Differential effects of EGFR ligands on endocytic sorting of the receptor

Abstract

Endocytic downregulation is a pivotal mechanism turning off signalling from the EGF receptor (EGFR). It is well established that whereas EGF binding leads to lysosomal degradation of EGFR, transforming growth factor (TGF)-alpha causes receptor recycling. TGF-alpha therefore leads to continuous signalling and is a more potent mitogen than EGF. In addition to EGF and TGF-alpha, five EGFR ligands have been identified. Although many of these ligands are upregulated in cancers, very little is known about their effect on EGFR trafficking. We have compared the effect of six different ligands on endocytic trafficking of EGFR. We find that, whereas they all stimulate receptor internalization, they have very diverse effects on endocytic sorting. Heparin-binding EGF-like growth factor and Betacellulin target all EGFRs for lysosomal degradation. In contrast, TGF-alpha and epiregulin lead to complete receptor recycling. EGF leads to lysosomal degradation of the majority but not all EGFRs. Amphiregulin does not target EGFR for lysosomal degradation but causes fast as well as slow EGFR recycling. The Cbl ubiquitin ligases, especially c-Cbl, are responsible for EGFR ubiquitination after stimulation with all ligands, and persistent EGFR phosphorylation and ubiquitination largely correlate with receptor degradation.

Figure 1. EGFR ligands differentially affect EGFR endocytosis and recycling

A) EGFR internalization following stimulation with increasing concentrations of various EGFR ligands. HEp2 cells were incubated on ice with increasing concentrations of ligand for 1 h, washed, and incubated at 37°C for 15 min. Subsequently, the amount of EGFR at the cell surface was determined by FACS analysis. Data points represent mean + /− SEM for three independent experiments. B) Time–course of EGFR internalization and recycling following stimulation with different EGFR ligands. Cells were incubated on ice with 10 or 100 nm of ligands as indicated, washed, and incubated at 37°C for different time periods. The amount of EGFR present at the cell surface was determined by FACS analysis. Data points represent mean + /− SEM for four independent experiments.

Figure 2. EGFR localization to early endosomes following ligand stimulation

HEp2 cells were incubated on ice with 10 nm (EGF, TGF-α, HB-EGF, and BTC) or 100 nm (AR and EPI) of ligand, washed, and incubated at 37°C for different time periods. Cells were fixed and labelled for EGFR and the early endosome marker EEA1. (A) shows confocal microscopy images of representative cells after 15 min of EGFR internalization. The lower right panel shows a magnified field of the area boxed in the panel to the left. Bars, 10 μm. (B) shows a quantification of the amount of EGFR colocalizing with EEA1 in an average of 50–58 cells for each time-point + /− SEM.

Figure 3. EGFR ligands differentially stimulate EGFR degradation

Cells were incubated with ³⁵S-methionine/cysteine for 1–2 h followed by unlabelled medium for 3 h. The cells were subsequently incubated on ice with 10 nm (EGF, TGF-α, HB-EGF, and BTC) or 100 nm (AR and EPI) of ligand, washed, and incubated at 37°C for 2 or 6 h. Cells were lysed, immunoprecipitated EGFR was separated by gel electrophoresis (upper image), and the amount of radioactive EGFR was quantified by PhosphorImager. The column bar graph shows mean + /− SEM for quantification of four independent experiments. Statistical difference from the 0 h control as determined by Student's t-test is indicated by stars (*: p < 0.05; ** p < 0.01).

Figure 4. EGFR localization to lysosomes following ligand stimulation

A) HEp2 cells were incubated on ice with 10 nm (EGF, TGF-α, HB-EGF, and BTC) or 100 nm (AR and EPI) of ligand, washed, and incubated at 37°C for 60 min. Cells were fixed and labelled for EGFR and the lysosomal marker Lamp1. Representative confocal microscopy images are shown. Note that in the case of TGF-α and EPI, relatively little EGFR is found inside the cell, whereas EGFR is distinct at the plasma membrane (arrow heads). The lower panel shows a magnified field of the areas boxed in the panels above. Arrows show colocalization. Bars, 10 μm. B) HEp2 cells were incubated on ice with 10 nm BTC, washed, and incubated at 37°C in the presence of 500 nm bafilomycin A1 for 120 min. Cells were fixed and labelled for EGFR and Lamp1. Note that bafilomycin A1 induces an up-concentration of EGFR in lysosomes following BTC stimulation.

Figure 5. EGFR binding of HB-EGF, BTC, and AR is acid-resistant

HEp2 cells were incubated with 10 nm (EGF, TGF-α, HB-EGF, and BTC) or 100 nm (AR) of unlabelled ligand on ice for 6 h followed by a brief wash with buffers of various pH. The number of ligand-free EGFRs was subsequently determined by incubation with ¹²⁵I-EGF for 1 h on ice. The graph shows the relative increase in number of free binding sites following acid wash at different pH. Data points represent mean + /− SEM for three to six independent experiments.

Figure 6. Differential effects of EGFR ligands on EGFR ubiquitination and interaction with c-Cbl

A) HEp2 cells were incubated 1 h on ice with 10 nm (EGF, TGF-α, HB-EGF, and BTC) or 100 nm (AR and EPI) ligand, washed, and incubated at 37°C for 5 min. Cells were lysed in RIPA buffer, EGFR was immunoprecipitated and the amount of EGFR ubiquitination determined by western blotting for ubiquitin. The column bar graph shows the average intensity of ubiquitin signal quantified from six to seven independent experiments + /− SEM. B–C): HEp2 cells were incubated 1 h on ice with 10 nm (EGF, TGF-α, HB-EGF, and BTC) or 100 nm (AR and EPI) ligand, washed, and incubated at 37°C for 0–10 or 15 min (B and C, respectively). Cells were lysed in co-IP buffer; EGFR was immunoprecipitated; and co-precipitation of c-Cbl or Grb2 was determined by western blotting. The western blots shown are representative of three to four independent experiments.

Figure 7. Ubiquitination of EGFR is Cbl ubiquitin ligase-dependent for all ligands

HEp2 cells were transfected with scrambled siRNA or c-Cbl and/or Cbl-b siRNA twice with 48 h interval. S = scrambled siRNA, C = c-Cbl siRNA, B = Cbl-b siRNA, and C + B = c-Cbl and Cbl-b siRNA. A): 48 h after the last siRNA transfection, HEp2 cells were incubated for 1 h on ice with 10 nm (EGF, TGF-α, HB-EGF, and BTC) or 100 nm (AR) ligand, washed, and incubated at 37°C for 15 min. Cells were lysed in RIPA buffer, EGFR was immunoprecipitated, and the amount of EGFR ubiquitination determined by western blotting for ubiquitin. The column bar graph shows the average intensity of ubiquitin signal quantified from three independent experiments + /− SEM. B): 48 h after last siRNA transfection, HEp2 cells were lysed and the amounts of c-Cbl, Cbl-b, and transferrin receptor (TfR) were determined by western blotting. It is seen that c-Cbl is knocked down after treatment with c-Cbl siRNA, but c-Cbl siRNA also has an effect on Cbl-b. Cbl-b siRNA is specific for Cbl-b. TfR is used as a marker of protein levels.

Figure 8. Differential effects of EGFR ligands on Tyr 1173 EGFR phosphorylation kinetics

HEp2 cells were incubated 1 h on ice with 10 nm (EGF, TGF-α, HB-EGF, and BTC) or 100 nm (AR and EPI) ligand, washed, and incubated at 37°C for 0, 1.5, 5, 10 or 20 min. Cells were lysed in RIPA buffer, and the amount of EGFR phosphorylation determined by western blotting for phosphorylation on Tyr1173. TfR is used as a marker of protein levels. The western blots shown are representative of three independent experiments.

Figure 9. Model for EGFR trafficking after stimulation with the six ligands

The model shows how EGFR is either recycled or transported to lysosomes after stimulation with the different ligands. See discussion for further details. PM, plasma membrane.