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: 6869255
• 负责人: ALAN S KOPIN
• 金额: $ 16.3万
• 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/6869255
• 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。为了更好地理解决定哺乳动物受体介导功能的过程，我们将利用果蝇作为模式生物。苍蝇适应于一种独特的遗传方法，先进的研究工具（如微阵列，突变蝇系库）很容易获得。值得注意的是，果蝇和哺乳动物调节多巴胺能神经传递的蛋白质之间存在高度的结构和功能守恒。我们的实验室最近通过果蝇多巴胺2样受体（DD2R）的克隆和药理学特征扩展了这些已知的相似之处，DD2R是一种公认的哺乳动物蛋白的同源物，是运动功能的关键调节因子。果蝇和人类D2受体的体外药理学比较显示激动剂（内源性胺和选定的合成配体）诱导的信号传导具有相似的特征。同时，一组抗帕金森病D2R药物优先结合并激活人类（相对于果蝇）受体同源物。在Aim 1中，将产生受体突变体，其中在人类D2R和DD2R之间交换不同的残基。这些结构将用于鉴定赋予临床重要激动剂功能活性的人类氨基酸。在Aim 2中，我们建议定义我们已经确定的多个DD2R剪接变体的组织特异性表达和药理学特征。作为这些研究的基本原理，哺乳动物D2受体异构体在体内具有不同的细胞分布和调节不同的功能。DD2R剪接变异的评估将依赖于多种方法的组合，包括定量PCR来评估相对转录物丰度，用DD2R抗体共聚焦显微镜来评估表达谱，以及基于体外细胞的检测来评估药理学特性。为了研究其在体内的功能，我们培育了DD2R表达降低的转基因RNA干扰（RNAi）果蝇。这些果蝇的特征揭示了运动功能的高度显著下降。在Aim 3中，我们建议利用两种互补的方法来鉴定与多巴胺能信号通路相关的新基因(i) DD2R RNAi果蝇的转录组分析（与对照），以及（ii）筛选现有的插入轴承果蝇系的运动活动/多巴胺能信号通路的改变。一旦确定，这些基因将进行功能评估，以确定它们在多巴胺能信号传导中的潜在作用。