Effects Of Ganglioside GM₃ On The Distribution Of Epidermal Growth Factor Receptor In The Lipid Rafts Of Human Leukemia J6-2 Cell Line

Ganglioside GM3 has been proved to be a physiological celldifferentiation inducer in several human leukemia cell lines. However, theinducing differentiation mechanism of GM3 remains unclear. Our previouswork has shown that GM3 can induce human monocytoid leukemiaJ6-2 cell line to differentiate along the monocyte/macrophageroute ,and simultaneously inhibit the phosphorylation of EGFR anddownregulate the PLCγ/DAG/ PKCα signaling pathway ,but themechanism is still unknown.Recently, the finding of lipid raft ,which isthe microdomain enriched in sphingolipids and cholesterol in cellplasma membrane ,give rise to a new strategy for the study ofmolecular mechanism of transmembrane signaling. Activation of EGFRcan be downregulated by trapping it into lipid rafts.So in this paper,inorder to investigate the mechanism of GM3 inhibit the phosphorylation ofEGFR,we mainly observe the effects of GM3 on the distribution of EGFRin the lipid rafts and the phosphorylation of EGFR. In order to study the effects of GM3 on the distribution of EGFR inlipid rafts, the first thing to be settled is preparation and identification oflipd rafts . The lipid rafts are prepared by extracting with cold non-ionicdetergent and then by density gradient ultracentrifugation.GM1,aglycosphinglipid (GSL) that highly enriched in the lipid rafts,is often usedas a molecular marker for lipid rafts.The cholera toxin B subunit labelledwith fluorescence or biotin, which has high affinity with GM1,can be usedas a probe for identifying lipid rafts. But this procedure for lipid raftsidentification is complicated,time-consuming and prone to environmentalpollution..So we want to find out the high specific and widely appliedmolecules which can be used as lipid rafts maker. The lipid rafts from human monocytoid leukemia J6-2 cells and rathepatoma CBRH-7919 cells were prepared by lysis with 1% Triton X-100TNE buffer and sucrose density gradient ultracentrifugation ,and thedistribution of sphingomyelin,cholesterol,GPI-anchord protein(CD14) inand out lipid rafts are compared with GM1 by high-performance thin-layerchromatography(HPTLC),Western blotting and cholera toxin B subunitlabeled with biotin dot blotting.The result shown that sphingomyelin can beused as specific marker for lipid rafts identification. Using SDS-PAGE, Western blotting and Enzyme-linkedimmunosorbent stain combined with computer quantiscan technology, weinvestigated the effect of GM3 on the distribution of EGFR in the lipid rafts.The results showed that the relative content of EGFR in the lipid rafts ofJ6-2 cells treated with GM3 was higher than that of control cells. The resultssuggested that GM3 could promote the EGFR to distribute in the lipidrafts.We further investigated the effects of GM3 on the phosphorylation ofEGFR. The results showed that the content of phosphorylation EGFR in thenon-lipid raft fractions of J6-2 cells treated with GM3 was lower thanthat of control cells, and the phosphorylation EGFR was not found in thelipid rats.The results suggested that the phosphorylation of EGFR wasinhibited in the lipid rafts. From the findings above, it was postulated that GM3 couldinhibit the phosphorylation of EGFR possibly via two ways. One isincreased the area of lipid rafts in the membrane,and increase thedistribution of EGFR in the lipid rafts. On the other hand, the GM3 caninduce the distribution of EGFR in lipid raft by identifyingand binding to the N-carbohydrate chain on IV domain EGFR. Theaffinity between EGFR in the lipid raft and ligand was reduced. As a result,the dimerization,.autophosphorylation and activation of EGFR weresurpresed . This may be the main mechanism that GM3 inhibitthe phosphorylation of EGFR.