Figure 7. EHD3 increases ligand-induced EGFR ubiquitination and lysosomal degradation.

(A) Blockade of recycling events shifts the peak of EGFR activation only in absence of EHD3. U251tetEHD3 cells, induced (+Dox) or not (−Dox) to express EHD3 by Dox treatment, and serum starvation-primed overnight, were pre-treated with Mon A and CHX for 30 min prior to performing an EGF stimulation kinetic. (B) Densitometric quantitation of a representative immunoblot. The results are shown as relative units (r.u.) of the pEGFR levels in Dox-induced versus the control without Dox treatment. (C) Pulse-chase analysis of EGFR recycling. U251tetEHD3 cells were induced (+Dox) or not (−Dox) to express EHD3 by Dox treatment, serum starvation-primed overnight. Cells were subsequently stimulated with EGF and pulsed for 15 min, followed by a 60 min chase, before a second EGF stimulation for the indicated times. (D) The histogram depicts the densitometric quantitation of a representative immunoblot. The results are shown as relative units (r.u.) of the pEGFR levels in Dox-induced versus the control without Dox treatment, after either the pulse or the 15 min ligand stimulation. (E) Blockade of lysosomal degradation significantly blocks attenuation of EGFR signal. Similar to C, the lysosome inhibitor Chloroquine was used in presence of CHX, to pre-treat U251tetEhd3 cells prior to EGF stimulation. In A–C, whole cell lysates were analyzed by immunoblotting for the levels of total EGFR, tyrosine phosphorylated EGFR (pEGFR), EHD3 and GAPDH. (F) EHD3 promotes ligand-dependent ubiquitination of EGFR. Dox-induced and control U251tetEhd3 cells were stimulated with EGF for different time points and whole cell extracts were used to immunoprecipitate EGFR and immunoblot with an anti-Ubiquitin antibody.