GM3-enriched microdomain involved in cell adhesion coupled with signaling in mouse melanoma B16 cells

Mouse melanoma B16 cell membranes are characterized by predominant presence of ganglioside GM3. These cells adhere, spread, and display enhanced motility on LacCer or Gg3-coated plates, and on mouse endothelial relis which express LacCer or Gg3, through interaction of GM3 with these glycolipids. This process is considered the initial step in B16 metastasis. We now report thai >90% of GM3 present in original B16 cell membrane is organized with vari ous transducer molecules such as c Src, FAK, Ras, and Rho in a low-density membranous fraction hereby termed "glycosphingolipid-enriched microdomain (GEM)." Approximately the same composition of GM3 and transducers is found for fractions obtained by sucrose density gradient centrifugation either in Tris buffer containing 1% Triton X 100, or in hypertonic sodium carbonate. c-Srr. Rho, and FAK were co-immunoprecipitated with anti-GM3 antibody, indicating a close association of these transducers with GM3- GM3-dependent adhesion of B16 cells to Gg3-coated plates, but not to GM3 or GlcOer-coated plates, greatly enhanced tyrosine phosphorylation in FAK within 15-30 min. This enhancement was specifically inhibited when the Gg3-coated plates were pretreated with anti-Gg3 antibody. GM3-dependent adhesion to Gg3 coated plates led to significant increase in GTP loading on Ras and Rho. These data indicate that GEM is a structural and functional unit for initiation of GM3 dependent adhesion coupled with signal transduction. Supported by Nil/ N(T. OIG CA-42505.