Molecular Analysis of 5HT Receptor Mediated Signaling

描述（由申请人提供）： 在哺乳动物中，血清素 (5HT) 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血清素信号的遗传修饰剂将是一个重要的进步。众所周知，哺乳动物和果蝇蛋白质之间存在显着的结构相似性，这些蛋白质构成血清素能途径（即 5HT 合成酶、转运蛋白和受体）。与哺乳动物相比，果蝇中 5HT 受体介导的功能尚不明确。我们最近证明 5HT 可以调节果蝇的食物摄入量，从而在果蝇和哺乳动物之间建立了功能上的相似之处。我们已经证明，给予 5HT 受体激动剂和拮抗剂分别显着减少和增加食物摄入量。 作为最初的目标，我们将确定果蝇 5HT 受体亚型中的哪一种调节食物摄入。将建立基于体外细胞的测定法来筛选 5HT 配体，以确定化合物对四种果蝇 5HT 受体中每一种的亚型特异性。这些配体随后将用于幼虫食物摄入范例。体外药理学特异性将与体内受体介导的效应相关，以确定哪种 5HT 受体亚型是进食行为的血清素抑制的基础。下一步，将评估相关受体基因下调的果蝇（即 RNAi 果蝇）的食物摄入量变化。最后，将对这些果蝇进行基因表达谱分析，以确定血清素信号传导的新型遗传修饰剂。在该遗传模型中转录发生改变的基因将优先使用体外和体内测定进行表征。

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.