Signaling by the EGF Receptor from Endosomes

来自内体的 EGF 受体发出信号

基本信息

批准号：
10004683
负责人：
ALEXANDER D SORKIN
金额：
$ 34.43万
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/10004683
关键词：
Abnormal Cell Acute Address Adhesions Adult Affect Automobile Driving Catalogs Cell Adhesion Cell Line Cell Nucleus Cell division Cell membrane Cell surface Cells Cessation of life Chemicals Development Endocytosis Endosomes Engineering Ensure Epidermal Growth Factor Epidermal Growth Factor Receptor Experimental Models Family Fluorescence Microscopy Gene Proteins Genes Genetic Transcription Goals Growth Homeostasis Human Label Laboratories Ligand Binding Ligands MAPK3 gene Malignant Neoplasms Mass Spectrum Analysis Mediating Metabolic Methodology Methods Molecular Outcome Pathogenesis Pathway interactions Pattern Phosphorylation Physiological Play Process Prognostic Marker Protein Tyrosine Kinase Proteins RAS genes RNA Interference Receptor Activation Receptor Protein-Tyrosine Kinases Receptor Signaling Regulation Research Resolution Role Shapes Signal Pathway Signal Transduction Signaling Protein Stimulus System Systems Biology Testing Time Tissues Work cancer type carcinogenesis cell growth cell motility functional outcomes imaging modality inhibitor/antagonist knock-down member metastatic process microscopic imaging optical imaging phosphoproteomics programs receptor receptor internalization spatiotemporal therapeutic target time use tissue regeneration trafficking wound healing

Abnormal Cell Acute Address Adhesions Adult Affect Automobile Driving Catalogs Cell Adhesion Cell Line Cell Nucleus Cell division Cell membrane Cell surface Cells Cessation of life Chemicals Development Endocytosis Endosomes Engineering Ensure Epidermal Growth Factor Epidermal Growth Factor Receptor Experimental Models Family Fluorescence Microscopy Gene Proteins Genes Genetic Transcription Goals Growth Homeostasis Human Label Laboratories Ligand Binding Ligands MAPK3 gene Malignant Neoplasms Mass Spectrum Analysis Mediating Metabolic Methodology Methods Molecular Outcome Pathogenesis Pathway interactions Pattern Phosphorylation Physiological Play Process Prognostic Marker Protein Tyrosine Kinase Proteins RAS genes RNA Interference Receptor Activation Receptor Protein-Tyrosine Kinases Receptor Signaling Regulation Research Resolution Role Shapes Signal Pathway Signal Transduction Signaling Protein Stimulus System Systems Biology Testing Time Tissues Work cancer type carcinogenesis cell growth cell motility functional outcomes imaging modality inhibitor/antagonist knock-down member metastatic process microscopic imaging optical imaging phosphoproteomics programs receptor receptor internalization spatiotemporal therapeutic target time use tissue regeneration trafficking wound healing

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项目摘要

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.

阐明内吞运输调节信号转导过程的机制要了解多种刺激如何通过细胞表面传播信号非常重要保守的胞质机械以及导致刺激和上下文特异性信号的细胞核结果。表皮生长因子（EGF）受体（EGFR）是一种原型受体酪氨酸激酶，一直是研究信号网络时空调节的主要实验模型。 EGFR扮演着必不可少的在成年人中的哺乳动物发育和组织稳态中的作用，并参与人类发病机理特别是癌症。但是，尽管已知EGFR信号网络的主要成分，但它们如何在EGFR内吞作用和随后靶向受体以降解为降解的过程中的协同功能内体确保信号传导过程的适当强度和持续时间是大部分未知的。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。