A NOVEL ROLE OF THE ENDOCYTIC ADAPTOR PROTEINS EPS15 AND EPS15L1 IN THE REGULATION OF NOTCH SIGNALING

Eps15 and Eps15L1 are endocytic adaptor proteins involved in both clathrin-mediated and clathrin-independent endocytosis of receptor tyrosine kinases. Eps15/Eps15L1 double knockout mice are embryonic lethal and display a Notch loss-of-function phenotype, accompanied by downregulation of Notch target genes. The Notch pathway is an evolutionary conserved signal transduction pathway that regulates multiple aspects of the development of multicellular organisms. Notch signaling activation requires direct contact between a signal-sending cell, expressing the ligand, and a signal-receiving cell, expressing the receptor. Both Notch ligands and receptors are transmembrane proteins, tightly regulated by endocytosis and membrane trafficking. Given these premises, the overall aim of this thesis was to understand whether Eps15 and Eps15L1 have a direct role in the regulation of Notch signaling, and, if so, whether this regulation takes place in the signal-receiving cell or in the signal-sending cell. Our final goal was to understand the molecular mechanisms by which Eps15 and Eps15L1 affect Notch signaling. To investigate the role of Eps15 and Eps15L1 in Notch signaling, we set-up a co-culture model system composed of the signal-sending cell OP9-Dll1 that overexpresses the ligand Dll1, and the signal-receiving cell C2C12-Notch1 that overexpresses the receptor Notch1. To assess the role of Eps15 and Eps15L1 in Notch signaling regulation, we developed an in vitro Notch transactivation reporter assay based on our co-culture model system. We observed no reduction in Notch activity after knockdown (KD) of Eps15 or Eps15L1 in the signal-receiving cell, indicating that the two proteins do not have a role in the regulation of Notch receptors. However, when we performed KD of Eps15 and/or Eps15L1 in the signal-sending cell, we observed a 40-50% reduction in Notch activity. This reduction was observed for all the Notch ligands tested, Dll1, Dll4, Jag1 and Jag2, indicating that Eps15 and Eps15L1 have a general role in the modulation of Notch ligand activity/signaling. Since Eps15 and Eps15L1 are known to act in combination with Epsin, a fundamental regulator of Notch ligand internalization, we investigated whether removal of Eps15 and Eps15L1 from the signal-sending cell affected the early steps of Dll1 endocytosis. To do so, we set-up a Dll1 internalization assay to measure induced or constitutive Dll1 endocytosis. Using this assay, we observed a strong reduction in Dll1 endocytosis following KD of known endocytic regulators of Notch ligands, Epsin and Mindbomb1. However, we did not detect any alterations in Dll1 internalization after KD of Eps15, Eps15L1 or both Eps15 and Eps15L1, indicating that the two proteins do not participate in the regulation of the early steps of Notch ligand endocytosis, both induced and constitutive. Eps15 and Eps15L1 have also been implicated in endocytic recycling. Therefore, we asked whether a recycling function of Eps15/Esp15L1 could be involved in Notch ligand regulation. We attempted to assess Dll1 recycling in OP9-Dll1 cells, however, results suggested that Dll1 does not undergo significant recycling in our experimental conditions, therefore a change of strategy will be necessary. We also assessed Dll1 localization in plasma membrane lipid rafts following KD of Eps15 and Eps15L1, but did not score any change in ligand membrane localization. In conclusion, we showed that Eps15 and Eps15L1 are key regulators of Notch ligands in the signal-sending cell. However, in contrast to their known role as endocytic adaptors, the two proteins do not appear to be involved in the early steps of Dll1 endocytosis. Given that Eps15 and Eps15L1 have been implicated in other pathways and cellular processes, such as recycling, secretion, degradation, cell-matrix adhesion and cell-cell connection, it is possible that Eps15/Esp15L1 might mediate Notch ligand regulation through one of these other pathways. However, the precise molecular mechanisms by which Eps15 and Eps15L1 regulate Dll1 activity remain to be defined, and will be the focus of future studies.