Roles for Cbl and ESCRT Proteins in 5-HT Receptor Function

描述(由申请人提供)： 本研究的目的是揭示5-羟色胺(5-HT)受体调节的新分子机制，5-羟色胺(5-HT)受体是一种将细胞外信号转化为细胞内信号的细胞表面蛋白。5-羟色胺受体是典型的G蛋白偶联受体，是人类基因组的重要组成部分。它们几乎参与了生理学的方方面面，也参与了许多疾病状态。然而，我们对这些受体如何传递信号的理解仍然不完整。特别是，它们在细胞内运输的许多方面，以及决定GPCRs命运的特定蛋白质，仍然是个谜。这将使用以下方法来完成：共聚焦显微镜、亚细胞分离、生化方法、融合蛋白和对Hek293细胞中表达的基因的转基因或沉默。这项工作所基于的假设是，已知的参与生长因子受体降解的两个关键蛋白质家族-Cb1和HRS-是5-HT2A受体循环的关键介质。第一个目标是c-Cbl，第二个目标是HRS。这项工作意义重大，因为我们的初步数据表明，c-Cbl和HRS在5-HT2A受体的循环(而不是降解)过程中发挥了意想不到的作用。这项工作具有临床意义，因为5-羟色胺调节情绪、攻击性、焦虑、性行为、睡眠-觉醒、血管紧张性、免疫功能、细胞生长和增殖以及液体和电解质的动态平衡。这项工作与退伍军人群体特别相关，因为这些障碍经常出现在退伍军人管理局的临床环境中。重要的是，随着我们的退伍军人人口继续老龄化，其中许多疾病的发病率和严重性将会恶化。这项提议的主题也很重要，因为了解GPCR信号转导的基本重要性，因为它与人类细胞的所有功能有关。GPCRs是已知的最大的整合膜受体蛋白家族。GPCRs是治疗退伍军人许多疾病的关键治疗靶点，包括高血压、动脉粥样硬化、中风、心力衰竭、癌症、哮喘、肾脏疾病、糖尿病、创伤性脑损伤、精神疾病、神经退行性疾病和包括艾滋病在内的免疫疾病。最终，这项申请中提出的基础研究类型可能会导致新的治疗见解和策略，这些将形成对多种疾病状态的临床干预的基础。 公共卫生相关性： 研究与退伍军人管理局患者护理任务的相关性本提案所涵盖的工作与退伍军人管理局患者护理任务在两个方面相关。首先，它将增加我们关于5-羟色胺如何调节情绪、攻击性、焦虑、性行为、睡眠-觉醒、血管紧张性、免疫功能、细胞生长和增殖以及液体和电解质动态平衡的知识。这项工作与退伍军人群体特别相关，因为在退伍军人管理局的临床环境中，焦虑、睡眠障碍、抑郁、血管性头痛、动脉粥样硬化-血栓形成血管疾病和高血压经常出现。重要的是，随着我们的退伍军人人口继续老龄化，其中许多疾病的发病率和严重性将会恶化。这项提议主题重要的第二个原因涉及理解GPCR信号转导的基本重要性，因为它与人类细胞的所有功能有关。GPCRs是已知的最大的整合膜受体蛋白家族。最近的估计表明，GPCR代表了H1000个成员，GPCRs家族占人类基因组的1%。GPCRs对各种各样的刺激做出反应，包括激素、神经递质、光、气味、味道和多肽。因此，它们在所有器官系统和荷尔蒙功能的调节中起着关键作用。GPCRs在人类生理和疾病中的核心重要性因以下事实而突显出来：目前开出的所有药物中有一半以上是针对GPCRs的。GPCRs是治疗退伍军人许多疾病的关键治疗靶点，包括高血压、动脉粥样硬化、中风、心力衰竭、癌症、哮喘、肾脏疾病、糖尿病、精神疾病、神经退行性疾病和包括艾滋病在内的免疫疾病。最终，这项申请中提出的基础研究类型可能会导致新的治疗见解和策略，这些将形成对多种疾病状态的临床干预的基础。

DESCRIPTION (provided by applicant): Project Summary/Abstract The objective of this proposal is to unravel novel molecular mechanisms of regulation of serotonin (5-HT) receptors, which are cell surface proteins that transduce extracellular signals into intracellular signals. 5-HT receptors are prototypical G protein-coupled receptors (GPCRs), constitute a significant portion of the human genome. They participate in nearly every aspect of physiology and in many disease states. Nevertheless, our understanding of how these receptors transfer signals remains incomplete. In particular, many aspects of their intracellular trafficking, and the specific proteins involved in determining the fate of GPCRs, remain mysterious. This will be accomplished using the following methodologies: confocal microscopy, subcellular fractionation, biochemical methods, fusion proteins and transfections into -- or silencing of genes expressed in -- Hek293 cells. The hypothesis upon which this work is based is that two key protein families known to be involved in degradation of growth factor receptors - Cbl and HRS - are critical mediators of recycling of 5-HT2A receptors. The first aim focuses on c-Cbl, and the second on HRS. The work is significant in that our preliminary data demonstrate unexpected roles for c-Cbl and HRS in recycling (rather than degradation) of 5-HT2A receptors. The work is clinically relevant in that 5-HT regulates mood, aggression, anxiety, sexual behavior, sleep-wakefulness, vascular tone, immune function, cell growth and proliferation, and fluid and electrolyte homeostasis. This work is particularly germane to the Veteran population in that those disorders are frequently encountered in VA clinical settings. Importantly, the incidence and severity of many of those maladies will worsen as our Veteran population continues to age. The topic of this proposal also is significant because of the fundamental importance of understanding GPCR signal transduction as it relates to all functions of human cells. GPCRs comprise the largest known family of integral membrane receptor proteins. GPCRs represent key therapeutic targets for the treatment of many diseases of Veterans including hypertension, atherosclerosis, stroke, heart failure, cancer, asthma, renal diseases, diabetes mellitus, traumatic brain injury, psychiatric diseases, neurodegenerative diseases, and immune disorders including AIDS. Ultimately, the type of fundamental research proposed in this application will likely lead to new therapeutic insights and strategies that will form the basis for clinical interventions in multiple disease states. PUBLIC HEALTH RELEVANCE: Relevance of the Research to the VA Patient Care Mission The work covered in this proposal is relevant to the VA patient care mission in two respects. First, it will add to our knowledge about how 5-HT regulates mood, aggression, anxiety, sexual behavior, sleep-wakefulness, vascular tone, immune function, cell growth and proliferation, and fluid and electrolyte homeostasis. This work is particularly germane to the Veteran population in that anxiety, sleep disorders, depression, vascular headache, atherosclerotic-thrombotic vascular diseases and hypertension are frequently encountered in VA clinical settings. Importantly, the incidence and severity of many of those maladies will worsen as our Veteran population continues to age. The second reason why the topic of this proposal is significant involves the fundamental importance of understanding GPCR signal transduction as it relates to all functions of human cells. GPCRs comprise the largest known family of integral membrane receptor proteins. Recent estimates suggest that the GPCRs represent H1,000 members, and the GPCR family makes up > 1% of the human genome. GPCRs respond to a wide variety of stimuli including hormones, neurotransmitters, light, odor, taste, and peptides. Consequently, they play critical roles in the regulation of all organ systems and hormonal functions. The central importance of GPCRs in human physiology and diseases is highlighted by the fact that more than half of all medications prescribed today are targeted at GPCRs. GPCRs represent key therapeutic targets for the treatment of many diseases of Veterans including hypertension, atherosclerosis, stroke, heart failure, cancer, asthma, renal diseases, diabetes mellitus, psychiatric diseases, neurodegenerative diseases, and immune disorders including AIDS. Ultimately, the type of fundamental research proposed in this application will likely lead to new therapeutic insights and strategies that will form the basis for clinical interventions in multiple disease states.