The present study indicated that gene and protein expression levels of EGFR before EGF stimulation did not differ significantly between RBE and MMNK-1 cell lines. However, whereas EGFR protein expression was markedly down-regulated under sustained EGF stimulation in MMNK-1 cells, a considerable amount of endocytosed EGFR was retained in early endosomes in RBE cells. EGFR over-expression on the plasma membrane was observed in this cell line as well.
Clathrin-coated vesicles containing EGF-EGFR complexes release their coat and fuse with early endosomes quickly following endocytosis. Since the EGF-EGFR complexes do not dissociate in the early endosome, EGFR remains phosphorylated and associated with Cbl . Cbl mediates the interaction between EGFR and endosomal sorting complex required for transport (ESCRT) machinery and promotes EGFR sorting in multi-vesicular bodies (MVB) . An early dissociation of the EGF-EGFR complex because of abnormally low pH in endosomes or an unstable association between EGF and EGFR-HER2 heterodimers enhances the recycling of unoccupied EGFR . In our study, EGFR was retained in early endosomes and kept phosphorylated at Tyr1068 in RBE cells. Thus, early dissociation of the EGF-EGFR complex was not considered to be the mechanism of impaired degradation and enhanced recycling of EGFR in RBE cells.
Cbl-mediated ubiquitination is critical for EGFR early endosome exit, lysosome sorting, and degradation [18, 19]. Since low levels of EGFR ubiquitination and poor Cbl association were observed in RBE cells, we considered these factors to be attributed to the dramatically diminished EGFR degradation in RBE cells. Reduced Cbl association and/or impaired EGFR ubiquitination could be linked with a Cbl mutation in the RING Finger domain, especially cysteine 381 (C381), which is the first cysteine of the C3HC4 zinc finger motif , or at the RING finger C-terminal flank, especially valine 431 (V431) and phenylalanine 434 (F434) . Apart from mutations in c-Cbl, the loss of the Cbl docking site on EGFR (pY1068 and pY1045) for numerous reasons, such as EGFR mutations in the tyrosine kinase domain [32, 33] or mutations at ubiquitination sites of EGFR , could also lead to reduced Cbl association and/or impaired EGFR ubiquitination. In RBE cells, no mutations at C381, V431 or F434 of Cbl, the tyrosine kinase domain or Tyr1068 or 1045 residues of EGFR, or of the extracellular domain that binds to ligands (exon 2 ~ 16)  were identified. Meanwhile, we observed that transfected wtEGFR behaved similarly to endogenous EGFR; Myc-tagged wtEGFR in pcDNA3.1, a kind gift from Dr. Tokuzou Arao , was transfected into RBE cells and revealed that Myc-tagged wtEGFR was retained in early endosomes and was not sorted into late endosomes/lysosomes (data not shown). This verified that endogenous EGFR was not the reason for impaired EGFR degradation in RBE cells. However, unlike Tyr1068 that could be phosphorylated normally, Tyr1045 could not be phosphorylated following EGF stimulation. Combining this with the data from Grøvdal et al. , who described that a direct association of c-Cbl with EGFR pY1045 was important for MVB sorting of EGFR, we surmised that aberrant EGFR sorting into lysosomes in RBE cells was caused by an impaired association between c-Cbl and EGFR through pY1045.
Hypophosphorylation of Tyr1045 has been reported in non-small cell lung cancers (NSCLCs) bearing EGFR mutations in the tyrosine kinase domain  and in an EGFRvIII variant bearing an internal in-frame deletion in the extracellular domain . However, no EGFR mutation was identified in our RBE cells, nor was Tyr1045 of the transfected wtEGFR seen to be phosphorylated (data not shown). Willmarth et al.  reported similar findings caused by stimulation with one member of the EGF family, Amphiregulin (AR), in SUM149 human breast cancer cells. In their study, AR activation of EGFR resulted in increased steady-state levels of the receptor that accumulated at the cell surface as a result of decreased phosphorylation of Tyr1045 on EGFR (wild type) and a resultant failure to ubiquitinate . However, the mechanism of Tyr1045 hypophosporylation without mutation as in RBE cells remains unclear. Apart from an abnormality of EGFR or its ligand, a diminished EGF-EGFR interaction affinity and reduced receptor-associated tyrosine kinase activity caused by phosphorylation of EGFR threonine residues through protein kinase C (PKC)-dependent  or independent pathways  may be speculated as the mechanism of Tyr1045 hypophosphorylation in RBE cells. Uncovering the mechanism of Tyr1045 hypophosphorylation is of great importance in restoring EGFR degradation and negatively controlling EGFR over-activation in RBE cells.
Lastly, we employed two methods to verify the role of EGFR recycling in cell membrane EGFR over-expression: monensin treatment [24, 42] and Rab11a protein depletion . Rab proteins regulate various steps in recycling: Rab4 regulates fast/direct recycling from the early endosome to the cell membrane , and Rab11a regulates recycling from the deeper perinuclear recycling compartment . Monensin treatment blocks recycling from both the early endosome and perinuclear recycling compartment . Suppressed cell surface EGFR expression by monensin treatment or Rab11a depletion indicated that enhanced recycling occurred at least in the perinuclear recycling compartment in RBE cells with EGF stimulation. Furthermore, early endosome retainment of EGFR without recycling inhibition showed that recycling in RBE cells did not significantly take place through fast/direct recycling from early endosomes.