GPCR-mediated nongenomic steroid actions in control of alcohol-induced behavior

描述(由申请人提供)：类固醇激素对神经系统的发育和功能有重大影响，并在各种神经和神经精神障碍中发挥关键作用。虽然类固醇主要通过与核激素受体结合和激活转录来发挥作用，但越来越多的证据表明，类固醇激素也可以迅速地产生生物学效应，并且不依赖于其核受体。尽管这种“非基因组”类固醇作用具有潜在的重要性，但人们对其功能意义和潜在的分子机制仍知之甚少。为了解决我们知识中的这一差距，我们将使用果蝇特有的遗传工具来研究最近发现的G蛋白偶联受体(G-Protein Couted Receptor，GPCR)--多普ECR。多巴ECR是一种非经典类固醇受体的有力候选者，因为它可以直接与昆虫类固醇激素蜕皮激素和儿茶酚胺多巴胺结合并产生反应。虽然已知多巴受体与脊椎动物肾上腺素能受体具有最高的序列相似性，并且优先在神经系统中表达，但其在完整动物中的功能尚不清楚。本研究的目的是揭示多巴酚丁胺受体在行为调控中的作用并确定其潜在的作用机制。这项研究的中心假设是，多巴酚丁胺受体介导的蜕皮激素信号通路通过调节多巴胺、cAMP和MAPK通路的作用，在控制乙醇刺激和抑制的平衡中发挥作用。这一假说是基于我最近的发现，即新发现的多巴受体突变体在乙醇诱导的行为中表现出有趣的表型。本项目将追求的两个具体目标是：1)确定多巴ECR的内源性表达模式和功能作用部位，2)确定多巴ECR调节乙醇行为反应的机制。对于目标1，将以神经元亚群特异性的方式跟踪多巴受体野生型cDNA或RNAi的表达后进行行为分析。对于目标2，将检查多巴受体的遗传变异与多巴胺、cAMP和MAPK通路的相互作用。这些目标的成功完成有望揭示GPCR介导的非基因组类固醇信号如何控制果蝇的乙醇诱导行为。这种应用的结果将产生重要的积极影响，因为它们有望为类固醇激素对行为调节的非基因组效应提供强有力的基于体内机制的证据。由于类固醇信号和酒精反应行为的高度功能性和机械性保守，了解这些影响将为未来酒精使用障碍的预防和治疗提供新的见解。

DESCRIPTION (provided by applicant): Steroid hormones have significant impact on the development and function of the nervous system and play crucial roles in various neurological and neuropsychiatric disorders. While steroids primarily act through the "genomic" mechanism by binding nuclear hormone receptors and activating transcription, accumulated evidence indicates that steroid hormones also induce biological effects rapidly and independent of their nuclear receptors. Despite the potential importance of such "nongenomic" steroid actions, their functional significance and underlying molecular mechanisms are still poorly understood. To address this gap in our knowledge, DopEcR, a recently discovered G-protein coupled receptor (GPCR), will be studied using genetic tools uniquely available in the fruit fly in Drosophila melanogaster. DopEcR is a strong candidate for a nonclassical steroid receptor, because this GPCR can directly bind and respond to the insect steroid hormone ecdysone as well as the catecholamine dopamine. Although DopEcR is known to have the highest sequence similarity with vertebrate ¿-adrenergic receptors and is preferentially expressed in the nervous system, its function in intact animals is not known. The objective of this proposed research is to reveal the roles of DopEcR in behavioral regulation and identify its underlying mechanism of actions. The central hypothesis of this proposed research is that DopEcR-mediated ecdysone signaling plays a role in controlling balance of excitation and inhibition upon exposure to ethanol by modulating the effects of dopamine, cAMP and MAPK pathways. This hypothesis is based on my recent discovery that newly identified DopEcR mutants display intriguing phenotypes in ethanol-induced behaviors. The two specific aims that will be pursued in this project are: 1) Determine the endogenous expression patterns and functional sites of action of DopEcR, and 2) Determine the mechanisms by which DopEcR regulates behavioral responses to ethanol. For Aim 1, behavioral analyses will be carried out following the expression of wild type cDNA or RNAi for DopEcR in a neuronal subset-specific manner. For Aim 2, genetic variants of DopEcR will be examined for their interactions with dopamine, cAMP and MAPK pathways. Successful completion of these aims is expected to reveal how GPCR-mediated nongenomic steroid signaling controls ethanol-induced behavior in Drosophila. The outcomes of this application will have an important positive impact because they are expected to provide strong mechanism-based in vivo evidence for the nongenomic effects of steroid hormones on behavioral regulation. Due to the high functional and mechanistic conservation of steroid signaling and alcohol response behaviors, understanding these effects should provide novel insights into the future prevention and treatment of alcohol use disorders.