Administrative Supplement-Signaling by the EGF Receptor from Endosomes

行政补充-内体 EGF 受体发出的信号

基本信息

批准号：
10381939
负责人：
ALEXANDER D SORKIN
金额：
$ 13.77万
依托单位：
UNIVERSITY OF PITTSBURGH AT PITTSBURGH
依托单位国家：
美国
项目类别：
财政年份：
2017
资助国家：
美国
起止时间：
2017-09-15 至 2022-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10381939
关键词：
Abnormal Cell Address Administrative Supplement Adult Cell Adhesion Cell Nucleus Cell surface Cells Development Endocytosis Endosomes Ensure Epidermal Growth Factor Receptor Experimental Models Fluorescence Microscopy Gene Proteins Grant Growth Homeostasis Human Label Malignant Neoplasms Mass Spectrum Analysis Methodology Methods Outcome Pathogenesis Pathway interactions Phosphorylation Physiological Play Process Proteins Receptor Protein-Tyrosine Kinases Receptor Signaling Regulation Research Resolution Role Shapes Signal Pathway Signal Transduction Stimulus System Systems Biology Testing Time Tissues cell growth cell motility confocal imaging functional outcomes imaging system metastatic process microscopic imaging optical imaging phosphoproteomics programs receptor receptor internalization spatiotemporal time use tissue regeneration trafficking tumorigenesis wound healing

项目摘要

Abstract of Parental Grant GM124186 Elucidation of the mechanisms by which endocytic trafficking regulates signal transduction processes remains to be profoundly important for understanding how diverse stimuli propagate signals from the cell surface through the conserved cytosolic machinery and to the nucleus leading to a stimulus- and context-specific signaling outcome. Epidermal growth factor (EGF) receptor (EGFR), a prototypic receptor tyrosine kinase, has been the major experimental model to study spatiotemporal regulation of signaling networks. EGFR plays an essential role in mammalian development and tissue homeostasis in the adult, and is involved in human pathogenesis, in particular, cancer. However, while the main constituents of the EGFR signaling network are known, how they coordinately function during EGFR endocytosis and subsequent targeting of receptors for degradation to endosomes to ensure proper intensity and duration of signaling processes is for the large part unknown. Addressing this fundamental question has now become possible owing to the availability of new cutting- edge methodologies. Using time-resolved quantitative mass-spectrometry of cellular phosphoproteomes we found that after the majority of active EGFRs are internalized into endosomes, phosphorylation of a multitude of proteins, known to be involved in the regulation of signaling to growth, survival, cell motility and adhesion, is maintained by EGFR. Therefore, we hypothesize that the pathways involving these putative signaling effectors of EGFR operate in endosomes through the sustained activity of endosomal EGFR. We further hypothesize that by maintaining the activity along some signaling pathways while down-regulating other pathways, EGFR endocytosis shapes the overall functional outcome of EGFR signaling. To test these hypothesis in the physiological experimental system (cells that are growth-dependent on EGFR) we will: 1) examine whether putative substrates and signaling effectors of endocytosed EGFR identified by the phosphoproteomic analysis are located in EGFR-containing endosomes by labeling endogenous effectors with fluorescent proteins by gene-editing and dissecting their time-dependent localization dynamics using multi- dimensional fluorescence microscopy imaging; and 2) examine whether endocytosis and localization in endosomes control downstream signaling activity of putative phosphorylation substrates and effectors of endosomal EGFR, and define the mechanisms of this regulation. Combination of the high-throughput method of labeling endogenous proteins by gene-editing with various methods of quantitative live-cell optical imaging at all levels of resolution will allow us to apply a systems biology approach to untangling the entire endosomal signaling program.

父母摘要GM124186 阐明内吞运输调节信号转导过程的机制对于理解各种刺激如何从细胞表面传播信号仍然非常重要通过保守的胞质机械和核导致刺激和上下文特异性的细胞核信号结果。表皮生长因子（EGF）受体（EGFR）是一种原型受体酪氨酸激酶，具有是研究信号网络时空调节的主要实验模型。 EGFR扮演在成年人中的哺乳动物发育和组织稳态中的重要作用，并参与人类发病机理，尤其是癌症。但是，尽管EGFR信号网络的主要成分是已知，它们如何在EGFR内吞作用和随后的受体靶向时如何协同起作用降解向内体降解以确保信号传导过程的适当强度和持续时间是很大的部分未知。由于新替代的可用性，解决这个基本问题现已成为可能边缘方法。使用细胞磷酸蛋白质组的时间分辨定量质谱法我们发现在大多数活性EGFR被内部化为内体之后，众多磷酸化蛋白质，已知与信号调节对生长，生存，细胞运动和粘附的调节，由EGFR维护。因此，我们假设涉及这些推定信号效应子的途径 EGFR通过内体EGFR的持续活性在内体中运行。我们进一步假设通过在下调其他途径的同时保持某些信号通路的活动，EGFR 内吞作用塑造了EGFR信号传导的总体功能结果。在生长实验系统中检验这些假设（生长依赖于 egfr）我们将：1）检查鉴定的内吞EGFR的假定底物和信号传导效应子是否已确定通过磷酸化蛋白质组学分析，通过标记内源性效应子来定位在含EGFR的内体中通过基因编辑并解剖其时间依赖性定位动力学，并使用多种多样的蛋白质进行荧光蛋白尺寸荧光显微镜成像； 2）检查内吞和定位是否在内体控制推定磷酸化底物和效应子的下游信号传导活性内体EGFR，并定义该调节的机制。高通量方法的组合通过基因编辑各种定量活细胞光学成像的基因编辑来标记内源性蛋白分辨率水平将使我们能够采用系统生物学方法来解开整个内体信号传导程序。