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性疾病的重要治疗靶点。为了更好地了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型生物。果蝇适合一系列独特的遗传方法，先进的研究工具（例如微阵列、突变果蝇品系库）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保守性。我们的实验室最近将这些已知的相似之处扩展到果蝇多巴胺 2 样受体 (DD2R) 的克隆和药理学表征，DD2R 是一种成熟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 受体之间的体外药理学比较揭示了激动剂（内源胺和选定的合成配体）诱导的信号传导的相似特征。同时，一部分抗帕金森病 D2R 药物优先结合并激活人类（相对于果蝇）受体同系物。在目标 1 中，将产生受体突变体，其中人 D2R 和 DD2R 之间交换不同的残基。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类氨基酸。在目标 2 中，我们建议定义我们已鉴定的多个 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 受体亚型具有不同的细胞分布并在体内调节不同的功能，这一点已得到证实。 DD2R 剪接变体的评估将依赖于多种方法的组合，包括评估相对转录本丰度的定量 PCR、评估表达谱的 DD2R 抗体共聚焦显微镜以及评估药理特性的体外细胞测定。为了检查体内功能，我们生成了 DD2R 表达降低的转基因 RNA 干扰 (RNAi) 果蝇。这些果蝇的特征表明，它们的运动功能显着下降。在目标 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（与对照）果蝇的转录组分析，以及（ii）筛选现有的插入轴承果蝇集合运动活动/多巴胺能信号改变的线路。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传导中的潜在作用。