EGF Receptor Endocytosis: Mechanisms and Role in Signaling

受体酪氨酸激酶（RTK）信号传导控制发育和成年真核生物的主要生命过程 并且与多种人类病理学有关。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 以及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 关于信号传导是否由内化的EGFR从核内体触发以及这种信号传导是如何 未涉及由内体分选机制调节的蛋白质。反过来，EGFR信号传导是否 调节内吞贩运机制，仍然很少研究和有争议。此外，程度和 EGFR信号传导复合物掺入多泡内体的腔内囊泡的速率， 终止信令的过程也是未知的。在NIGMS资助的研究中，我们开发了 单细胞和高通量方法来定量监测内源性基因的内吞运输， 使用pH敏感的比率荧光激发模型对编辑的EGFR进行分析。我们使用这些方法， 阐明应激诱导的p38-MAP激酶激活引起EGFR内吞的机制， 确定该途径与配体诱导的内吞途径的串扰机制。我们有 开发了一种用于产生表达内源性荧光标记EGFR的基因编辑细胞的管道， 其下游信号效应器，以及检查这些成分在高水平下动态的新方法 活细胞的空间和时间分辨率。我们进行了全面的磷酸化蛋白质组学和邻近 蛋白质组质谱筛选以鉴定内体EGFR的信号传导效应物和调节物。 最后，我们使用一种新的活细胞测定法进行的初步实验提示我们假设EGFR： 复合物迅速掺入多泡内体的腔内囊泡中， 这些复合物的信号能力。这些进步使我们处于一个独特的位置， 网格蛋白介导的EGFR内化的分子机制，EGFR在体内的内吞途径，以及 解决了EGFR信号是否来自内体以及这种信号是如何被激活的关键基本问题。 终止我们将利用肌动蛋白调节因子VAV 2和蛋白质的内体定位的发现， TFG（Trk-基因融合蛋白），以确定它们在来自内体的EGFR信号传导中的功能。 使用我们的新测定法测量活细胞中多泡内体内信号复合物的分布， 细胞，我们将确定终止信号从内体EGFR复合的动态 各种内源性荧光标记的衔接子和酶。总之，拟议的研究将导致 全面了解内吞作用和信号时空调控的机制 过程中的内吞作用，并将作为一个范例，以类似的分析，整个RTK家庭。

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.