A novel GPCR-mediated steroid signaling that controls alcohol-induced behavior

描述(由申请人提供)：类固醇激素在各种神经和精神疾病中起着关键作用。除了涉及核类固醇激素受体的“基因组”机制外，类固醇的生物效应也是由“非基因组”机制介导的，这些机制发生迅速且独立于新的mRNA合成。这种非常规类固醇信号的功能和分子机制，特别是在神经系统中的作用和分子机制，仍然知之甚少。知识基础中的这一差距代表了对与类固醇激素有关的神经系统疾病的以机制为基础的理解的重要的未得到满足的需求。长期目标是了解类固醇激素如何调节神经系统功能，从而控制行为。这一特殊应用的目的是阐明非基因组类固醇信号控制果蝇乙醇诱导行为的基本机制。申请人实验室最近的遗传学研究表明，多普ECR，一种昆虫类固醇激素蜕皮激素的G蛋白偶联受体(GPCR)，在对乙醇的行为反应中发挥着重要作用。这一新的发现提供了一个前所未有的机会，通过利用果蝇的遗传学来研究一种新的GPCR介导的类固醇信号的基础，这种信号对酒精诱导的行为很重要。核心假设是，多巴受体是一种独特的蜕皮激素和多巴胺的双重受体，它通过负向调节表皮生长因子受体(EGFR)信号来调节对乙醇镇静作用的抵抗。为了验证这一假说，并达到这一应用的目的，我们将追求以下两个具体目标：1)确定多巴ECR的表达模式和作用部位；2)确定参与多巴受体介导的信号转导的分子成分。在第一个目标下，将使用免疫学和遗传学方法来确定多巴ECR蛋白在成人脑中的解剖定位，而营救性和表型实验将用于确定参与多巴ECR介导的类固醇作用的脑神经元。在第二个目标下，将使用药理学和遗传学方法研究两种配体(蜕皮激素和多巴胺)和潜在的下游信号(EGFR信号)在多巴-ECR作用中的作用。遗传方法是创新的，因为它不仅利用零等位基因，而且利用果蝇特有的各种遗传工具来研究非基因组类固醇信号的机制。预期的结果将为理解在酒精诱导行为调控中发挥关键作用的新的非基因组类固醇信号奠定必要的基础。这项研究意义重大，因为它有望为神经系统中一种新的类固醇信号提供强有力的基于体内机制的证据，最终可能有助于开发预防和治疗酒精使用障碍以及其他受类固醇激素影响的常见疾病的创新策略。

DESCRIPTION (provided by applicant): Steroid hormones play a critical role in various neurological and psychiatric conditions. In addition to the "genomic" mechanisms involving nuclear steroid hormone receptors, biological effects of steroids are also mediated by "nongenomic" mechanisms that occur rapidly and independently of new mRNA synthesis. The functions and molecular mechanisms for such unconventional steroid signaling, particularly in the nervous system, are still poorly understood. This gap in the knowledge base represents an important unmet need for a mechanism-based understanding of nervous system disorders related to steroid hormones. The long-term goal is to understand how steroid hormones modulate nervous system functions and thereby control behavior. The objective of this particular application is to elucidate the fundamental mechanisms for nongenomic steroid signaling that controls ethanol-induced behaviors using the fruit fly Drosophila. Recent genetic studies in the applicant's lab demonstrate that DopEcR, a G-protein coupled receptor (GPCR) for the insect steroid hormone ecdysone, plays a significant role in behavioral responses to ethanol. This new finding provides an unprecedented opportunity to investigate the underpinnings for a novel GPCR-mediated steroid signaling that is important for alcohol-induced behavior, by taking advantage of Drosophila genetics. The central hypothesis is that DopEcR is a unique dual receptor for ecdysone and dopamine, and that it modulates resistance to the sedative effects of ethanol by negatively regulating epidermal growth factor receptor (EGFR) signaling. To test this hypothesis and attain the objective of this application, we will pursue the following two specific aims: 1) Determine the expression pattern of DopEcR and sites of its actions; and 2) Identify molecular components involved in DopEcR-mediated signaling. Under the first aim, immunological and genetic methods will be used to determine the anatomical localization of DopEcR protein in the adult brain, whereas rescue and phenocopy experiments will be performed to identify brain neurons involved in DopEcR-mediated steroid actions. Under the second aim, roles of two ligands (ecdysone and dopamine) and potential downstream signaling (EGFR signaling) in DopEcR actions will be investigated using pharmacological and genetic methods. The genetic approach is innovative, because it utilizes not only a null allele, but also various genetic tools uniquely available in Drosophila to study the mechanisms responsible for nongenomic steroid signaling. The expected outcomes will form an essential foundation for understanding the novel nongenomic steroid signaling that plays critical roles in regulation of alcohol-induced behavior. This study is significant, because it is expected to provide strong mechanism-based in vivo evidence for a novel steroid signaling in the nervous system, which may ultimately contribute toward the development of innovative strategies for the prevention and treatment of alcohol use disorders as well as other common diseases that are affected by steroid hormones.