Molecular Analysis of 5HT Receptor Mediated Signaling

描述(由申请人提供)： 在哺乳动物中，5-羟色胺(5-羟色胺)受体调节多种生理功能，包括渴望、成瘾和进食行为。鉴于这些受体的重要作用，建立一个简化的模型系统来识别5-羟色胺能信号的遗传修饰物将是一个重要的进步。已知哺乳动物和果蝇中组成5-羟色胺能途径(即5-羟色胺合成酶、转运体和受体)的蛋白质在结构上存在显著的相似性。与哺乳动物不同，5-羟色胺受体在果蝇中的作用尚未明确。我们最近证明了5-羟色胺调节果蝇的食物摄取，从而在果蝇和哺乳动物之间建立了功能上的平行。我们已经证明，给予5-羟色胺受体激动剂和拮抗剂分别显著减少和增加食物摄入量。 作为最初的目标，我们将确定果蝇的哪一种5-羟色胺受体亚型(S)调节食物摄入量。在体外，将建立基于细胞的分析来筛选5-羟色胺配体，以确定四种苍蝇5-羟色胺受体上每一种化合物的亚型特异性。然后，这些配体将被用于幼虫摄食范例。体外药理特异性将与体内受体介导的效应相关联，以确定哪一种5-羟色胺受体亚型(S)是5-羟色胺能抑制摄食行为的基础。下一步，将对相关受体基因下调的苍蝇(即RNAi苍蝇)进行食物摄入量变化的评估。最后，将对这些果蝇进行基因表达谱分析，以确定5-羟色胺能信号的新遗传修饰物。在这个遗传模型中，转录发生变化的基因将优先用于体外和体内分析。

DESCRIPTION (provided by applicant): In mammals, serotonin (5HT) receptors modulate a variety of physiologic functions including craving, addiction, and feeding behavior. Given the important role of these receptors, the establishment of a simplified model system in which to identify genetic modifiers of serotonergic signaling would be an important advance. It is known that significant structural similarities exist between the mammalian and Drosophila proteins that comprise the serotonergic pathway (i.e. 5HT synthetic enzymes, transporters, and receptors). In contrast to mammals, 5HT receptor mediated function in the fly is not well defined. We have recently demonstrated that 5HT modulates food intake in flies thus establishing a functional parallel between Drosophila and mammals. We have shown that the administration of 5HT receptor agonists and antagonists significantly decreases and increases food intake, respectively. As an initial goal, we will determine which of the Drosophila 5HT receptor subtype(s) regulates food intake. In vitro cell based assays will be established to screen 5HT ligands to determine the subtype specificity of the compounds at each of the four fly 5HT receptors. These ligands will then be utilized in a larval food intake paradigm. The in vitro pharmacologic specificity will be correlated with in vivo receptor mediated effects to define which of the 5HT receptor subtype(s) underlies the serotonergic inhibition of feeding behavior. As a next step, flies in which the relevant receptor genetically down-regulated (i.e. RNAi flies) will be assessed for changes in food intake. Finally, these flies will be analyzed with gene expression profiling to identify novel genetic modifiers of serotonergic signaling. Genes with altered transcription in this genetic model will be prioritized for characterization using both in vitro and in vivo assays.