Hrs and Endocytic Regulation of Notch and Wingless

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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.