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Brian P. Ceresa, Jennifer McClintock, Eric D. Phelps, Jamie Rush; Termination Of Epidermal Growth Factor Receptor Signaling In Corneal Epithelial Cells Is Regulated By Receptor Sequestration. Invest. Ophthalmol. Vis. Sci. 2011;52(14):1948.
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© ARVO (1962-2015); The Authors (2016-present)
The epidermal growth factor receptor (EGFR) is a cell surface receptor tyrosine kinase that plays a critical role in the homeostasis and healing of the corneal epithelium. We have previously reported that EGFR trafficking to the lysosome negatively regulates EGFR signaling. We are interested in identifying which step in the endocytic pathway is the limiting step for signal attenuation. The goal is to disrupt this step to prolong EGFR signaling and potentiate corneal epithelial wound healing.
RNAi was used to attenuate expression of the small molecular weight guanine nucleotide binding protein, RAB7, or TSG101, which is part of the endosomal sorting complex required for transport (ESCRT) machinery. Both proteins regulate the trafficking of the EGFR, but at different points in the endocytic pathway. Knockdown of TSG101 causes the EGF:EGFR complex to accumulate on the limiting membrane of the early/late endosome. Knockdown of RAB7 enriches the EGF:EGFR complex in the intraluminal vesicles of the late endosome/multivesicular bodies. The consequence on EGFR-mediated signaling was monitored using modified Boyden Chamber cell migration assays.
Knockdown of TSG101 or RAB7 delayed the endocytic trafficking of the EGF:EGFR complex as indicated by biochemical and immunofluorescent analysis. Cell migration was enhanced when the ligand:receptor complex was enriched on the limiting membrane of the endosomal compartment, but not when it was in the intraluminal vesicles of the late endosome/multivesicular body.
Using corneal epithelial cell migration as a readout, preventing the degradation of the EGF:EGFR complex is not sufficient to prolong EGFR signaling. EGFR signaling is only enhanced when the receptor is localized in the proper subcellular orientation that allows the carboxyl terminus to interact with downstream signaling molecule. Thus, EGFR sequestration into the intraluminal vesicles of the late endosome is what limits receptor signaling.
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