Extended Data Fig. 2. Cross-linking studies of ligand-induced sEGFR dimerisation.

a. Quantitation and summary of SAXS I(0)/c measurements reported in Fig. 2 across multiple repeats. For sEGFR^WT, only EGF (black) doubles the I(0) value, representing selective EGF-induced dimerisation. By contrast, both EREG (magenta) and EGF (black) induce dimerisation of sEGFR harbouring L38R (red), R84K (green), A265V (blue) or A265T (gold) mutations – with EREG-induced dimerisation of A265 variants appearing slightly less robust. Data represent mean I(0)/c ± SD for 10 repeats (WT + EGF), 6 repeats (WT + EREG), 4 repeats (A265V + EGF and A265V + EREG), 3 repeats (L38R + EREG, R84K + EGF, R84K + EREG, A265T + EGF, A265T + EREG), and 2 repeats (L38R + EGF) – where a repeat corresponds to a biologically independent sample. An additional single experiment was undertaken for T239P + EREG, which showed an elevation of I(0)/c by 1.44 fold. The degree of sEGFR dimerisation for EGF and EREG is significantly different only for WT (p < 0.0001) and A265T (p < 0.0001). p values are from unpaired two-tailed Student’s t-tests.

We estimate based on the SAXS data in Figs. 2a-e that the GBM mutations studied here strengthen dimerisation of EREG-bound sEGFR by several hundred fold based on the following considerations. Since sEGFR^WT at 70 μM shows no dimerisation when saturated with EREG, the dissociation constant (K_D) for dimers of the EREG:sEGFR complex must be >450 μM (assuming that we could detect a minimum of 10% dimer by SAXS). By contrast, the complete dimerisation seen for the EREG:sEGFR complex with mutated variants (when corrected for differences in ligand-binding affinities) places a lower limit of ~0.7 μM on K_D for these dimers. Thus, GBM mutations must enhance dimerisation of the EREG:sEGFR complex by at least ~650-fold.

b. Representative crosslinking analysis of sEGFR dimerisation (n = 3 biologically independent samples for each mutated variant). Different sEGFR variants at 5 μM were incubated alone or with the noted ligand (EGF or EREG) at 6 μM, and subjected to 100 μM DSS for 30 min (see Methods). Samples were then subjected to SDS-PAGE and stained with Coomassie Blue. Dimer and monomer bands are marked (note the shift in monomer band position following ligand cross-linking). EGF promotes dimerisation of all variants. EREG fails to increase sEGFR^WT dimerisation above that seen without ligand, but detectably enhances dimerisation of all variants with GBM mutations, consistent with the SAXS data shown in Fig. 2. See Supplementary Figure 1 for gel source data, and (c) for quantitation and reproducibility information.

c. Quantitation of data in (b), including additional repeats for each variant. For sEGFR^WT, EGF induces substantially more dimerisation than EREG (p < 0.0001), whereas the difference between EGF and EREG is not significant for L38R (p = 0.0522) or A265T (p = 0.0577), and only just reaches statistical significance for R84K (p = 0.0410) and A265V (p = 0.0377). p values are for unpaired two-tailed Student’s t-tests. Data in the graph represent mean ± SD for 6 repeats (WT + EGF and WT + EREG), or 3 repeats (A265V + EGF, A265V + EREG, R84K + EGF, R84K + EREG, A265T + EGF, A265T + EREG, L38R + EGF, and L38R + EREG), where repeats refer to biologically independent samples.

d. Top panel: WT and R84K-sEGFR (5 μM) were crosslinked alone or with the noted ligands (EGF, AREG EREG, or EPGN) at 6 μM. Middle panel: as in Top panel, but with 60 μM ligand. Bottom panel: Crosslinking studies were performed with 60 μM ligand added to sEGFR^WT and 6 μM added to sEGFR^R84K, to account for affinity differences. Data are representative of three biologically independent samples. See Supplementary Figure 1 for gel source data.

e. As for (d), but using sEGFR^A265V with 6 μM or 60 μM ligand as marked, for 5 biologically independent samples of sEGFR^A265V with EGF and EREG, but n = 2 for AREG and EPGN. See Supplementary Figure 1 for gel source data.