Molecular Analysis of Dopamine 2 Like Receptor Function

多巴胺 2 样受体功能的分子分析

基本信息

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
项目类别：
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

项目摘要

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，因此是包括帕金森氏病在内的神经病理疾病的重要治疗靶标。为了更好地了解确定哺乳动物受体介导的功能的过程，我们将利用果蝇作为模型生物。苍蝇可以适应一种独特的遗传方法，该方法很容易获得高级研究工具（例如微阵列，突变蝇线的存储库）。值得注意的是，果蝇和哺乳动物蛋白之间具有高度的结构和功能保护，可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，其克隆和药理表征是果蝇多巴胺2（例如受体（DD2R）），这是一种已建立的哺乳动物蛋白质的同源物，这是运动机能功能的关键调节剂。苍蝇和人D2受体之间的体外药理比较揭示了激动剂（内源性胺和选定的合成配体）诱导的信号传导相似的特征。同时，一部分抗Parkinsonian D2R药物优先结合并激活人（与苍蝇）受体同源物。在AIM 1中，将产生受体突变体，其中在人D2R和DD2R之间交换了不同的残基。这些构建体将用于鉴定赋予临床重要激动剂功能活性的人氨基酸。在AIM 2中，我们建议定义我们已经鉴定出的多个DD2R剪接变体的组织特异性表达和药理学谱。作为这些研究的基本原理，已经很好地确定哺乳动物D2受体同工型具有不同的细胞分布，并在体内调节了不同的功能。 DD2R剪接变体的评估将依赖于包括定量PCR在内的方法的组合，以评估相对转录的丰度，与DD2R抗体的共聚焦显微镜评估表达谱以及基于体外细胞的测定法以评估药理特性。为了检查体内功能，我们已经产生了跨齿RNA干扰（RNAi）苍蝇，DD2R的表达降低。这些苍蝇的表征表明运动功能的降低极为显着。在AIM 3中，我们建议鉴定与多巴胺能信号通路相关的新型基因，利用两种互补方法（i）对DD2R RNAi（与对照）蝇的转录组分析，以及（ii）筛选现有的插入插入系列，以在插入式轴承线上收集，以改变运动型蝇类活性/多波巴尼信号的改变。一旦确定，这些基因将在功能上进行评估，以确定它们在多巴胺能信号传导中的潜在作用。