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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 affect EGFR endocytosis and recyclingA) 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.
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fig01: EGFR ligands differentially affect EGFR endocytosis and recyclingA) 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.

Mentions: To compare the ability of the different EGFR ligands to induce internalization of EGFR, we used the common approach of pre-binding ligand on ice, thereby looking at a synchronized wave of receptor internalization (7,14,15). We initially determined the incubation time necessary to obtain maximal ligand-binding. First,125I-EGF binding to HEp2 cells as a function of time was tested, and in Figure S1A it is seen that the equilibrium is reached after only 15 min. To investigate the binding kinetics of the other ligands to EGFR, cells were incubated with unlabelled ligand on ice for up to 2 h. Thereafter, cells were briefly washed followed by binding of 125I-EGF for 30 min. The level of 125I-EGF binding reflects unbound EGFRs. In Figure S1B it is seen that all ligands have reached maximal binding well before 1 h. Thus, to investigate EGFR internalization, HEp2 cells were incubated with increasing ligand concentrations on ice for 1 h followed by washing and 15 min chase at 37°C. Subsequently, the amount of EGFR present at the cell surface was determined by fluorescence-activated cell sorter (FACS) analysis. As seen in Figure 1A, all six ligands induced EGFR internalization. However, the degree of internalization varied between the ligands. HB-EGF and BTC were very efficient at inducing EGFR internalization. At saturating concentrations of ligand, 70–80% of cell surface EGFR was internalized upon the 15 min chase. EGF and TGF-α both induced internalization of approximately 50% of the receptors at saturating concentrations of ligand. In case of EPI and AR, the internalization did not reach saturation with the ligand concentrations used. This is in accordance with the fact that these two ligands have a lower affinity for EGFR compared with the other ligands (16,17).


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 affect EGFR endocytosis and recyclingA) 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.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig01: EGFR ligands differentially affect EGFR endocytosis and recyclingA) 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.
Mentions: To compare the ability of the different EGFR ligands to induce internalization of EGFR, we used the common approach of pre-binding ligand on ice, thereby looking at a synchronized wave of receptor internalization (7,14,15). We initially determined the incubation time necessary to obtain maximal ligand-binding. First,125I-EGF binding to HEp2 cells as a function of time was tested, and in Figure S1A it is seen that the equilibrium is reached after only 15 min. To investigate the binding kinetics of the other ligands to EGFR, cells were incubated with unlabelled ligand on ice for up to 2 h. Thereafter, cells were briefly washed followed by binding of 125I-EGF for 30 min. The level of 125I-EGF binding reflects unbound EGFRs. In Figure S1B it is seen that all ligands have reached maximal binding well before 1 h. Thus, to investigate EGFR internalization, HEp2 cells were incubated with increasing ligand concentrations on ice for 1 h followed by washing and 15 min chase at 37°C. Subsequently, the amount of EGFR present at the cell surface was determined by fluorescence-activated cell sorter (FACS) analysis. As seen in Figure 1A, all six ligands induced EGFR internalization. However, the degree of internalization varied between the ligands. HB-EGF and BTC were very efficient at inducing EGFR internalization. At saturating concentrations of ligand, 70–80% of cell surface EGFR was internalized upon the 15 min chase. EGF and TGF-α both induced internalization of approximately 50% of the receptors at saturating concentrations of ligand. In case of EPI and AR, the internalization did not reach saturation with the ligand concentrations used. This is in accordance with the fact that these two ligands have a lower affinity for EGFR compared with the other ligands (16,17).

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