Mouse
melanoma B16 cells are characterized by the predominant presence of
ganglioside GM3 and adhere to
lactosylceramide- or Gg3-coated plates through interaction of GM3 with
lactosylceramide or Gg3, whereby not only adhesion but also spreading and enhancement of cell motility occur (Kojima, N., Hakomori, S. (1991) J. Biol. Chem. 266, 17552-17558). We now report that the adhesion process is based essentially on a
glycosphingolipid-enriched microdomain (GEM) at the B16 cell surface, since >90% of GM3 present in the original cells is found in GEM, and GEM is also enriched in several signal transducer molecules, e.g. c-Src, Ras, Rho, and
focal adhesion kinase (FAK). GEM was isolated as a low density membranous fraction by homogenization of B16 cells in lysis
buffer under two different conditions (i.e.
buffer containing 1%
Triton X-100, or hypertonic
sodium carbonate without
detergent), followed by
sucrose density gradient centrifugation. A close association of GM3 with c-Src, Rho, and FAK was indicated by co-immunoprecipitation of GM3 present in GEM by anti-GM3
monoclonal antibody DH2, followed by Western blotting with
antibodies directed to these transducer molecules. The following data indicate that GEM is a structural and functional unit for initiation of GM3-dependent cell adhesion coupled with signal transduction. 1)
Tyrosine phosphorylation in FAK was greatly enhanced in B16 cells adhered to Gg3-coated plates but was minimal in cells adhered to GM3-coated, GlcCer-coated, or noncoated plates. 2)
GTP loading on Ras and Rho increased significantly when cells were adhered to Gg3-coated plates, compared with GM3-coated, GlcCer-coated, or noncoated plates. Since Ras and Rho are closely associated with GM3 in GEM, cell adhesion/stimulation through GM3 in GEM may induce activation of Ras and Rho through enhanced
GTP binding.