Molecular Analysis of Dopamine 2 Like Receptor Function

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学病症的重要治疗靶点。为了更好地理解哺乳动物中决定受体介导功能的过程，我们将利用果蝇作为模式生物。苍蝇服从于一系列独特的遗传方法，先进的研究工具（如微阵列，突变苍蝇线库）是现成的。值得注意的是，在调节多巴胺能神经传递的果蝇和哺乳动物蛋白质之间存在高度的结构和功能保守性。我们的实验室最近扩展了这些已知的相似之处与果蝇多巴胺2样受体（DD 2 R）的克隆和药理学表征，该受体是一种成熟的哺乳动物蛋白质的同系物，是运动功能的关键调节因子。果蝇和人D2受体之间的体外药理学比较揭示了激动剂（内源性胺和选择的合成配体）诱导的信号传导的类似概况。同时，抗帕金森病D2 R药物的一个子集优先结合并激活人类（与苍蝇相比）受体同系物。在目的1中，将产生受体突变体，其中在人D2 R和DD 2 R之间交换不同的残基。这些构建体将用于鉴定赋予临床上重要的激动剂功能活性的人氨基酸。在目标2中，我们建议定义我们已经确定的多种DD 2 R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，已充分确定哺乳动物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。DD 2 R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、使用DD 2 R抗体进行共聚焦显微镜检查以评估表达谱以及评估药理学特性的体外细胞分析。为了检查体内功能，我们产生了DD 2 R表达减少的转基因RNA干扰（RNAi）果蝇。对这些果蝇的表征揭示了运动功能的显著下降。在目标3中，我们提出鉴定与多巴胺能信号传导途径相关的新基因，利用两种互补的方法（i）DD 2 R RNAi（与对照）果蝇的转录组分析，和（ii）筛选携带果蝇品系的插入物的现有集合以改变运动活性/多巴胺能信号传导。一旦确定，这些基因将进行功能评估，以确定其在多巴胺能信号传导中的潜在作用。

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.