GM3-ENRICHED MICRODOMAIN INVOLVED IN CELL-ADHESION AND SIGNAL-TRANSDUCTION THROUGH CARBOHYDRATE-CARBOHYDRATE INTERACTION IN MOUSE MELANOMA B16 CELLS

Mouse melanoma B16 cells are characterized by the predominant presence of ganglioside GM3 and adhere to lactosylceramide- or Gg3-coated plat es through interaction of GM3 with lactosylceramide or Gg3, whereby no t only adhesion but also spreading and enhancement of cell motility oc cur (Kojima, N., Hakomori, S. (1991) J. Biol. Chem. 266, 17552-17558). We now report that the adhesion process is based essentially on a gly cosphingolipid-enriched microdomain (GEM) at the B16 cell surface, sin ce >90% of GMS present in the original cells is found in GEM, and GEM is also enriched in several signal transducer molecules, e.g. c-Src, R as, Rho, and focal adhesion kinase (FAK). GEM was isolated as a low de nsity membranous fraction by homogenization of B16 cells in lysis buff er under two different conditions (i.e. buffer containing 1% Triton X- 100, or hypertonic sodium carbonate without detergent), followed by su crose density gradient centrifugation. A close association of GM3 with c-Src, Rho, and FAK was indicated by co-immunoprecipitation of GM3 pr esent in GEM by anti-GMS monoclonal antibody DH2, followed by Western blotting with antibodies directed to these transducer molecules. The f ollowing data indicate that GEM is a structural and functional unit fo r initiation of GM3-dependent cell adhesion coupled with signal transd uction. 1) Tyrosine phosphorylation in FAK was greatly enhanced in B16 cells adhered to Gg3 coated plates but was minimal in cells adhered t o GM3-coated, GlcCer-coated, or noncoated plates. 2) GTP loading on Ra s and Rho increased significantly when cells were adhered to Gg3-coate d plates, compared with GM3-coated, GlcCer-coated, or noncoated plates . Since Ras and Rho are closely associated with GM3 in GEM, cell adhes ion/stimulation through GM3 in GEM may induce activation of Ras and Rh o through enhanced GTP binding.