Investigating the Molecular Mechanisms Underlying Endocytic Trafficking of the Epidermal GrowthFactor Receptor

Science Center, Bent Corridor

Endocytosis is a general term used to describe a ubiquitous cellular processes required for the uptake of essential extracellular nutrients, regulation of cell-membrane bound receptors, antigen presentation, and plasticity. Following internalization, membrane proteins proceed through a series of compartments where they proceed through one of two paths: recycled back to the cellular membrane or degraded following internalization in multivesicular bodies (MVBs) and later sent to the lysosome. The epidermal growth factor receptor (EGFR) is a transmembrane protein that activates many downstream pathways involved in regulating cellular proliferation, differentiation, and survival. Previous studies have implicated EGFR overexpression to several neurological diseases and cancers where around 84% of tumors were positive for EGFR. Recognizing the deficiencies in EGFR degradation and overexpression, we hoped to investigate which cytosolic components, more specifically proteins, affect endosomal sorting of EGFR. We examined EGFR sorting through the endocytic pathway using a cell-free reconstitution assay, which allowed us to assay a single membrane protein without the uncertainty of other factors affecting EGFR internalization or recycling. A yeast genetic knockout library was utilized in order to analyze the effects the absence of candidate proteins implicated as regulators of MVB sorting and/or recycling had on EGFR sorting. We report EGFR internalization into MVBs was decreased by 50% following the deletion of an Endosomal Sorting Complexes Required for Transport (ESCRTS) protein. Other tested proteins did not seem to affect EGFR inward budding into MVBs. However, there is a potential effect on recycling, which we hope to further investigate.