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。一旦与细胞外配体结合，受体就会触发一系列决定细胞命运和行为的反应。在我们对受体激活持续时间是如何控制的理解上存在着严重的差距，这是一个对于开发控制信号障碍的药物策略至关重要的问题。然而，有一种图景显示，内体转运是调节信号持续时间的主要机制：如果内化的激活受体留在内体或循环到表面，则信号保持不变，但如果受体靶向溶酶体并被降解，则信号被下调。这个项目将确定这种分类决定是如何发生的。受体信号的内吞控制是普遍存在的，但表皮生长因子受体(EGFR)是一个例子，因为它具有巨大的临床/药理学重要性。EGFR也是解剖决定受体命运的局部信号事件的杰出模型，因为它根据其所结合的生理配体的不同而表现不同。EGF促进EGFR的降解，从而促进短期的信号反应，而转化生长因子(TGF)α诱导EGFR循环和延长信号转导。导致EGFR降解的关键分选步骤发生在内体，因为EGFR进入管腔内小泡(ILV)形成多囊泡体(MVB)并进入溶酶体。目前的模型通过促进EGFR参与ESCRT-0(运输所需的内体分选复合体)，将EGFR泛素化确定为指导MVB分选的关键因素，ESCRT-0是一系列ESCRT复合体中的第一个，这些复合体结合在一起产生ILV。然而，令人惊讶的是，Francavilla对EGFR信号的定量、质谱学(MS)分析表明，泛素化不足以指导MVB分选：在EGF或TGFpha刺激后，EGFR同样泛素化。此外，我们发现，EGFR与ESCRT-0的结合与配体无关，但只有在EGF刺激下才能传递给后来的ESCRT。因此，ESCRT-0作为一个支点，泛素化的EGFR的命运在其上平衡，但一个未知的EGFR信号网络作用于ESCRT-0下游，对MVB的分选产生控制影响。这个网络将是未来药物的关键靶点。这个网络的可能焦点是ESCRT辅助因子和肿瘤抑制因子，His区域蛋白酪氨酸磷酸酶(HD-PTP)。Woodman已经证明，HD-PTP结合多个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分类的影响提供了一个框架。微软领导的一种不偏不倚的方法来寻找这个网络的组成部分，并剖析这些组成部分是如何运作的，这是全面评估MVB分类控制方式的唯一现实途径。我们处于进行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相互作用的动态，这些相互作用是在EGF激活的EGFR被分类到MVB时选择性形成的。这将识别与ESCRT依赖的排序映射的候选效应器和翻译后修饰，这基本上建立在Francavilla之前的发现基础上，即ESCRT-0在EGF下游被磷酸化，而不是TGFpha。仔细挑选关键候选人，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了另一种RTK，成纤维细胞生长因子受体(FGFR)的MVB分类，来测试它是否具有普遍性。综上所述，我们将开发新的方法来观察MVB的分类途径，并阐明如何最好地控制受体酪氨酸激酶信号，从而开发新的药物。

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.