A dramatic change of binding kinetics was observed when the cells were treated with AG1478, an ATP competitive inhibitor of EGFR [25], before exposure to EGF. AG1478 specifically inhibits EGFR kinase activity through binding to the ATP
AR A014418 situated in the cytoplasmic domain of EGFR thereby blocking subsequent downstream signaling molecules from docking to the EGFR. Therefore, it is anticipated that AG1478 should not have a major impact on the EGF–EGFR binding behaviors on the cell surface. However, AG1478 dramatically changed the EGF binding behavior in HeLa cells (Fig. 4D and E). The experimental data cannot be explained by the same EGF–EGFR binding kinetics developed for the two cell lines. Apparently in this case, monomeric forms of EGFR are present in very small numbers, which quickly dropped to almost zero upon EGF treatment. In order to explain the observed data, ligand binding pathway 2 has to be applied. In this scheme, the major difference is that upon pre-treatment with AG1478, pre-formed EGFR dimers no longer go through an intermediary activation step, and EGF can subsequently bind to the same dimer without the receptor going through further conformational changes. An excellent match was achieved between experimental data and pathway 2 ( Fig. 4D and E), but not with pathway 1 ( Supplementary Fig. 3). It is clear that the intermediate, activation step is no longer observed upon AG1478 treatment. It is possible that upon AG1478 binding, EGFRs tend to form dimers that have both EGF
binding sites well exposed, resulting in a rapid 2:2 EGF–EGFR complex formation. One thing to be expected in this scenario is that the number of pre-formed EGFR dimers on the cell surface could be greatly increased after the AG1478 pre-treatment. This is indeed confirmed by data fitting. The percentage of pre-formed dimers now is ～50% ( Fig. 4E) compared to the 3.75% pre-formed dimers with no AG1478 pretreatment ( Table 1). AG1478 induces massive formation of EGFR–EGFR dimers, which display a different molecular conformation from normally-functioning dimers where an intermediate activation step is no longer required prior to the second EGF binding.
