Cholesterol suppresses spontaneous activation of EGFR-mediated signal transduction

Epidermal growth factor receptor (EGFR)-mediated signal transduction controls cell growth and proliferation. The signaling pathway is regulated so that it is activated only by external EGF stimuli, but the mechanisms that prevent EGF-independent spontaneous activation of EGFR-mediated signaling are unknown. Here we report cholesterol depletion activates EGFR-mediated signaling without EGF. We applied automated single-molecule imaging to EGFR and characterized the lateral diffusion and cluster formation on cholesterol-depleted and cholesterol-supplemented membranes. In cells in which cholesterol was depleted by methyl-β-cyclodextrin (MβCD) treatment, EGFR exhibited a reduction in lateral diffusion, an acceleration of cluster formation, and autophosphorylation without EGF. Concurrently, extracellular signal-regulated kinase (ERK), which is regulated by EGFR-mediated signaling, exhibited phosphorylation and nuclear translocation without EGF. These cholesterol depletion-induced changes were similar, albeit less efficient, to those that occurred with EGF stimulation in normal cells without MβCD, indicating the spontaneous activation of EGFR signaling. The exogenous supplementation of cholesterol suppressed the MβCD-induced spontaneous activation of EGFR and ERK nuclear translocation. Single-molecule imaging of EGFR in a large number of cells revealed cell-to-cell heterogeneity, with a sub-population showing a high ability for spontaneous activation. These results provide evidence that EGFR-mediated signaling is properly regulated by cholesterol metabolism to prevent uncontrolled spontaneous activation.