Figure 5.

EGF-induced p38-independent and p38-dependent internalization target ligand-occupied and free EGFRs to the same endosomes. (A) HeLa/FAP-EGFR cells were treated with 1 ng/ml EGF for indicated times at 37°C and lysed. Lysates were probed by Western blotting with pY1068, pS1046/47, phospho-p38 (p-p38), phospho-ERK1/2 (pERK), and α-actinin antibodies (loading control). (B) HeLa/FAP-EGFR cells were incubated with DMSO (vehicle, black) or 100 nM BIRB796 (red) for 90 min and then treated with 0.48–100 ng/ml EGF for 15 min. FAP-EGFR internalization was measured using the FERI assay. Mean values with SEM of duplicates are plotted against log of EGF concentrations. Mean values of the 561/640 ratio in cells treated with BIRB796 were subtracted from those values in vehicle-treated cells to estimate the relative contribution of the p38-dependent internalization (dashed violet). This experiment is representative of three independent experiments. (C) HeLa/FAP-EGFR cells were pretreated with DMSO (vehicle, black) or 100 nM BIRB796 (red) for 90 min, and then incubated with 1 ng/ml ¹²⁵I-EGF for indicated times at 37°C. Surface-bound and internalized ¹²⁵I-EGF was measured, and the ratio of the amounts of internalized and surface ligand is plotted against time. The data are normalized to the maximum value of the internalized/surface ¹²⁵I-EGF ratio at the 10-min time point. Mean values with SDs from two independent experiments are presented. The difference between internalization rates in vehicle- and BIRB796-treated cells is not statistically significant. (D–F) HeLa/FAP-EGFR cells were pretreated for 90 min with DMSO (vehicle) or 100 nM BIRB796, labeled with MG-B-Tau, and stimulated with 0.5 ng/ml or 20 ng/ml EGF-Rh for 15 min. Cells were fixed, and 3D imaging through 640-nm (green, EGFR) and 561-nm (red, EGF-Rh) channels was performed. In D, single confocal sections are shown. Scale bars, 10 µm. In E, the ratio of EGF-Rh and MG-B-Tau fluorescence (EGF-Rh/FAP-EGFR) in individual (ind.) endosomes was calculated in 3D images generated as in D. Median and quartiles are shown on the violin graph; n is >5,000 endosomes. In F, the interpretation of the data in E is proposed. Stimulation with 0.5 ng/ml EGF results in internalization of EGF:EGFR dimers and monomeric ligand-free receptors (in a p38-dependent manner) to the same endosomes. Inhibition of p38 results in endocytosis of only EGF:EGFR complexes but not ligand-free receptors, which leads to an apparent increase of the EGF/EGFR ratio per endosome. When cells are stimulated with the saturating concentration of EGF (20 ng/ml), p38-dependent internalization of free EGFR is negligible, and therefore, BIRB796 does not change the EGF:EGFR ratio in endosomes. A considerable fraction of EGF:EGFR dimers with 1:2 stoichiometry may exist in cells treated with 0.5 ng/ml EGFR (Macdonald and Pike, 2008).