A comprehensive approach to reveal how EGFR signalling controls endosomal sorting

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.

The endocytic pathway exerts decisive control over the longevity of signalling by cell surface receptors, exemplified by epidermal growth factor receptor (EGFR). Internalised receptors are recycled and retain activity, or are sorted to the multivesicular body (MVB) and then degraded. The MVB sorting step is thus a key therapeutic target.

Current models state that ubiquitination is the crucial signal that directs MVB sorting by promoting the receptor to engage ESCRT-0, the first of a series of ESCRT complexes that generate the MVB. We show, however, that EGFR is ubiquitinated and binds ESCRT-0 whether activated by EGF or TGFa, ligands that promote EGFR degradation or recycling respectively. Hence ubiquitination is not the controlling influence over MVB sorting.

Instead, we propose that EGFR fate is determined primarily by local EGFR signaling events (as-yet largely unknown) that activate ESCRT-dependent sorting, and thus control whether EGFR passes from ESCRT-0 through the ESCRT pathway or is offloaded to other destinations. This signalling is most likely focussed on His Domain PTPase (HD-PTP), a tumour suppressor that coordinates ESCRT function.

To understand comprehensively how EGFR controls MVB sorting, we will exploit our proven expertise in quantitative proteomics to identify the ESCRT landscape as EGFR is sorted. Analysis of dynamic ESCRT-0 and HD-PTP "interactomes" (i.e. binding partners and their post-translational modifications) after EGF stimulation will highlight candidate signaling networks and key effectors.

Taking these new leads, and building on our work showing that ESCRT-0 is selectively modified by EGF stimulation, we will exploit our range of assays and reagents to dissect the molecular basis for ESCRT pathway activation by EGFR. We will then test whether these mechanisms are universal and thus impact on fibroblast growth factor receptor sorting. Our work will reassess which signals drive MVB sorting and find new therapeutic targets.

This proposal is centred on the control of receptor tyrosine kinase (RTK) endocytic trafficking pathways, a process that is fundamental to the lifetime of the active RTK and thus the duration of RTK signalling. The immediate academic impact of this project will be with research scientists working in related areas of the life sciences, including RTK signalling and receptor trafficking. Our work will provide new knowledge, insight and scientific advances that will be relevant to all those that are active in these areas, irrespective of whether their research is being carried out from a basic or a medical perspective. Clearly, however, our work will also be of relevance to clinical scientists, particularly those working in the field of cancer biology where RTK signalling is aberrant.
A major stakeholder with a fundamental interest in this proposal will be the pharmaceutical industry, since modulating RTKs such as EGFR and FGFR is a highlight area for new clinical targets, and the endocytic pathway plays an important role in cancer cell resistance to current antibody-based therapies. Our proposal seeks to identify at a comprehensive level new mechanisms that control the down-regulation of EGFR and other RTKs, and will therefore bring to light new targets for pharmacological control of RTK signalling. At present, much is made of the potential of receptor ubiquitination as a "druggable" target with which to modulate RTK degradation. Our approach will highlight potential new targets that act as alternatives or complements to this strategy.
This project fits precisely with the BBSRC priority areas, systems approaches to bioscience and data driven biology. In particular, this project will exploit how a systems-biology approach (i.e. quantitative proteomics) allows key discoveries to be made in addressing a focused scientific question (RTK trafficking and endosome sorting). We will achieve these advances in "functional proteomics" by using a broad spectrum of cutting-edge data analysis and bioinformatics approaches, exploiting the proven expertise of Chiara Francavilla. Therefore, this project is strategically important for its training and hence in building capacity.
This is a multidisciplinary proposal, combining cell biology with cutting edge quantitative proteomics. As such, it builds on very broad links between the investigators and other researchers within Manchester University, as well as both national and international colleagues. These cross-disciplinary links will be essential for promoting the academic impact of the work, and furthermore in developing new ties with clinical and pharmaceutical end-users. With these goals in mind, we will be aided in particular by the strong track record of University of Manchester Intellectual Property (UMIP) and business engagement teams in generating links with Pharma and other industrial partners.
The applicants are strongly committed to engaging with the general public about the importance of Science and the specific goals of their research, actively participating in a variety of activities, including Faculty and Institute open days. This project is of high public impact because of its relevance to disease and use of new technological approaches. We will therefore undertake outreach activities during the course of the project to communicate the importance of the work and the merits of our approach.