Exploring the roles of Gi and S1Pr1 in endothelial barrier function in vivo

描述（由申请人提供）：该职业发展奖的目标是为Wilsbacher博士培养必要的技能，使其成功成为学术中心的独立内科科学家。Wilsbacher博士在西北大学（Northwestern University）的医学家培训项目（Medical Scientist Training Program）获得了医学博士和博士学位，在那里她研究了驱动昼夜节律的遗传和分子机制。她曾在加州大学旧金山分校接受内科和心脏病学培训。Wilsbacher博士目前的研究目标是研究正常生理和疾病期间血管屏障功能的机制，她的长期职业目标是弥合昼夜生理和内皮功能之间的差距，以帮助推进对心血管生物学和疾病控制机制的理解。培训计划包括由Shaun Coughlin博士指导，他是止血、血栓形成和血管完整性中G蛋白偶联受体（GPCR）信号的世界级研究者，在培养年轻研究者独立方面有着良好的记录。由GPCR信号传导（Mark von Zastrow博士）、炎症（Donald McDonald博士）和蛋白质化学（James Wells博士）领域的领导者科学家组成的专家组将提供额外指导。优秀的研究设施和设备随时可用。培训计划还包括高级教学课程，重点是细胞信号，膜运输，蛋白质相互作用和显微镜；参加系列讨论会；参加地方和国家会议并作发言；并指导指导稿件和拨款准备。治疗或预防炎症需要彻底了解内皮屏障功能的机制。该研究计划旨在揭示调节血管完整性的信号机制，特别是通过鞘氨醇-1-磷酸受体1 （S1Pr1）和Gi信号传导。目的1研究内皮Gi和S1Pr1信号在屏障功能中的作用。具体来说，在内皮中表达百日咳毒素（抑制Gi），在内皮中表达百日咳不敏感的Gi，或在成人内皮中有条件地删除S1Pr1的遗传小鼠模型将被检测血管通透性的变化。Aim 2使用一种新的GPCR激活检测系统来探测体内炎症过程中S1Pr1在何时何地被激活。新的报告系统，转录报告GPCR的激活，是一个创新的工具，将广泛适用于其他GPCR在不同的组织。目的3探讨小泡是否影响S1Pr1- gi偶联，以及S1Pr1在小泡中的定位是否影响2arrestin介导的内化。Wilsbacher博士的职业发展奖提案包括一个有前途的候选人，优秀的培训环境，严格的培训计划，以及令人兴奋的研究提案，其中包括一个创新的新工具，并专注于与人类健康和疾病直接相关的主题。在完成本奖项后，Wilsbacher博士应具备作为独立研究者取得成功所必需的技能。

DESCRIPTION (provided by applicant): The goal of this Career Development Award is for Dr. Wilsbacher to develop the skills necessary to successfully become an independent physician-scientist at an academic center. Dr. Wilsbacher earned her M.D. and Ph.D. degrees in the Medical Scientist Training Program at Northwestern University, where she investigated genetic and molecular mechanisms that drive circadian rhythms. She trained in Internal Medicine and Cardiology at the University of California, San Francisco. Dr. Wilsbacher's current research objective is to investigate mechanisms of vascular barrier function during normal physiology and disease, and her long-term career goal is to bridge the gap between circadian physiology and endothelial function to help advance understanding of mechanisms governing cardiovascular biology and disease. The training plan includes mentorship by Dr. Shaun Coughlin, a world-class investigator of G protein- coupled receptor (GPCR) signaling in hemostasis, thrombosis, and vascular integrity who has an established record of training young investigators to independence. Additional guidance will be provided from an expert group of scientists who are leaders in GPCR signaling (Dr. Mark von Zastrow), inflammation (Dr. Donald McDonald), and protein chemistry (Dr. James Wells). Outstanding research facilities and equipment are readily available. The training plan also incorporates advanced didactic coursework that focuses on cell signaling, membrane trafficking, protein interactions, and microscopy; attendance at seminar series; participation and presentation in local and national conferences; and mentored guidance with manuscript and grant preparation. Treating or preventing inflammation requires thorough understanding of the mechanisms of endothelial barrier function. The research proposal aims to uncover signaling mechanisms that regulate vascular integrity, particularly through sphingosine-1-phosphate receptor 1 (S1Pr1) and Gi signaling. Aim 1 investigates the role of endothelial Gi and S1Pr1 signaling in barrier function. Specifically, genetic mouse models that express pertussis toxin (which inhibits Gi) in endothelium, express a pertussis-insensitive Gi in endothelium, or conditionally delete S1Pr1 in adult endothelium will be tested for changes in vascular permeability. Aim 2 uses a new GPCR activation detection system to probe when and where S1Pr1 is activated in vivo during inflammation. The new reporter system, which transcriptionally reports on GPCR activation, is an innovative tool that will be widely applicable to other GPCRs in diverse tissues. Aim 3 probes whether caveolae influence S1Pr1-Gi coupling, and whether S1Pr1 localization in caveolae affects 2arrestin-mediated internalization. Dr. Wilsbacher's Career Development Award proposal comprises a promising candidate, outstanding training environment, rigorous training plan, and exciting research proposal that includes an innovative new tool and focuses on a topic with direct relevance to human health and disease. At the completion of this Award, Dr. Wilsbacher should have the skills necessary to succeed as an independent investigator. PUBLIC HEALTH RELEVANCE: A diverse range of acute and chronic human diseases involve abnormal endothelial permeability, including asthma, acute lung injury, inflammatory bowel disease, diabetic retinopathy, rheumatic disease, multiple sclerosis, atherosclerosis, and sepsis. Treating or preventing inflammation and its sequelae requires thorough understanding of the mechanisms of endothelial barrier function. The overall goal of this proposal is to uncover signaling mechanisms regulating vascular integrity that might be utilized in the prevention or treatment of these important human conditions.