EGF Receptor Signaling in Time and Space in Tumor Cells

描述（申请人提供）：ErbB家族的受体酪氨酸激酶在各种类型的人类癌症的发生和进展中发挥重要作用。阐明该家族的一个典型成员表皮生长因子（EGF）受体（EGFR/ErbB1）和其他erbb的生理调控，是理解其致癌激活机制的关键。生长因子结合到EGFR导致启动无数的信号转导过程。丝裂原活化蛋白激酶1/2 （MAPK/ERK1/2）的激活是EGFR触发的主要信号通路，它决定了EGFR信号转导的最终结果。最近的研究表明，EGFR信号传导并不局限于细胞表面的信号传导，相反，该途径的所有组分都在细胞内移动，并可能与各种细胞内区室相关并发挥作用。活化的EGFR被迅速内化到核内体中，并被分类到溶酶体中降解。在信号传导和内吞机制之间存在着大量的双向交流。有人提出，内吞作用是MAPK激活的必要条件，尽管这一问题仍存在争议。

DESCRIPTION (provided by applicant): Receptor tyrosine kinases of the ErbB family play important roles in the development and progression of various types of human carcinoma. Elucidation of the physiological regulation of a prototypical member of the family, epidermal growth factor (EGF) receptor (EGFR/ErbB1), and other ErbBs, is the key to understanding the mechanisms causing their oncogenic activation. Growth factor binding to the EGFR results in initiation of a myriad of signal transduction processes. Activation of the mitogen-activated protein kinase 1/2 (MAPK/ERK1/2) is the major signaling pathway triggered by EGFR, which determines the ultimate outcome of the EGFR signaling. Recent studies revealed that EGFR signaling is not limited by signaling at the cell surface, but, rather, all components of the pathway move within the cell and may associate with and function in various intracellular compartments. The activated EGFR is rapidly internalized into endosomes and sorted to lysosomes for degradation. There are numerous bi-directional cross-talks between signaling and endocytic machineries. It has been proposed that endocytosis is necessary for MAPK activation, although this issue remains controversial. The overall goal of this proposal is to reconstitute the signaling process in space and time in cancer cells and to integrate the new experimental data into a computational model of the dynamics of the EGFR-MAPK pathway. Firstly, the components of the MAPK signaling pathway will be tagged with various fluorescent proteins (XFPs) and stably expressed at physiological levels in human carcinoma cells, in which the corresponding endogenous protein had been knocked-down by vector-mediated RNA interference. The patterns of the dynamic localization and trafficking of XFP-tagged proteins involved in ERK1/2 activation will then be analyzed in these new cell lines using quantitative fluorescence microscopy. Secondly, using a combination of RNA interference, live-cell fluorescence imaging, multi-chromophore fluorescence resonance energy transfer microscopy and biochemical techniques, the mechanisms of the formation of the multi-protein complexes during MAPK activation and the localization of these complexes will be investigated. Thirdly, a systematic analysis of the role of endocytosis and endosomes in the regulation of EGFR signaling to MAPK will be performed in a range of human cancer cell lines expressing various levels of EGFR. Using the experimental data describing the spatial and temporal overlap of the components of the EGFR-MAPK pathway, their diffusion rate parameters, routes of trafficking and protein-protein interactions, a new computational model of spatial and temporal organization of EGFR-MAPK signaling will be generated, experimentally validated and used to predict the effects of different EGFR expression levels, concentrations of EGFR ligands and various perturbations of the system on the intensity and duration of MAPK signaling. PUBLIC HEALTH RELEVANCE: The EGF receptor has become the major prognostic and diagnostic marker and an important therapeutic target in human carcinoma. However, the benefits from using the EGF receptor as a marker and a therapeutic target are currently highly limited. The studies proposed here will enhance our understanding of the regulation of EGF receptors in cancer cells and should help in developing new strategies of down regulating the activity of EGF receptors and similar receptors in cancer cells and may also reveal new potential prognostic markers and therapeutic targets.