Chiara Francavilla, Phd
PI, Manchester Breast Centre,
The University of Manchester
Trafficking routes after internalization regulate the signalling outputs of ligand-bound Receptor Tyrosine Kinases (RTKs), such as Fibroblast Growth Factor and Epidermal Growth Factor Receptors (FGFR2, EGFR). RTK recycling to the plasma membrane counterbalances lysosome-mediated receptor degradation, induces sustained signalling activation, and regulates cell proliferation and motility. Dysregulated recycling and/or signalling are associated with human diseases, such as breast cancer. However, it is not known through which signalling players recycling endosomes fine-tune downstream responses.
Here, we used FGFR2b as a model system to study signalling modules which depend on the presence or on the permanence of RTKs in the recycling endosomes upon ligand stimulation. We combined traditional Mass Spectrometry-based quantitative phosphoproteomics with a novel method to detect signalling proteins in proximity of FGFR2b during receptor recycling (local phosphoproteomics), bioinformatics, imaging, and functional assays in breast cancer cells.
We showed that recycling endosomes integrate the signalling outputs of FGFR2b and of EGFR in response to their respective ligands in breast cancer cell models. Furthermore, signalling partners in the proximity of recycling endosomes which regulated autophagy, cell growth, and stress responses were identified only by the local phosphoproteomics approach. Therefore, this approach could revolutionize the way in which we study how signalling architecture changes depending on the spatio-temporal regulation of RTKs.
Overall, these findings increase our scarce understanding of recycling endosomes as fine-tune regulators of signalling outputs and reveal how manipulating specific signalling players during receptor recycling maintains cell survival, such as in breast cancer cells.