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3.2.2.2
Syntenin-1 modulates ephrinB2 internalization
To analyze ephrinB2 internalization, we performed biotinylation assay. We decided to perform the experiments in the presence or absence of EphB4/Fc stimulation, as the latter leads to ephrinB2 internalization and degradation (see section 1.1.4.7 of the Introduction). The biotinylation experiment is used to determine the percentage of internalized cell surface protein compared to the overall amount at any given time-point (figure 3.13A). Hence, we stimulated cells with 5 µg/ml of EphB4/Fc or anti-Fc (as a control) to maintain the same experimental settings used in the signalling experiments. However, we did not serum starve cells prior to stimulation to avoid indirect effects on ligand internalization. In these experiments we stimulated cells for 15 and 60 minutes. The earlier time-point allowed us to analyse changes in EphB4-driven ligand internalization, while the later time-point was chosen to correlate ligand phosphorylation and ephrinB2 internalization.
The WB data in figure 3.13A show the levels of total biotinylated ephrinB2 (lanes 1-4) and of internalized biotinylated ephrinB2 (lanes 5-8) after EphB4/Fc or anti-Fc stimulation. As expected, EphB4/Fc stimulation in MCF7 Tet-ephrinB2 cells increased the internalization levels of ephrinB2 (lanes 7 and8) compared to non-stimulated conditions (lanes 5 and 6). Surprisingly, ephrinB2/G internalization levels were not affected by EphB4/Fc stimulation (lanes 7 and 8) and remained unchanged when compared to non-stimulated condition (lanes 5 and 6). EphrinB2/∆V internalization levels appeared similar to ephrinB2 under non- stimulated conditions (lanes 5 and 6), but did not notably increase after EphB4/Fc stimulation (lanes 7 and 8). Interestingly, in syntenin-1 depleted cells, ephrinB2 internalization levels appeared higher in both non-stimulated (lanes 5 and 6) and EphB4/Fc stimulated conditions (lanes 7 and 8). The histogram in figure 3.13B illustrates the WB densitometric analysis
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presented as percentage of internalized relative to total protein. In control samples, we noticed that ligand internalization occurred at a slow speed. At 15 minutes, ephrinB2 internalization was 2% and barely increased to 3% after 60 minutes. Similarly, ephrinB2/G internalization was 1% at 15 minutes and less than 2% at 60 minutes. Interestingly, internalization levels of both ephrinB2/ΔV and ephrinB2 expressed in syntenin-1 depleted cells were slightly higher under non-stimulated conditions, reaching 3% after 15 minutes and over 4% after 60 minutes. Overall, these results on control samples suggest that wild type and ephrinB2 mutants remain stable on the cell surface and do not undergo rapid internalization under non-stimulated conditions. As expected, EphB4/Fc stimulation notably increased ephrinB2 internalization, which reached 4% at 15 minutes and 8% at 60 minutes. Surprisingly, ephrinB2/G internalization was not affected by EphB4/Fc stimulation, as internalization levels remained significantly lower, compared to ephrinB2, at 1% after 15 minutes and 2% after 60 minutes. In MCF7 Tet-ephrinB2/shSyntenin-1 cells, EphB4/Fc stimulation increased ephrinB2 internalization to only 4% after 15 minutes, but more than 10% after 60 minutes. EphrinB2/ΔV internalization was mostly unaffected by EphB4/Fc stimulation, as internalization levels remained at 3% after 15 minutes and barely rose over 5% after 60 minutes, thus remaining significant lower compared to ephrinB2. Summarizing, ephrinB2 internalization doubled at 15 minutes and nearly quadrupled at 60 minutes upon EphB4/Fc stimulation, while ephrinB2/G internalization remained unchanged. In MCF7 Tet- ephrinB2/shSyntenin-1 cells, ephrinB2 internalization barely rose after 15 minutes of EphB4/Fc stimulation, but increased 2.5 fold after 60 minutes. Following a similar pattern, ephrinB2/ΔV internalization remained unchanged after 15 minutes of EphB4/Fc stimulation, but rose nearly 1.5 fold after 60 minutes.
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Taken together, these results suggest that syntenin-1 modulates ephrinB2 internalization, especially upon EphB4 interaction. In particular, the pattern of ephrinB2 internalization in syntenin-1 depleted cells and the absent EphB4/Fc induced internalization of ephrinB2/G seemed to suggest that syntenin-1 might be involved in stabilizing ephrinB2 on the cell surface by binding to its PDZ binding motif and preventing internalization. Moreover, this effect was significantly observed at 60 minutes after EphB4/Fc stimulation. Additionally, disruption of the PDZ binding motif impairs ephrinB2 internalization, thus suggesting that other PDZ domain-containing partners might be involved in this process. However, the mechanism behind this regulation remains unclear.
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B)
Figure 3.13: EphB4/Fc-induced internalization of ephrinB2 is strongly impaired by the /G PDZ mutation; mildly impaired by the /ΔV PDZ mutation; and slightly increased in the presence of syntenin-1 knock down.
Analysis of internalization levels of wild type and ephrinB2 mutants under basal or EphB4/Fc stimulated conditions measured using biotinylation assay. MCF7 Tet-ephrinB2, MCF7 Tet-ephrinB2/G, MCF7 Tet- ephrinB2/ΔV and MCF7 Tet-ephrinB2/shSyntenin-1 cells were stimulated with 1 µg/ml doxycycline for 48 hours and subsequently labelled with sulfo-NHS-SS-biotin for 1 hour and stimulated with EphB4/Fc or anti-Fc antibody for 15 and 60 minutes at 37ºC. The experiment was conducted in duplicate: in the control set of samples, cells were lysed after stimulation and lysates were incubated with NeutrAvidin-conjugated agarose beads to pull down all biotinylated proteins; in the other set of samples (internalized) cells were treated with MESNA to remove biotin from cell surface proteins and lysates were incubated with NeutrAvidin-conjugated agarose beads to pull down internalized biotinylated proteins. A) Pulled-down proteins from both set of samples were separated on SDS-PAGE and ephrinB2 was detected with anti-ephrinB pAb. B) Densitometric analysis was conducted on blots of three independent experiments for each cell line and the ratio between control and MESNA-treated samples was calculated. Mean values and standard deviations of ratios were calculated and presented as percentage of internalized ephrinB2 (MESNA-treated) compared to overall biotinylated ephrinB2 (control) in the histogram. Statistical significance was determined by the two-way ANOVA multiple comparison test with the Tukey-Kramer post-test, using GraphPad Prism software, whereby significance was set at 0.05 (* P<0.05; **** P<0.0001). 0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00
15 min 60 min 15 min + EphB4/Fc 60 min + EphB4/Fc
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Internalization time points