EGF Receptor Endocytosis: Mechanisms and Role in Signaling

EGF 受体内吞作用：机制及其在信号传导中的作用

• 批准号: 10552100
• 负责人: ALEXANDER D SORKIN
• 金额: $ 39.75万
• 依托单位: UNIVERSITY OF PITTSBURGH AT PITTSBURGH
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-06-01 至 2028-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10552100
• 关键词: Actins; Address; Adult; Biological Assay; Cells; Cellular Assay; Chimeric Proteins; Clathrin; Complex; Development; Endocytosis; Endosomes; Enzymes; Epidermal Growth Factor Receptor; Eukaryota; Family; Fluorescence; Funding; Gene Fusion; Generations; Genes; Homeostasis; Human Pathology; Label; Ligands; Mass Spectrum Analysis; Measures; Mediating; Methods; Modeling; Molecular; Monitor; National Institute of General Medical Sciences; Pathology; Pathway interactions; Play; Positioning Attribute; Process; Proteins; Proteome; Receptor Protein-Tyrosine Kinases; Receptor Signaling; Regulation; Research; Role; Signal Pathway; Signal Transduction; Sorting; Stress; VAV2 gene; Vesicle; carcinogenesis; experimental study; in vivo; metastatic process; novel; novel strategies; p38 Mitogen Activated Protein Kinase; phosphoproteomics; ratiometric; screening; spatiotemporal; temporal measurement; tissue regeneration; trafficking; wound healing

Signaling by receptor tyrosine kinases (RTK) controls major vital processes in developing and adult eukaryotes and is involved in a variety of human pathologies. Epidermal growth factor receptor (EGFR) is the classic RTK, whose signaling and trafficking have been extensively studied, but the molecular mechanisms of endocytosis and spatiotemporal regulation of signaling processes by endocytosis remain poorly understood. Fundamental questions of whether signaling is triggered from endosomes by internalized EGFR and how such signaling is regulated by the endosomal sorting machinery are not addressed. Reciprocally, whether EGFR signaling regulates endocytic trafficking machinery, remains sparsely studied and controversial. Further, the degree and rates of the incorporation of EGFR signaling complexes into intralumenal vesicles of multivesicular endosomes, a process that terminates signaling, are also unknown. In the NIGMS funded research, we have developed single-cell and high-throughput methods to quantitatively monitor endocytic trafficking of endogenous gene- edited EGFR using a pH-sensitive ratiometric fluorescence excitation model. We used these methods to elucidate the mechanism of EGFR endocytosis caused by activation of stress-induced p38-MAP kinase and to determine mechanisms of the crosstalk of this pathway with ligand-induced endocytic pathways. We have developed a pipeline for generation of gene-edited cells expressing endogenous fluorescently tagged EGFR and its downstream signaling effectors, and new approaches to examine the dynamics of these components at high spatial and temporal resolution in living cells. We performed comprehensive phosphoproteomic and proximity proteome mass-spectrometry screenings to identify signaling effectors and regulators of endosomal EGFR. Finally, our preliminary experiments using a novel live-cell assay prompted us to hypothesize that EGFR:adaptor complexes are rapidly incorporated into intralumenal vesicles of multivesicular endosomes which diminishes the capacity of those complexes to signal. These advances have placed us in a unique position to fully define molecular mechanisms of clathrin-mediated internalization of EGFR, an endocytic pathway of EGFR in vivo, and address key fundamental questions of whether EGFR signals from endosomes and how this signaling is terminated. We will exploit the discoveries of the endosomal localization of an actin regulator VAV2 and a protein of uncertain function, TFG (Trk-gene fusion protein), to define their functions in EGFR signaling from endosomes. Using our new assay measuring distribution of signaling complexes within multivesicular endosomes in living cells, we will determine the dynamics of the termination of signaling from endosomal EGFR complexed with various endogenous fluorescently labeled adaptors and enzymes. Together, proposed studies will lead to a comprehensive understanding of the mechanisms of endocytosis and spatiotemporal regulation of signaling processes by endocytosis, and will serve as a paradigm to a similar analysis of the whole RTK family.

受体酪氨酸激酶（RTK）信号传导控制发展和成人真核生物的主要重要过程 并参与各种人类病理。表皮生长因子受体（EGFR）是经典的RTK， 其信号传导和运输已被广泛研究，但内吞作用的分子机制 并且通过内吞作用对信号传导过程的时空调节仍然知之甚少。基本的 内在eGFR触发信号的问题以及这种信号是如何触发的。 未解决由内体分类机制调节的。互惠，EGFR是否信号传导 调节内吞贩运机制，仍然对研究和有争议。此外，学位和 将EGFR信号复合物掺入到多虫内体的毫米内囊泡中的速率， 终止信号传导的过程也未知。在NIGMS资助的研究中，我们已经开发了 单细胞和高通量方法用于定量监测内源基因的内吞运输 使用pH敏感的比率荧光激发模型编辑了EGFR。我们使用这些方法来 阐明了由应激诱导的p38-MAP激酶激活引起的EGFR内吞作用的机制 确定该途径与配体诱导的内吞途径的机理。我们有 开发了一种用于生成基因编辑的细胞，表达内源性荧光标记的EGFR和 它的下游信号效应子，以及在高度检查这些组件的动态的新方法 活细胞中的空间和时间分辨率。我们进行了全面的磷光蛋白质组学和接近性 蛋白质组质谱筛选，以识别内体EGFR的信号传导效应子和调节剂。 最后，我们使用新颖的活细胞测定法的初步实验促使我们假设EGFR：适配器 络合物迅速掺入多​​囊内体的囊内囊泡中，从而减少 这些复合物发出信号的容量。这些进步使我们处于完全定义的独特位置 网格蛋白介导的EGFR的内在化的分子机制，EGFR，EGFR在体内的内吞途径和 解决了eGFR信号是否来自内体和该信号的关键基本问题 终止。我们将利用肌动蛋白调节剂VAV2和蛋白质的内体定位的发现 TFG（TRK-Gene融合蛋白）的功能不确定的功能，以定义其在内体EGFR信号中的功能。 使用我们的新测定测量在生物中多尖内体内信号传导复合物的分布 单元格，我们将确定来自内体EGFR的信号传导终止的动力学 各种内源性荧光标记的适配器和酶。共同提出的研究将导致 对内吞作用的机制和信号传导的时空调节机制的全面理解 通过内吞作用进行过程，并将作为对整个RTK家族的类似分析的范式。