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Differential effects of EGFR ligands on endocytic sorting of the receptor.

Roepstorff K, Grandal MV, Henriksen L, Knudsen SL, Lerdrup M, Grøvdal L, Willumsen BM, van Deurs B - Traffic (2009)

Bottom Line: 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.Amphiregulin does not target EGFR for lysosomal degradation but causes fast as well as slow EGFR recycling.

View Article: PubMed Central - PubMed

Affiliation: Department of Cellular and Molecular Medicine, The Panum Building, Faculty of Health Sciences, University of Copenhagen, Copenhagen, Denmark.

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.

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EGFR ligands differentially stimulate EGFR degradationCells 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).
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fig03: EGFR ligands differentially stimulate EGFR degradationCells 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).

Mentions: To test how the various EGFR ligands affect receptor degradation, two different methods were applied. Cells were stimulated with ligand for 1 h on ice, washed, and chased for 0–8 h in the presence of cycloheximide to inhibit de novo EGFR synthesis. The cells were subsequently lysed, and the amount of EGFR determined by ELISA (Figure S3). Alternatively, to avoid the use of cycloheximide, cells were pulse-labelled with 35S-methionine, stimulated with ligand for 1 h on ice, washed, and incubated for 2 or 6 h at 37°C. EGFR was subsequently immunoprecipitated and the amount of 35S-labelled EGFR quantified by PhosphorImaging of an SDS-PAGE gel (Figure 3). As can be seen, stimulation with TGF-α, EPI, or AR does not lead to significant degradation of EGFR. Stimulation with either EGF or HB-EGF leads to degradation of 40–60% of the cellular EGFR, whereas stimulation with BTC leads to degradation of approximately 70% of the cellular EGFR.


Differential effects of EGFR ligands on endocytic sorting of the receptor.

Roepstorff K, Grandal MV, Henriksen L, Knudsen SL, Lerdrup M, Grøvdal L, Willumsen BM, van Deurs B - Traffic (2009)

EGFR ligands differentially stimulate EGFR degradationCells 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).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2723868&req=5

fig03: EGFR ligands differentially stimulate EGFR degradationCells 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).
Mentions: To test how the various EGFR ligands affect receptor degradation, two different methods were applied. Cells were stimulated with ligand for 1 h on ice, washed, and chased for 0–8 h in the presence of cycloheximide to inhibit de novo EGFR synthesis. The cells were subsequently lysed, and the amount of EGFR determined by ELISA (Figure S3). Alternatively, to avoid the use of cycloheximide, cells were pulse-labelled with 35S-methionine, stimulated with ligand for 1 h on ice, washed, and incubated for 2 or 6 h at 37°C. EGFR was subsequently immunoprecipitated and the amount of 35S-labelled EGFR quantified by PhosphorImaging of an SDS-PAGE gel (Figure 3). As can be seen, stimulation with TGF-α, EPI, or AR does not lead to significant degradation of EGFR. Stimulation with either EGF or HB-EGF leads to degradation of 40–60% of the cellular EGFR, whereas stimulation with BTC leads to degradation of approximately 70% of the cellular EGFR.

Bottom Line: 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.Amphiregulin does not target EGFR for lysosomal degradation but causes fast as well as slow EGFR recycling.

View Article: PubMed Central - PubMed

Affiliation: Department of Cellular and Molecular Medicine, The Panum Building, Faculty of Health Sciences, University of Copenhagen, Copenhagen, Denmark.

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.

Show MeSH
Related in: MedlinePlus