表皮生长因子从反式高尔基网络运输到细胞膜的分子机制及表皮生长因子受体新靶点的探索

The epidermal growth factor receptor (EGFR) is a cell surface localized receptor for epidermal growth factor (EGF) and transforming growth factor-α (TGF-α). Upon ligand binding, EGFR activates downstream signaling pathways, leading to cell growth and proliferation. EGFR plays important roles in cancer progression and is one of the major drug targets for targeted cancer therapy. . Newly synthesized EGFR needs to be delivered to the cell surface in order to receive its ligands. Our preliminary results indicate that surface delivery of newly synthesized EGFR follows the conventional steps in the biosynthetic transport pathway. In the biosynthetic transport pathway, after being synthesized from ribosomes, EGFR is translocated in the membranes of the endoplasmic reticulum (ER) and then delivered to the Golgi apparatus. EGFR exits the Golgi at the trans Golgi network (TGN). At the TGN, EGFR is packaged into transport vesicles and these transport vesicles are transported along sophisticated post-Golgi trafficking routes to the plasma membrane. Post-Golgi biosynthetic trafficking is an important process to ensure that newly synthesized proteins are delivered to correct localizations where they perform their physiological functions. Despite their importance, the molecular mechanisms regulating post-Golgi trafficking of EGFR remain unclear.. Here we propose to analyze the molecular mechanism that regulates transport of EGFR from the TGN to the plasma membrane. EGFR is localized at the basolateral membranes in polarized MDCK cells. Our preliminary analysis indicates that EGFR and p75, an apical-plasma membrane-localized protein, are transported in separate transport carriers after exiting the trans Golgi network (TGN). In addition, TGN export of EGFR but not p75 is regulated by the adaptor complex-1, a major cargo sorting complex at the TGN. We thus hypothesize that EGFR is sorted into specific transport vesicles at the TGN and these transport vesicles are delivered along a specific post-Golgi biosynthetic trafficking route leading to the basolateral plasma membrane. To test this hypothesis, we will first utilize light sheet microscopy to elucidate the post-Golgi trafficking routes of EGFR in polarized MDCK cells. We will then analyze how EGFR is packaged into transport vesicles at the TGN and reveal the identity of proteins, such as motor proteins, that regulate delivery of EGFR-enriched transport vesicles from the TGN to specific acceptor compartments. .Currently, all cancer patients who initially benefit from EGFR-targeted therapy eventually develop resistance. One of the major causes of resistance is that tumor cells acquire additional mutations in EGFR that weaken the interaction between EGFR and the drug. Thus, to overcome drug resistance it is essential to explore a novel strategy that acts by an entirely different mechanism. Our preliminary results indicate that blocking the biosynthetic pathway inhibits EGF-induced EGFR autophosphorylation. Thus, blocking surface delivery of newly synthesized EGFR is a novel way to downregulate EGFR signing. Here we aim to test whether interfering with EGFR post-Golgi trafficking can effectively affect EGFR signaling and cancer cell proliferation.. These analyses will provide novel insights into the biosynthetic trafficking of EGFR. These analyses will also provide important information for rational design of inhibitors that block specific steps of post-Golgi trafficking of EGFR as a novel method to modulate EGFR signaling.

表皮生长因子受体（EGFR）是一个主要的肿瘤诊断标志分子及治疗靶点。新合成的EGFR通过合成转运途径运输到细胞质膜。在细胞膜上EGFR与配体结合后诱导EGFR自磷酸化从而激活下游信号通路。目前新合成的EGFR被运输到细胞膜特别是从反式高尔基体网络（TGN）运输到细胞膜的机制仍然大部分未知。本课题研究的目标是：1）阐明EGFR从TGN运输到细胞膜的路径； 2）探索EGFR在TGN 上被分选入囊泡的分子机制； 3）寻求调控EGFR后高尔基体运输的方向性的重要因子。我们的前期研究表明抑制合成转运途径能够抑制配体诱导的EGFR自磷酸化。基于此结果，我们将测试干扰EGFR从TGN到细胞膜的运输是否调节EGFR信号传导和癌细胞增殖。这些分析将为EGFR的合成转运途径的分子机制提供新的见解。本课题还将为设计阻碍EGFR后高尔基体运输的抑制剂提供有效的信息,为抑制EGFR介导的信号通路提供新的方法。

表皮生长因子受体（EGFR）在哺乳动物的发育中起到至关重要的作用，且参与维持多种机体组织的正常功能。EGFR在很多癌症患者的肿瘤组织中有过表达或突变的现象，因此EGFR是一个主要的肿瘤诊断标志分子以及治疗靶点。野生型EGFR必须运输到细胞膜上才能接收信号行驶其信号传导功能。目前关于EGFR细胞内部运输的研究集中于激活后的EGFR的内吞过程，而对新合成的EGFR被运输到细胞膜的运输机制，特别是从反式高尔基网络（TGN）运输到细胞膜的机制仍然大部分未知。本课题着力于研究EGFR从TGN运输到细胞膜的运输的分子机制以及其对EGFR信号通路的影响。通过系统的细胞学，生物化学和活细胞成像的研究手段，我们发现EGFR通过和p75不一样的路径从TGN运输到细胞膜。我们的研究表明AP-1识别EGFR上的998号酪氨酸基序将其装入囊泡离开TGN。并且我们发现了调控EGFR后高尔基体运输的重要因子Rab12和Arfrp1。此外我们发现通过去除AP-1和Rab12来下调EGFR从高尔基体到细胞膜的运输可以抑制EGFR信号传导和癌细胞增殖。同时，我们发现去除Rab12和AP11没有影响与肿瘤发生密切相关的EGFR突变体EGFR(L858R)从TGN向细胞膜的运输，说明EGFR(L858R)从高尔基体输出依赖于和野生型EGFR不一样的细胞因子。这些分析不仅为EGFR的合成转运途径的分子机制提供新的见解，而且还为以EGFR为代表的位于细胞的基底侧质膜的蛋白如何被运输到其在细胞膜上特殊的位置提供新的见解。本课题将为未来合理设计阻碍EGFR后高尔基体运输的抑制剂，从而为抑制EGFR介导的信号通路来治疗癌症的可行性提供有效的信息。