Hrs and Endocytic Regulation of Notch and Wingless

本项目的重点是研究内吞作用在发育信号通路调控中的作用。我们将专注于高度保守的Notch和Wingless信号通路。这些通路在发育和成年期调节细胞数量和细胞命运方面起着关键作用。考虑到这些重要功能，这些通路的失调与包括癌症在内的许多人类疾病有关也就不足为奇了。我们感兴趣的是这些信号通路是如何被如此精确地调节的。我们的初步数据集中于Hrs，一种介导内体分选和多泡体形成的内吞蛋白。我们已经发现，改变的Hrs功能导致Notch和Wingless信号传导成员的错误定位，并导致异常的Notch和Wingless信号传导。这些发现证明了蛋白质细胞内运输和信号调节之间的关键联系。在本提案中，我们计划研究这种内吞调节发生的机制。首先，将进行详细的结构-功能分析的Hrs，包括Hrs定位和蛋白质协会的功能意义的研究。这些研究不仅将为Notch和Wingless信号的调节提供深入了解，而且还将为多泡体形成的机制提供深入了解。对于Notch通路，我们将通过共聚焦和电子显微镜对蛋白质细胞内定位进行详细分析。目前对Notch信号蛋白的细胞内运输知之甚少。我们的研究将扩展目前已知的内容，并研究蛋白质定位对信号传导的影响。已知无翅蛋白形成形态发生梯度，其根据形态发生浓度诱导不同的发育命运。我们将通过研究对Wingless转录、分泌、传播和降解的影响来表征内吞作用在形成形态梯度和调节Wingless信号传导中所起的作用。这些发现不仅将阐明无翼信号的调节，而且还将阐明长期以来一直存在争议的问题-无翼梯度是如何形成的。总之，这项工作将有助于我们确定关键信号蛋白的细胞内运输以及这些运输事件在调节信号中发挥的作用。由于Notch和Wingless通路的失调与人类疾病有关，我们预计从这项研究中获得的见解将有助于更好地理解这些疾病的分子发病机制。

The focus of this project is to study the role that endocytosis plays in regulating of developmental signaling pathways. We will be focusing on the highly conserved Notch and Wingless signaling pathways. These pathways play critical roles in regulating cell number and cell fate specification during development and adulthood. Given these important functions, it is not surprising that misregulation of these pathways has been implicated in numerous human diseases including cancer. We are interested in how these signaling pathways are so precisely regulated. Our preliminary data focuses on Hrs, an endocytic protein that mediates endosomal sorting and multivesicular body formation. We have found that altered Hrs function results in mislocalization of Notch and Wingless signaling members and results in aberrant Notch and Wingless signaling. These findings demonstrate the critical link between protein intracellular trafficking and signaling regulation. In this proposal, we plan to investigate the mechanism through which this endocytic regulation occurs. First, a detailed structure-function analysis of Hrs will be performed, including a study of the functional implications of Hrs localization and protein associations. These studies will provide insight not only into the regulation of Notch and Wingless signaling, but also the mechanism of multivesicular body formation. For the Notch pathway, we will then perform detailed analyses of protein intracellular localization by confocal and electron microscopy. Very little is currently known about the intracellular trafficking of Notch signaling proteins. Our studies will expand on what is current known and investigate the implications of protein localization on signaling. The Wingless protein is known to form morphogen gradients that induce different developmental fates depending on morphogen concentration. We will characterize the role that endocytosis plays in forming the morphogen gradient and regulating Wingless signaling by investigating the effects on Wingless transcription, secretion, spread, and degradation. These findings will elucidate not only the regulation of Wingless signaling, but also what has long been an issue of controversy - how the Wingless gradient forms. Taken together, this work will help us determine the intracellular trafficking of key signaling protein and the effect that these trafficking events play in regulating signaling. Since misregulation of the Notch and Wingless pathways is implicated in human disease, we anticipate that insights gained from this study will aid in better understanding the molecular pathogenesis of these diseases.