ADAM10 as a gatekeeper of receptor tyrosine kinase activation and trafficking: comparative analysis of cell-morphogenetic and mitogenic Eph and EGF receptor signalling pathways
Final Report Abstract
In this study, trafficking and signalling of the EphA3 receptor was elucidated and the interaction and crosstalk between EphA3 and EGFR identified: these findings were elaborated and discussed in the context of downstream cancer cell signalling and cell-biological outcomes. Agonist (ephrin-A5) stimulation triggers the internalisation of EphA3 clusters via clathrin-mediated endocytosis, where EphA3 is rapidly transported to early endosomes and MVBs and finally recycles back to the PM to enter a second round of internalisation by further activation of ephrinA5. Based on the fact that EGFR in HN5 cells forms direct or indirect physical interaction with kinases such as EphA2 and EphB4, a possible role of EGFR in EphA3 signalling was investigated. EphA3 was found to associate with EGFR, when endogenously expressed or following transient transfection in various cell lines. However, so far the exact protein domains facilitating this interaction remain to be elucidated. The presented results suggest that both receptors internalise and traffic into different endocytic compartments. Moreover the data clearly demonstrates that EGFR cross-phosphorylates EphA3, even in the absence of EGF stimulation. However whether the increased EphA3 phosphorylation is mediated directly by the EGFR kinase or indirectly via the non-RTK Src cannot be ascertained at this point. Most intriguingly, EphA3 negatively influences EGFR phosphorylation. The notable downregulation of EGFR signalling is in particular increased upon stimulation with ephrinA5. The balance of these receptors could thus have a significant impact on the ability of tumour cells survival and metastatic potential. If EphA3 indeed mediates metastasis, this function could be enhanced by the overexpression of EGFR. However, the mitotic function of EGFR might be inhibited by the presence of activated EphA3. These observations demonstrate the importance of signalling cascades and outcomes that can be influenced positively and negatively by the interaction and solace of receptors and opens a wide range of perspectives that have to be analysed in more detail. Nevertheless our findings have therapeutic implications, and we propose that individuals with EGFR-positive cancer might benefit from inhibition of EGFR activity by using anti-EphA3 antibody in combination with anti-EGFR therapies.
