The absence of an intact ligand-binding domain in ΔEGFR, as well as the lack of response to EGF stimulation, suggested that it is defective in dimerization either as a homodimer or with family members capable of being activated by EGF (33
). Nevertheless, under some experimental circumstances, dimers have been reported, and it has been suggested that dimerization is required for ΔEGFR function (23
). Here we report for the first time the experimentally induced dimerization of a chimeric version of ΔEGFR, and observed a very significant enhancement of its phosphorylation levels. It is worth noting that the strategy that we used to force dimerization of EGFRs, first established by Dr. David Spencer and colleagues, did not result in abnormally high levels of phosphorylation of chEGFR when compared to EGF stimulation, suggesting that at least the initial impact of dimerization was similar regardless of whether it was mediated by the natural, extracellular domain and ligand or an artificial, intracellular domain and ligand. We conclude that if chΔEGFR was dimerized to any significant degree in terms of duration and stability in the absence of AP20187, the addition of this compound would not have been able to induce such a significant increased in phosphorylation and hence propose that naturally occurring ΔEGFR does not dimerize to the extent of the ligand-activated wild-type receptor. Interestingly, a recent report showed that a small subpopulation of ΔEGFR is dimerized, via a disulfide bond, and that the dimerized receptors are the most active as judged by tyrosine phosphorylation (35
). Taking these data together allows us to suggest that forced dimerization increases the activity of the ΔEGFR pool overall by bringing more receptors into this state, and stabilizing them there.
That ΔEGFR generates a signal that is different in some aspect from its wild-type counterpart is likely, as it is a much more potent oncogene in many model systems and is associated with worse outcomes for patients with glioblastoma. Our previous analysis comparing the signal of ΔEGFR and EGFR using phosphotyrosine directed shotgun phosphoproteomics suggested that there are no absolute differences, in that we were not able to identify any downstream signaling components that were exclusive to mutant or wild-type receptor. However, we did identify several phosphorylated tyrosines in target proteins that showed a stronger association with ΔEGFR, with signals reaching levels similar to what was observed when wild-type EGFR was acutely stimulated in serum starved cells, a likely non-physiological maximization of its signal. One of these targets was Y699 on pSTAT5b (22
), and we saw a similar preference for phosphorylation of this residue here, in that the signal in chΔEGFR cells was higher than in serum starved chEGFR cells (Supplementary Fig. S1
). However, the level of pSTAT5 was not increased further by addition of AP20187 to cells expressing chΔEGFR, suggesting that it may be maximal, and not limited by the low level signal of unstimulated ch EGFR. In other words, ΔEGFR may signal at an overall lower level, but reach near-maximal levels with a few select downstream targets, including STAT5. Other signaling components, such as GAB1 may still be preferentially connected (18
) but not maximally so (Supplementary Fig. S1
). Comparison of overall patterns of phosphorylation of a spectrum of tyrosine residues by an open approach (Supplementary Fig. S4
and Table. S1
) did not reveal any profound differences, further suggesting that changing signal strength does not noticeably change signal content, and so that the two are not tightly coupled. Indeed several of phosphorylations that we did observe to increase with forced dimerization of ch EGFR, such as those on paxillin, might be related to signaling through established downstream targets, in this case c-Met. This conclusion is supported by the analysis of cell behaviors. Increasing the signal of ch EGFR by forced dimerization enhances the existing stimulation of mitosis, but does not convert a non-migration/invasion-inducing ΔEGFR signal to a migration/invasion-inducing EGFR signal. While the close connection of c-Met and EGFR observed by others and us might suggest that EGFR should promote cell motility, a behavior associated mesenchymal transition of epithelial tumors, we do not observe it in glioma cells.
ΔEGFR signaling is constitutive with little receptor internalization. We previously hypothesized that the lack of ΔEGFR downregulation is due to the inability of the weakly active ΔEGFR to recruit the CIN85-Cbl complex that initiates endocytosis in EGFR (36
). This hypothesis would predict that enhancing the signal by forced dimerization would reestablish the active downregulation of the receptor, but we did not observe this for chΔEGFR, while the controls for chEGFR behaved as expected (). Our results strongly suggest that increased ΔEGFR activity is not sufficient to reestablish ΔEGFR downregulation, and so we conclude that the impaired downregulation of ΔEGFR is not due to its lower level of activity or lack of dimerization.
In summary our data provide the first direct, experimental test of ΔEGFR dimerization, and imply that it does not form strong, durable dimers under normal circumstances, which may be related to its low level of activity. Increasing the ΔEGFR activity by forced dimerization of chΔEGFR also provided the first opportunity to compare signals of ΔEGFR and EGFR at similar amplitudes by an open phosphoproteomic approach, which revealed no major qualitative differences. This in turn suggests that the differences that were observed by others and us previously (21
) related to the signals that the naturally occurring, overall low-activity ΔEGFR maintains at an elevated, near-maximal level; they stood out because they were high by comparison to the bulk of EGFR targets. Increasing the chΔEGFR signal did increase its ability to stimulate entry into S-phase and tumor growth rate as measured by survival of tumor bearing mice. Interestingly, however, the lack of downregulation of chΔEGFR was not altered by enhancing its activity, suggesting that this may be a fundamental characteristic of this mutant. The picture of ΔEGFR that emerges is of a receptor whose oncogenicity relies on a sustained signal, which although overall of low intensity, is capable of focusing on several select downstream nodes which are significantly activated. Raising its activity enhances the signal overall profoundly, but only to a modest degree on some of the already very active partners. The key to ΔEGFR’s oncogenicity may therefore very well be its inability to be downregulated, and this in turn suggests that altering this behavior may be key to targeting it therapeutically.