Control of the Basal Recycling and Surface Expression of Epidermal Growth Factor Receptor by the Endocytic Recycling Regulator EHD1 Utilizing a Pathway Shared by RUSC2

The epidermal growth factor receptor (EGFR) is a prototype receptor tyrosine kinase (RTK) and oncogene aberrantly expressed or mutated in solid tumors. Its surface expression is dynamically regulated. Display of an activation-competent pool is essential for response to ligands. Internalization and degradation of EGFR following stimulation has received the most attention, fewer studies have characterized the recycling arm of EGFR transit, basal traffic and surface display are poorly understood. Thus, we evaluated the endocytic recycling of EGFR for its therapeutic potential in EGFR driven cancers. The C-Terminal Eps15 homology (EH) domain-containing proteins have emerged as regulators of surface receptor recycling but their roles in EGFR traffic are not known. Studies presented here demonstrate EHD1 has a critical role in the positive regulation of total EGFR levels and surface display under unstimulated, basal conditions. Reduced EGFR surface expression due to EHD1 knockdown resulted in a reduction of ligand-induced signal transduction, and reduced EGF-dependent cell proliferation in two- and three-dimensional culture. Under EHD1 knockdown, EGFR accumulated in a juxta-nuclear compartment where it co-localized with markers for the Golgi apparatus. Single-cohort labeling of cell surface EGFR indicated this pool co-localizes with Rab11a, a marker of recycling endosomes, implicating EHD1 in the anterograde transport of recycling unstimulated EGFR. We have further determined that the RUN and SH3 domain containing protein (RUSC2) associates with EHD1, with both proteins co-localizing to vesicles and tubular membranes. RUSC2 knockdown led to a phenotype similar to EHD1 knockdown with reduction in total and surface EGFR expression. Our results show EHD1 is required for the basal recycling and plasma membrane targeting of EGFR under unstimulated conditions in a pathway shared with RUSC2. This study furthers our understanding of the mechanisms by which EGFR is transported to the plasma membrane for activation, provides a new function for EHD family members in regulating the itinerary of receptor tyrosine kinases and helps validate the endocytic recycling of RTKs as an approach to augmenting existing RTK-targeted therapies