Involvement of lipid rafts in adhesion-induced activation of Met and EGFR.
BACKGROUND
Cell adhesion has been shown to induce activation of certain growth factor receptors in a ligand-independent manner. However, the mechanism for such activation remains obscure.


METHODS
Human epidermal carcinoma A431 cells were used as a model to examine the mechanism for adhesion-induced activation of hepatocyte growth factor receptor Met and epidermal growth factor receptor (EGFR). The cells were suspended and replated on culture dishes under various conditions. The phosphorylation of Met at Y1234/1235 and EGFR at Y1173 were used as indicators for their activation. The distribution of the receptors and lipid rafts on the plasma membrane were visualized by confocal fluorescent microscopy and total internal reflection microscopy.


RESULTS
We demonstrate that Met and EGFR are constitutively activated in A431 cells, which confers proliferative and invasive potentials to the cells. The ligand-independent activation of Met and EGFR in A431 cells relies on cell adhesion to a substratum, but is independent of cell spreading, extracellular matrix proteins, and substratum stiffness. This adhesion-induced activation of Met and EGFR cannot be attributed to Src activation, production of reactive oxygen species, and the integrity of the cytoskeleton. In addition, we demonstrate that Met and EGFR are independently activated upon cell adhesion. However, partial depletion of Met and EGFR prevents their activation upon cell adhesion, suggesting that overexpression of the receptors is a prerequisite for their self-activation upon cell adhesion. Although Met and EGFR are largely distributed in 0.04% Triton-insoluble fractions (i.e. raft fraction), their activated forms are detected mainly in 0.04% Triton-soluble fractions (i.e. non-raft fraction). Upon cell adhesion, lipid rafts are accumulated at the cell surface close to the cell-substratum interface, while Met and EGFR are mostly excluded from the membrane enriched by lipid rafts.


CONCLUSIONS
Our results suggest for the first time that cell adhesion to a substratum may induce a polarized distribution of lipid rafts to the cell-substratum interface, which may allow Met and EGFR to be released from lipid rafts, thus leading to their activation in a ligand-independent manner.
Graduate Institute of Biomedical Sciences, National Chung Hsing University, Taichung, Taiwan.
Ying-Che
YC
Lu
Hong-Chen
HC
Chen
D002448:Cell Adhesion; D002462:Cell Membrane / Q000378:metabolism; D066246:ErbB Receptors / Q000378:metabolism*; D057809:HEK293 Cells; D006801:Humans; D021962:Membrane Microdomains / Q000378:metabolism*; D008856:Microscopy, Fluorescence; D019859:Proto-Oncogene Proteins c-met / Q000378:metabolism*; D015398:Signal Transduction; D019061:src-Family Kinases / Q000378:metabolism
C512478:EGFR protein, human; D066246:ErbB Receptors; C491743:MET protein, human; D019859:Proto-Oncogene Proteins c-met; D019061:src-Family Kinases