物。苍蝇容易受到一系列独特的遗传方法的影响，先进的研究工具(如微阵列、突变苍蝇品系资料库)是现成的。值得注意的是，果蝇和哺乳动物之间存在高度的结构和功能保守的蛋白质，这些蛋白质调节多巴胺能神经传递。我们的实验室最近扩展了这些已知的相似之处，克隆了果蝇多巴胺2样受体(DD2R)，并对其进行了药理学表征，DD2R是一种成熟的哺乳动物蛋白质的同源物，是运动功能的关键调节因子。苍蝇和人类D2受体的体外药理学比较显示，激动剂(内源胺和选定的合成配体)诱导的信号传递情况相似。同时，抗帕金森病D2R药物的子集优先结合并激活人(对苍蝇)受体同系物。在目标1中，将产生受体突变体，其中分歧残基在人D2R和DD2R之间交换。这些结构将被用来识别人类氨基酸，赋予临床上重要的激动剂功能活性。在目标2中，我们建议定义我们已经确定的多种DD2R剪接变异体的组织特异性表达和药理学特征。作为这些研究的理论基础，众所周知，哺乳动物D2受体亚型在体内具有不同的细胞分布和调节不同的功能。对DD2R剪接变异体的评估将依赖于多种方法的组合，包括定量PCR评估相对转录丰度，使用DD2R抗体的共聚焦显微镜评估表达谱，以及体外细胞分析评估药理学特性。为了检测体内功能，我们产生了转基因RNA干扰(RNAi)果蝇，降低了DD2R的表达。对这些苍蝇的研究表明，它们的运动功能显著下降。在目标3中，我们建议利用两种互补的方法(I)对DD2R RNAi(与对照)果蝇进行转录组分析，以及(Ii)筛选现有的插入承载果蝇系的集合，以确定与多巴胺能信号通路相关的新基因。一旦确定，将对这些基因进行功能评估，以确定它们在多巴胺能信号中的潜在作用。