Internalization Mechanisms of the Epidermal Growth Factor Receptor after Activation with Different Ligands

<div><p>The epidermal growth factor receptor (EGFR) regulates normal growth and differentiation, but dysregulation of the receptor or one of the EGFR ligands is involved in the pathogenesis of many cancers. There are eight ligands for EGFR, however most of the research into trafficking of the receptor after ligand activation focuses on the effect of epidermal growth factor (EGF) and transforming growth factor-α (TGF-α). For a long time it was believed that clathrin-mediated endocytosis was the major pathway for internalization of the receptor, but recent work suggests that different pathways exist. Here we show that clathrin ablation completely inhibits internalization of EGF- and TGF-α-stimulated receptor, however the inhibition of receptor internalization in cells treated with heparin-binding EGF-like growth factor (HB-EGF) or betacellulin (BTC) was only partial. In contrast, clathrin knockdown fully inhibits EGFR degradation after all ligands tested. Furthermore, inhibition of dynamin function blocked EGFR internalization after stimulation with all ligands. Knocking out a number of clathrin-independent dynamin-dependent pathways of internalization had no effect on the ligand-induced endocytosis of the EGFR. We suggest that EGF and TGF-α lead to EGFR endocytosis mainly via the clathrin-mediated pathway. Furthermore, we suggest that HB-EGF and BTC also lead to EGFR endocytosis via a clathrin-mediated pathway, but can additionally use an unidentified internalization pathway or better recruit the small amount of clathrin remaining after clathrin knockdown.</p> </div

EGFR co-localization with EEA1 after clathrin knockdown.

<p>A: Cells treated with siRNA were incubated with 10 nM ligand for 15 minutes at 37°C. The cells were fixed and labeled for EGFR and EEA1. B: Quantitative analysis of the amount of EGFR co-localizing with EEA1 in an average of 32–51 cells for each ligand+SEM. Statistical analysis comparing each column to their relevant unstimulated control (normalized to 1) was performed using one-way ANOVA with Bonferroni posttest. ** = p<0.01, *** = p<0.001, ns = non significant.</p

EGFR internalization after clathrin knockdown.

<p>A: Cells treated with siRNA were incubated with 3.22 nM ligand for 15 minutes at 37°C. The amount of cell surface EGFR was determined by flow cytometry and data normalized to unstimulated cells. Data points represent mean+SEM. Statistical analysis comparing each column to their relevant unstimulated control (normalized to 100) was performed using one-way ANOVA with Bonferroni posttest. *** = p<0.001, ns = non significant. B: Knockdown cells were incubated with 10 nM (EGF, TGF-α, HB-EGF, BTC) or 100 nM (AR, EPI) ligand for 15 minutes at 37°C. The amount of cell surface EGFR was determined by flow cytometry and data normalized to unstimulated cells. Data points represent mean+SEM. Statistical analysis comparing each column to their relevant unstimulated control (normalized to 100) was performed using one-way ANOVA with Bonferroni posttest. * = p<0.05, ** = p<0.01, *** = p<0.001, ns = non significant. C: Knockdown cells were incubated with 10 nM ligand for 5 minutes at 37°C. The amount of cell surface EGFR was determined by flow cytometry and data normalized to unstimulated cells. Data points represent mean+SEM. Statistical analysis comparing CHC to mock-treatment for each ligand was performed using two-way ANOVA with Bonferroni posttest. * = p<0.05, ** = p<0.01, ns = non significant. D: Test of the clathrin knockdown and EGFR levels. Cells were lysed in RIPA buffer and resolved by SDS-PAGE and western blotting. Actin is used as a loading control.</p

EGFR internalization after caveolin1 knockdown.

<p>A: Cells treated with siRNA were incubated with 10 nM ligand for 15 minutes at 37°C. The amount of cell surface EGFR was determined by flow cytometry and data normalized to unstimulated cells. Data points represent mean+SEM. Statistical analysis comparing caveolin siRNA treated cells to mock treatment for each ligand was performed using two-way ANOVA with Bonferroni posttest. ns = non significant. B: Test of the caveolin1 knockdown and EGFR levels. Cells were lysed in RIPA buffer and resolved on SDS-PAGE and western blotting. Actin is used as a loading control.</p

EGFR degradation after knockdown with CHC siRNA and ligand stimulation.

<p>Cells were incubated with 10 nM of the indicated ligand at 37°C for different time periods. Cells were lysed and the amount of EGFR determined by ELISA. Data points represent mean +/− SEM. A: EGF, B: TGF- α, C: BTC. Statistical analysis comparing degradation in unstimulated cells to ligand-treated and ligand+CHC siRNA-treated cells was performed using two-way ANOVA with Bonferroni posttest. * = p<0.05, ** = p<0.01, *** = p<0.001, ns = non significant.</p

EGFR internalization after filipin treatment.

<p>A: Cells were incubated with or without 1 µg/ml filipin for 1 hour, and then treated with 10 nM ligand for 15 minutes at 37°C. The amount of cell surface EGFR was determined by flow cytometry and data normalized to unstimulated cells. Data points represent mean+SEM. Statistical analysis comparing filipin to control treatment for each ligand was performed using two-way ANOVA with Bonferroni posttest. ns = non significant. B: Cells were incubated with or without 1 µg/ml filipin for 1 hour, and then allowed to bind fluorescently labeled cholera toxin on ice for 30 minutes. The cholera toxin solution was removed and the cells were allowed to internalize the cholera toxin for 1 hour at 37°C. After uptake the cells were washed and cholera toxin uptake was determined using flow cytometry. Statistical analysis comparing filipin to control treatment was performed using t-test. *** = p<0.001. C: Test of EGFR levels. Cells were lysed in RIPA buffer and resolved by SDS-PAGE and western blotting. Actin is used as a loading control.</p