A comprehensive approach to reveal how EGFR signalling controls endosomal sorting

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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.