Enhanced dimerization drives ligand-independent activity of mutant epidermal growth factor receptor in lung cancer

Abstract

Epidermal growth factor receptor kinase mutations drive oncogenesis, but the molecular mechanism of pathological signal initiation is poorly understood. Using high-resolution microscopy methods, the authors reveal that these kinase mutations induce structural changes in the receptor ectodomain that lead to enhanced, ligand-independent dimerization.

EGFR Ligands Differentially Stabilize Receptor Dimers to Specify Signaling Kinetics

“…Analyzing trajectories from multiple single quantum dots (see Figure S5C for examples) gave the ensemble mean square displacement (MSD) plots shown in Figure 5D, from which diffusion coefficients ( D ) were calculated (see inset for 95% confidence intervals). Adding saturating EGF reduced the mean D value by 2.5-fold (from 0.036 to 0.014 μm 2 s -1 ), consistent with previous reports (Chung et al, 2010; Valley et al, 2015). Saturating the receptor with epiregulin or epigen (at 20 μM) also reduced EGFR mobility, but to a smaller extent – reducing D by just under 1.9-fold.…”

“…The same increase is seen for the other high-affinity EGFR ligands, TGFα, HB-EGF and BTC (Figures 4A and S4). By contrast, I (0)/c increases only ∼1.3-fold when saturating epiregulin or epigen is added, which is the result expected when ligand binds without dimerization – as seen when EGF is added to ‘dimarm*’ sEGFR501, a well-studied variant with six mutations that abolish dimerization (Dawson et al., 2005; Garrett et al, 2002; Valley et al, 2015). Failure to detect dimerization in this assay (with 70 mM sEGFR501) places a lower limit of ∼200 μM on K d , compared with ∼3 μM for TGFα/sEGFR501 (Dawson et al, 2005) – arguing that sEGFR501 dimers induced by epiregulin or epigen are >60-fold weaker than those induced by TGFα.…”

“…First, both ligands can activate EGFR in cells without the need for other ErbB receptors. Second, dimerization arm (dimarm*) mutations that abolish activation by EGF (Valley et al, 2015) also eliminate EGFR activation by epiregulin or epigen (Figure 4B), arguing that these ligands rely on the same EGFR dimerization interface. Third, despite the fact that both ligands bind EGFR ∼100-fold more weakly than EGF, and induce substantially weaker sEGFR dimers, epiregulin and epigen appear to be full agonists of receptor phosphorylation – arguing that their distinct signaling properties do not simply reflect altered potency.…”

“…We labeled N-terminally HA-tagged EGFR, stably expressed in CHO cells, with quantum dots (QD605 and QD655) for two-color single particle tracking (SPT) studies as previously described (Low-Nam et al, 2011; Valley et al, 2015). Analyzing trajectories from multiple single quantum dots (see Figure S5C for examples) gave the ensemble mean square displacement (MSD) plots shown in Figure 5D, from which diffusion coefficients ( D ) were calculated (see inset for 95% confidence intervals).…”

Activation of the EGF Receptor by Ligand Binding and Oncogenic Mutations: The “Rotation Model”

“…Using spectrally distinct quantum dots (Qdots) conjugated to either EGF or a monovalent Fab antibody, transition between monomeric and dimeric EGFR was observed [54,55]. This transition is likely to occur between EGFR dimers and tetramers, since only one Fab molecule conjugated with a Qdot seems to bind EGFR dimers because of its large size.…”