Investigating molecular mechanisms regulating the post-Golgi trafficking of EGFR and secretion of EGFR-enriched extracellular vesicles

Abstract

The epidermal growth factor receptor (EGFR) has crucial functions in cancer progression. EGFR-targeted drug treatment is one of the major targeted cancer therapies. Although fundamentally important, how newly synthesized EGFR is transported to the plasma membrane to perform its cellular functions remains to be further investigated. We found that the clathrin adaptor complex-1 (AP1) and Rab12 interact with EGFR and regulate the export of EGFR out of the trans-Golgi network (TGN). In addition, the tyrosine residue at the 998 position on human EGFR is critical to bind to AP-1, and this residue is important for TGN export of EGFR. AP-1 and Rab12 are important for EGF-induced cell elongation and proliferation, suggesting that AP-1- and Rab12 mediated post-Golgi trafficking is important for EGFR signaling. Moreover, TGN export of the constitutively activated mutant form of EGFR (EGFR^L858R) is dependent on clathrin but independent of AP-1 and Rab12. The clathrin adaptor epsinR shows a higher binding efficiency to EGFR^L858R than the wt EGFR. Our results reveal insights into the molecular mechanisms that mediate the TGN-to-cell surface delivery of EGFR and indicate that TGN export of wild-type EGFR and EGFRL858Rdepends on different cellular factors.

Extracellular vesicles (EVs) are vesicles released by the cells. They play important roles in tissue development, neuron communication, and cancer progression by delivering proteins, mRNA, and miRNA to recipient cells. EGFR is one of the proteins delivered by EVs. Although the function of EGFR in cancer has been well studied, the secretion mechanism of EGFR-enriched EVs is largely unclear. In this study, I isolated the EGFR-enriched EVs through differential centrifugation combined with floatation. EGFR-enriched EVs are efficiently taken up by the recipient cells in a clathrin-dependent manner. EGFR is shown to be present in a subpopulation of EVs that are different from EVs enriched with another protein, CD63. Depletion of AP-1 significantly reduces the secretion of EGFR-enriched EVs. Moreover, dysfunction of VPS4A does not affect the secretion of EGFR-enriched EVs, suggesting EGFR is directly sorted into EVs from the plasma membrane. Thus, we found important protein factors that regulate the secretion of EGFR-enriched EVs, providing novel insights into this cellular process.

Date of Award	2022
Original language	English
Awarding Institution	The Hong Kong University of Science and Technology
Supervisor	Yusong GUO (Supervisor)