Ligand-Independent Activation of the EGFR by Lipid Raft Disruption

Abstract

Normal and immortalized keratinocytes demonstrate large aggregates of lipid rafts, detectable by membrane staining with fluorescently tagged cholera toxin (CTx). As lipid rafts are known to regulate the function of many surface receptors, we wished to investigate their impact on the EGFR in HaCaT cells. When rafts were disrupted by cholesterol sequestration with methyl-beta-cyclodextrin (MbetaCD) or filipin III, EGFR rearranged into approximately micrometer large clusters outside the CTx(bright) raft aggregates. These clusters contained high concentrations of activated, tyrosine-phosphorylated EGFR exhibiting greatly reduced mobility in the fluorescence recovery after photobleaching experiments. EGFR activation led to the stimulation of extracellular signal-regulated kinase 2, the phosphorylated form of which translocated to the nucleus and stimulated growth of the MbetaCD-treated cells. Experiments with the specific antagonistic antibody proved that the activation of EGFR by lipid raft disruption occurred without the participation of the ligand. We hypothesize that cholesterol depletion leads to the release of EGFR from the damaged rafts into the small confined areas of the membrane, where the receptor molecules are likely to be spontaneously activated owing to a very high density and/or separation from the inhibitory factors remaining in the surrounding portions of the membrane.

Transforming Growth Factor-β1 (TGF-β1)-stimulated Fibroblast to Myofibroblast Differentiation Is Mediated by Hyaluronan (HA)-facilitated Epidermal Growth Factor Receptor (EGFR) and CD44 Co-localization in Lipid Rafts

“…In contrast, the late activation remained in cells pretreated with nystatin, suggesting that the late signaling response of ERK1/2 and CaMKII leading to proliferation was indicative of another originating source that was not in lipid raft domains. This has been suggested in research highlighting how EGFR can be susceptible to ligand-free activation and can produce a proliferation response when it has been released to nonlipid raft areas of the cell membrane (56,57). However, it is not known whether this occurs in fibroblasts, in the absence of HA or CD44.…”

EGF induces coalescence of different lipid rafts

“…For these experiments we used the polyene antibiotic nystatin, which sequesters free cholesterol without considerable extraction of this sterol . Since another cholesterol-extracting agent, methyl-β-cyclodextrin, has previously been found to stimulate EGFR activity, we checked the effect of nystatin on several cellular functions (Lambert et al, 2006;Ringerike et al, 2002). These control experiments showed that nystatin treatment did not affect EGFR activity, cell shape, or the distribution of GM1, GPI-GFP or EGFR (supplementary material Figs S3 and S4).…”

The Inhibitory Effect of (−)-Epigallocatechin Gallate on Activation of the Epidermal Growth Factor Receptor Is Associated with Altered Lipid Order in HT29 Colon Cancer Cells

“…Furthermore, cholesterol may play a crucial role in modulating signaling from plasma membrane-associated receptors. Thus, the cholesterol-depleting agent methyl h-cyclodextrin (MhCD) can cause ligand-independent activation of the EGFR, apparently by altering membrane lipid organization (11)(12)(13)24). By contrast, depletion of cholesterol from vascular smooth muscle cells by treatment with MhCD led to inhibition of angiotensin IIstimulated phosphorylation of the EGFR (25), and treatment of prostate cancer cells with MhCD to reduce cellular cholesterol levels inhibited phosphorylation of EGFR and the downstream protein AKT (10).…”