Figure 5. EHD3 modulates endosomal signaling and growth inhibitory effects of EGF ligand stimulation.

(A) EHD3 minimally affects total AKT and ERK signaling pathways upon ligand stimulation. U251tetEHD3 cells were induced (+Dox) or not (−Dox) to express EHD3 by Dox treatment, serum starvation-primed overnight prior to stimulation with EGF at different time points. Whole cell lysates were analyzed by immunoblotting for the levels of phosphorylated AKT (pAKT), phosphorylated ERK1/2 (pERK), EHD3 and GAPDH. (B) Selective activation of endosomal EGFR results in significant inhibition of AKT and ERK signaling. U251tetEhd3 cells were serum starved for 24 h, pre-treated with the EGFR tyrosine kinase inhibitor AG1478, prior to treatment on ice with EGF, thus allowing the endosomal internalization of non-activated EGF-EGFR complexes without triggering membrane-originated signaling. The endosomal EGFR signaling was subsequently activated by removing AG-1478 from the medium and incubating at 37C. Whole cell lysates from different time points were analyzed by immunoblotting for the levels of total EGFR, phosphorylated EGFR, pAKT, pERK, EHD3 and GAPDH. (C) Densitometric quantitation of a representative immunoblot. The results are shown as relative units (r.u.) of the pEGFR/EGFR ratio in Dox-induced versus the control without Dox treatment. (D) EHD3 sensitizes glioma cells to EGF-induced growth inhibition. U251tetEhd3 cells induced (+Dox) or not (−Dox) to express EHD3, were treated with an increasing dose of EGF for 48 h and an MTT cell growth assay was performed. The mean values and standard deviation are plotted as percentages of the levels in absence of EGF treatment. The assay was performed in triplicates and statistical significance was calculated with a two-tailed t test using GraphPad Prizm 5.0. *≤ 0.05; **≤ 0.01.