THESIS
2017
xiii, 79 pages : illustrations (some color) ; 30 cm
Abstract
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...[
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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.
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