Signaling by the EGF Receptor from Endosomes

来自内体的 EGF 受体发出信号

• 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
• 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

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.

内吞转运调节信号转导过程的机制仍有待阐明 对于理解不同的刺激如何从细胞表面传播信号， 保守的胞质机制和细胞核导致刺激和环境特异性信号传导 结果。表皮生长因子（EGF）受体（EGFR）是一种原型受体酪氨酸激酶，已成为研究的热点。 主要的实验模型来研究信号网络的时空调节。EGFR是一种重要的 在成年哺乳动物发育和组织稳态中的作用，并参与人类发病机制， 尤其是癌症。然而，虽然EGFR信号网络的主要成分是已知的，但它们如何在细胞内表达， 在EGFR内吞作用和随后靶向受体降解过程中协调发挥作用， 核内体以确保信号传导过程的适当强度和持续时间的机制在很大程度上是未知的。 解决这一基本问题现在已经成为可能，因为有了新的切割工具- 边缘方法学。使用时间分辨定量质谱的细胞磷酸化蛋白质组，我们 发现在大多数活性EGFR被内化到核内体后， 已知参与调节生长、存活、细胞运动和粘附的信号传导的蛋白质， 由EGFR维持。因此，我们假设，这些信号效应子的通路 的EGFR在内体中通过内体EGFR的持续活性起作用。我们进一步假设， 通过维持沿着一些信号通路的活性，同时下调其他通路，EGFR 胞吞作用塑造EGFR信号传导的总体功能结果。 为了在生理学实验系统中检验这些假设（细胞生长依赖于 我们将：1）检查是否确定了内吞EGFR的推定底物和信号效应子 通过磷酸化蛋白质组学分析，通过标记内源性效应物， 通过基因编辑和解剖它们的时间依赖性定位动力学， 三维荧光显微成像;和2）检查是否内吞和定位在 内体控制推定的磷酸化底物的下游信号传导活性， 内体EGFR，并定义这种调节的机制。结合高通量方法， 用各种定量活细胞光学成像方法通过基因编辑标记内源性蛋白质 分辨率的水平将使我们能够应用系统生物学方法来解开整个内体信号传导 程序.