Regulation of epidermal growth factor receptor trafficking by secretory carrier membrane protein 3

Following ligand stimulation, the epidermal growth factor receptor (EGFR) is internalized and targeted for lysosomal degradation in a process referred to as downregulation. Downregulation of EGFR involves its ubiquitylation, which can act as an endocytic signal at the plasma membrane and as a sorting signal in endosomes. The endosomal sorting complexes required for transport (ESCRTs) are recruited from the cytosol to endosomal membranes to sort and package ubiquitylated EGFRs into intraluminal vesicles (ILVs). Multivesicular bodies (MVBs) containing the ILVs then bud off or mature and eventually fuse with lysosomes which degrade the ligand-receptor complex. We have been interested in the possibility that in addition to the ESCRTs, intracellular trafficking of EGFR may also involve other proteins, specifically those that constitutively reside on endosomal membranes. In particular, we have found that the secretory carrier membrane protein, SCAMP3 localizes in part to endosomes that traffic EGFR and contain phosphatidylinositol-3-phosphate (PI3P). In the studies presented in this dissertation, I show that SCAMP3 negatively regulates EGFR trafficking to lysosomes and enhances receptor recycling through processes that function in concert and in parallel to ESCRTs. SCAMP3 can interact with the ESCRT proteins Hrs and Tsg101, as well as several Nedd4 HECT ubiquitin ligases. SCAMP3 can also be multi-monoubiquitylated and this modification may be further regulated by deubiquitylation and tyrosine phosphorylation. We demonstrate that SCAMP3 expression, its ability to be ubiquitylated and interact with Tsg101 affect degradation and recycling of EGF and EGFR. Dual knockdowns of SCAMP3 with other ESCRT proteins retard EGF and EGFR degradation compared to knockdown of SCAMP3 alone but accelerate it compared to knockdown of ESCRTs alone. A mutation within SCAMP3's transmembrane core also reduced its ability to elicit EGFR recycling. These results suggest that SCAMP3 has an additional function in parallel to ESCRTs to regulate receptor degradation and recycling. Immunoelectron microscopy analysis of EGFR-containing MVBs suggests that SCAMP3 affects EGFR trafficking in pre-lysosomal compartments, downstream of initial MVB formation. Taken together, we propose that SCAMP3, its ubiquitylation, and interactions with ESCRTs regulate the efficiency of EGFR downregulation.