Ligand-independent activation of the EGFR by lipid raft disruption.
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.
Lambert
Sylviane
S
Department of Dermatology, University of Copenhagen, Bispebjerg Hospital, Bispebjerg bakke 23, DK-2400 Copenhagen NV, Denmark.
Vind-Kezunovic
Dina
D
Karvinen
Susanna
S
Gniadecki
Robert
R
D002460:Cell Line; D049109:Cell Proliferation; D002784:Cholesterol / Q000502:physiology; D066246:ErbB Receptors / Q000378:metabolism*; D006801:Humans; D015603:Keratinocytes / Q000378:metabolism; D008024:Ligands; D021962:Membrane Microdomains / Q000187:drug effects / Q000502:physiology*; D019950:Mitogen-Activated Protein Kinase 1 / Q000502:physiology; D048052:Mitogen-Activated Protein Kinase 3 / Q000502:physiology; D047392:beta-Cyclodextrins / Q000494:pharmacology
D008024:Ligands; D047392:beta-Cyclodextrins; C108732:methyl-beta-cyclodextrin; D002784:Cholesterol; D066246:ErbB Receptors; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3