REGULATION AND PHYSIOLOGICAL ROLE OF NON-CLATHRIN ENDOCYTOSIS OF THE EPIDERMAL GROWTH FACTOR RECEPTOR

The epidermal growth factor receptor (EGFR) plays a pivotal role in physiological cellular processes, but its aberrant activation is linked to cancer progression. Endocytosis is a critical regulator of EGFR activity. We have previously identified a novel endocytic route of the EGFR, non-clathrin endocytosis (NCE), which is activated only after stimulation with high doses of the ligand epidermal growth factor (EGF). Unlike canonical clathrin-mediated endocytosis (CME) that directs receptor mainly to recycling, NCE leads mostly to receptor degradation, restricting EGFR signaling and protecting cells from overstimulation. The molecular characterization of the NCE pathway led to the identification of a key functional regulator of NCE: the endoplasmic reticulum (ER)-shaping protein, reticulon-3 (RTN3). This regulator is necessary for the formation of contact sites between the plasma membrane (PM) and the ER during NCE internalization. Localized Ca2+ release at these contact sites induces the fission of NCE vesicles, completing the internalization process. In addition, CD147, a member of the immunoglobulin superfamily, was identified as a cargo co-internalizing with the EGFR in NCE vesicles. CD147 has been validated as a specific marker of NCE, and together with RTN3, represents an invaluable experimental tool for investigating this pathway. In the present work, we have dissected the signaling pathway promoting Ca2+ release from the ER upon EGFR-NCE activation, uncovering a specific role of the phospholipase C G2 (PLCG2) enzyme. PLCG2-dependent Ca2+ release at NCE sites occurs through the activation of the inositol trisphosphate receptor (IP3R) on the ER membrane and is required for the fission of EGFR/CD147-positive NCE vesicles from the PM. At the functional level, we extended the relevance of NCE to alternative EGFR ligands, beyond the EGF. Saturating doses of the two EGFR ligands, amphiregulin (AREG) and transforming growth factor-α (TGF-α), differentially triggered EGFR/CD147 internalization via NCE: while AREG efficiently activated NCE and Ca2+ release at the PM, TGF-α was less effective at triggering this pathway. This finding led us to hypothesize that the differential ability of alternative EGFR ligands to activate NCE could be responsible for the differences in the EGFR fate and biological output exerted by these ligands. Interestingly, stimulation of cells with another growth factor, hepatocyte growth factor (HGF), induced CD147-NCE similarly to stimulation with EGF, implying a broader role of NCE in the regulation of surface proteins. As a model system to test the physiological relevance of NCE, we are using organoids prepared from the non-transformed breast epithelial cell line MCF10A and from primary mouse intestinal crypt and mammary gland epithelial cells. These cells are dependent on EGFR signaling for their growth and differentiation in Matrigel. Our data showed that treatment with compounds that inhibit EGFR-NCE increase growth of mice intestinal and mammary organoids. In conclusion, we have molecularly dissected the signaling pathway leading to EGFR-NCE and expanded its relevance to alternative EGFR ligands and other growth factors. Given its crucial role in downregulating signaling and mediating growth restriction, NCE could behave as a possible tumor suppressor pathway and its regulators could represent novel targets in cancer therapy.