Induced endoplasmic reticulum (ER) stress and binding of over-expressed ER specific chaperone GRP78/BiP with dimerized epidermal growth factor receptor in mammalian cells exposed to low concentration of N-methyl-N'-nitro-N-nitrosoguanidine.

Previously we have shown that alkylating agent N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) can induce the clustering of epidermal growth factor receptor (EGFR) in human amnion FL cells. However, the biological consequence of MNNG-induced clustering is different from that of epidermal growth factor (EGF)-induced clustering. In addition, MNNG strongly blocks the autophosphorylation of EGFR in response to its ligand, we speculate it might be due to the altered conformation of EGFR by MNNG alkylation, or the binding of some unknown suppressive molecules to EGFR, which could lead to the down-regulation of EGFR pathway. In this study, we further demonstrated that EGFR could not be phosphorylated by EGF in lysates prepared from MNNG-pretreated cell. In addition, it was found that the clustering of EGFR induced by low concentration (<or=1 microM) of MNNG on cell surface was indeed the dimerization of EGFR; however, unlike EGF treatment, the dimerization initiated by MNNG was irreversible upon mild-acid washing. Besides, in accordance with our previous results, the recruitment of adaptor proteins Grb-2/Sos1, which play key roles in activating ensuing RAS-MAPK pathway, was also suppressed. Interestingly, we found that endoplasmic reticulum (ER) stress participates in MNNG-induced down-regulation of EGFR signaling. It was demonstrated that the ER specific chaperone, glucose-regulated protein 78 (GRP78/BiP) formed a stable complex with EGFR in MNNG-treated cell. However, in the presence of 1mM ATP, EGF induced phosphorylation of tyrosine residues of EGFR can be revitalized in lysates prepared from MNNG pretreated cells. We also found that MNNG can induce ER stress or unfolded protein response (UPR) which is characterized by induced expression of ER-stress response proteins, such as GRP78/BiP, GADD153/CHOP, and activation of ER-localized caspase-12. Therefore, it is concluded MNNG is also an ER stress inducer. In MNNG-exposed cells, ER stress plays an important role in the blockage of EGFR-signaling pathway by forming a stable complex of EGFR/BiP.