CONTACT SITES BETWEEN THE ENDOPLASMIC RETICULUM AND THE PLASMA MEMBRANE CONTROL EGFR ENDOCYTOSIS

The epidermal growth factor receptor (EGFR) can be internalized through different routes. While clathrin-mediated endocytosis destines EGFR for recycling and signaling, internalization through non-clathrin endocytosis (NCE) targets the receptor for degradation. Since NCE appears to be a major negative regulator of EGFR levels, a more complete picture of this pathway would likely reveal new insights into aberrant EGFR signaling observed in many types of cancer. By combining a candidate gene approach with an unbiased proteomic approach, we have defined EGFR-NCE as molecularly distinct from other NCE pathways, relying on functional regulators not previously implicated in endocytosis. We found that reticulon 3 (RTN3), an endoplasmic reticulum (ER)-resident protein, is fundamental for NCE-mediated EGFR internalization, and that its ablation delays EGFR degradation, demonstrating that the NCE pathway is a critical regulator of the EGF-dependent cellular response. We show that, upon stimulation with high dose of EGF, RTN3 is localized in close proximity to EGFR and that it is crucial for the formation of contact sites between the ER and the plasma membrane (PM), which are needed for NCE to proceed efficiently. We also show that ER contact sites are involved in local Ca2+ release: high EGF doses induce a release of Ca2+ from the ER to the PM, which is strongly inhibited upon knockdown of RTN3. This calcium release depends on the inositol trisphosphate (IP3) cascade and is essential for the internalization of EGFR via NCE. In conclusion, we have discovered a new clathrin-independent endocytic pathway that relies on the action of RTN3. RTN3 is necessary for the formation of contact sites between the ER and EGFR-NCE sites at the PM, which are required for IP3R-dependent local calcium release and the completion of EGFR internalization through NCE.