Hyperactivation of Epidermal growth factor receptor (EGFR) signaling results in uncontrolled cell proliferation and cancer. Anti-cancer drugs have been developed by directly inhibiting the kinase activity of EGFR, but patients acquire drug resistance quickly. Therefore, it is desperate for identifying new therapeutic targets to manipulate EGFR signaling. Newly synthesized EGFR needs to be transport to the plasma membrane to initiate EGFR signaling. We hypothesize that the regulators involved in the surface delivery of EGFR can be an alternative therapeutic target for cancer treatment to inhibit EGFR signaling. To design such a therapeutic strategy, we first sought to elucidate molecular mechanisms that regulate transport of EGFR to the plasma membrane. Newly synthesized EGFR is...[ Read more ]

Hyperactivation of Epidermal growth factor receptor (EGFR) signaling results in uncontrolled cell proliferation and cancer. Anti-cancer drugs have been developed by directly inhibiting the kinase activity of EGFR, but patients acquire drug resistance quickly. Therefore, it is desperate for identifying new therapeutic targets to manipulate EGFR signaling. Newly synthesized EGFR needs to be transport to the plasma membrane to initiate EGFR signaling. We hypothesize that the regulators involved in the surface delivery of EGFR can be an alternative therapeutic target for cancer treatment to inhibit EGFR signaling. To design such a therapeutic strategy, we first sought to elucidate molecular mechanisms that regulate transport of EGFR to the plasma membrane. Newly synthesized EGFR is transported to the plasma membrane along the secretory transport pathway which includes two sequential steps: 1) endoplastic reticulum (ER) to the Golgi trafficking; 2) transport from the trans Golgi network (TGN) to the plasma membrane by an unknown molecular machinery. In this study, by an in vitro vesicle budding assay and immunofluorescence analyses, we found that newly synthesized EGFR was exported from the ER to the Golgi by COPII vesicles dependent on the Sar1 GTPase, which directly interacts with EGFR. At the TGN, clathrin and the clathrin adaptor AP-1 formed complexes with EGFR and sorted EGFR into clathrin-coated vesicles. Knockdown of clathrin or AP-1 by siRNA caused retention of EGFR at the TGN. Furthermore, the small GTPase Arf1 was found to regulate the TGN sorting of EGFR. Inhibition of Arf1 activity by the constitutively active mutant (Q71L) blocked budding of EGFR-containing vesicles from the TGN in the in vitro vesicle budding assay, whereas excess wild-type Arf1 protein stimulated the budding. Moreover, our data also show that the two cancer-related EGFR mutants L858R and double mutant T790M/L858R bypass the requirement of Arf1 in TGN export, suggesting alternative Arfs-mediated TGN export mechanisms for those mutants. Taken together, the results revealed several key regulators that mediate surface delivery of EGFR and uncovered potential targets to manipulate EGFR signaling for cancer treatments.