A comprehensive approach to reveal how EGFR signalling controls endosomal sorting

表面受体是细胞感知和与环境交流的主要途径。在结合细胞外配体时，受体触发决定细胞命运和行为的广泛反应。我们对受体激活的持续时间是如何控制的理解存在着关键的差距，这是一个对于开发控制信号转导障碍的药理学策略至关重要的问题。然而，正在出现这样一种情况，即内体运输是调节信号持续时间的主要机制：如果已内化的活化受体保留在内体中或再循环至表面，则信号传导得以维持，但如果受体靶向溶酶体并降解，则信号传导下调。受体信号的内吞控制是普遍的，但表皮生长因子受体（EGFR）是一个范例，因为它具有巨大的临床/药理学意义。EGFR也是一个杰出的模型，用于解剖决定受体命运的局部信号传导事件，因为它的行为根据其生理配体的结合而不同。EGF促进EGFR降解，从而促进较短期的信号传导反应，而转化生长因子（TGF）α诱导EGFR再循环和延长信号传导。导致EGFR降解的关键分选步骤发生在内体中，因为EGFR进入腔内囊泡（ILV）以在通往溶酶体的途中形成多泡体（MVB）。目前的模型通过促进EGFR与ESCRT-0（转运蛋白-0所需的内体分选复合物）结合，将EGFR泛素化鉴定为指导MVB分选的关键因素，ESCRT-0是一系列ESCRT复合物中的第一种，其联合收割机组合产生ILV。然而，令人惊讶的是，Francavilla对EGFR信号传导的定量质谱（MS）分析表明，泛素化不足以指导MVB分选：EGFR在EGF或TGF α刺激后也同样被泛素化。此外，我们发现EGFR与ESCRT-0无关，但仅在EGF刺激后才传递到后来的ESCRT。因此，ESCRT-0作为平衡泛素化EGFR命运的支点，但作用于ESCRT-0下游的未知EGFR信号传导网络对MVB分选产生控制性影响。该网络将成为未来药物的关键靶点，该网络的焦点可能是ESCRT辅助因子和肿瘤抑制因子，His结构域蛋白酪氨酸磷酸酶（HD-PTP）。Woodman已经证明HD-PTP结合多种ESCRT，并协调EGFR从ESCRT-0通过ESCRT途径。他的工作为研究EGFR信号对MVB分选的影响提供了一个框架。一个公正的，MS主导的方法来找到这个网络的组成部分，并解剖这些行为，提出了唯一现实的路线，全面评估如何MVB排序控制。我们处于开展这项工作的理想位置。我们将进行定量蛋白质组学实验，以跟踪ESCRT-0和HD-PTP相互作用组的动力学，这些相互作用组在EGF激活的EGFR被分选到MVB时选择性形成。这将确定与ESCRT依赖性分选映射的候选效应子和翻译后修饰，基本上建立在Francavilla先前的发现上，即ESCRT-0在EGF下游磷酸化，而不是TGF α。仔细选择关键候选人，然后我们将查明他们对EGFR贩运的影响。最后，在为控制EGFR的信号网络建立了“ESCRT指纹”之后，我们将通过检查它是否也驱动另一种RTK成纤维细胞生长因子受体（FGFR）的MVB分选来测试它是否具有普遍性。总之，我们将开发观察MVB分选途径的新方法，并阐明如何最好地控制受体酪氨酸激酶信号传导，以开发新型药物。

Surface receptors are the primary route by which cells sense and communicate with their environment. Upon binding extracellular ligands, receptors trigger a vast range of responses that determine cell fate and behaviour. There are crucial gaps in our understanding of how the duration of receptor activation is controlled, a question vital for developing pharmacological strategies to control signaling disorders. However, a picture is emerging that endosomal trafficking is the major mechanism that regulates signal duration: signaling is maintained if activated receptors that have been internalised remain in the endosome or recycle to the surface, but is down-regulated if receptors are targeted to the lysosome and degraded. This project will identify how this sorting decision occurs.The endocytic control of receptor signaling is universal, but epidermal growth factor receptor (EGFR) is an exemplar because of its huge clinical/pharmacological importance. EGFR is also an outstanding model for dissecting local signaling events that determine receptor fate, because it behaves differently according to which of its physiological ligands is bound. EGF promotes EGFR degradation and thus shorter-term signaling responses, whilst transforming growth factor (TGF) alpha induces EGFR recycling and prolonged signaling. The critical sorting step leading to EGFR degradation occurs in the endosome, as EGFR enters intralumenal vesicles (ILVs) to form the multivesicular body (MVB) en route to the lysosome. Current models identify EGFR ubiquitination as the critical factor that directs MVB sorting, by promoting EGFR to engage ESCRT-0 (Endosomal Sorting Complexes Required for Transport-0), the first of a series of ESCRT complexes that combine to generate ILVs. Strikingly, however, Francavilla's quantitative, mass spectrometry (MS)-based analysis of EGFR signaling shows that ubiquitination is NOT sufficient to direct MVB sorting: EGFR is similarly ubiquitinated after EGF or TGFalpha stimulation. Moreover, we show that EGFR engages ESCRT-0 irrespective of the ligand, but passes to later ESCRTs only upon EGF stimulation. Hence, ESCRT-0 acts as a fulcrum upon which the fate of ubiquitinated EGFR is balanced, but an unknown EGFR signaling network acting downstream of ESCRT-0 exercises the controlling influence over MVB sorting. This network will be the key target for future drugs.The likely focal point for this network is the ESCRT accessory factor and tumour suppressor, His Domain Protein Tyrosine Phosphatase (HD-PTP). Woodman has shown that HD-PTP binds multiple ESCRTs and coordinates the passage of EGFR from ESCRT-0 through the ESCRT pathway. His work provides a framework for interrogating the impact of EGFR signaling on MVB sorting. An unbiased, MS-led approach to find the components of this network, and dissection of how these act, presents the only realistic route to comprehensively evaluate how MVB sorting is controlled. We are ideally placed to conduct this work. We will perform quantitative proteomics experiments to follow the dynamics of the ESCRT-0 and HD-PTP interactomes that are formed selectively as EGF-activated EGFR is sorted to the MVB. This will identify candidate effectors and post-translational modifications that map with ESCRT-dependent sorting, building substantially on Francavilla's previous finding that ESCRT-0 is phosphorylated downstream of EGF but not TGFalpha. Carefully selecting key candidates, we will then pinpoint their influence over EGFR trafficking. Finally, having established an "ESCRT fingerprint" for the signaling network that controls EGFR, we will test if it is universal, by examining if it also drives the MVB sorting of another RTK, fibroblast growth factor receptor (FGFR). In summary, we will develop new ways of viewing the MVB sorting pathway and shed light on how best to control receptor tyrosine kinase signaling, towards developing novel pharmaceutics.