Separation of "glycosphingolipid signaling domain" from caveolin-containing membrane fraction in mouse melanoma B16 cells and its role in cell adhesion coupled with signaling

Two membrane subfractions, one enriched in GM3 ganglioside and the other co ntaining caveolin, were separated from low density detergent-insoluble memb rane fraction prepared by sucrose density gradient centrifugation of postnu clear fraction of mouse melanoma B16 cells. The GM3-enriched subfraction, s eparated by anti-GM3 monoclonal antibody DH2, contained sphingomyelin, chol esterol, c-Src, and Rho A but not caveolin. In contrast, the caveolin-conta ining subfraction, separated by anti-caveolin antibody, contained neither G M3, c-Src, nor Rho A but did contain glucosylceramide, has, a very small qu antity of sphingomyelin, and a very large quantity of cholesterol. The GM3/ c-Src-enriched membrane subfraction was characterized by (i) maintenance of GM3-dependent adhesion and (ii) susceptibility to being activated for sign al transduction through GM3. P-32-phosphorylation of c-Src (M-r 60,000) tog ether with two other components (M-r 45,000 and 29,000) was enhanced in the fraction bound to dishes coated with asialo-GM2 (Gg3) or with anti-GM3 mon oclonal antibody DH2, detected by incubation with [gamma-P-32]ATP 37 degree s C for 5 min. GM3-dependent adhesion of B16 cells to Gg3-coated dishes and associated signaling were not reduced or abolished in the presence of eith er filipin or nystatin, which are cholesterol-binding reagents known to abo lish caveolae structure and function. B16 melanoma cells incubated with fil ipin (0.16-0.3 mu g/ml) or with nystatin (25 mu g/ml) for 30 min showed dep letion of cholesterol in detergent-insoluble membrane fraction but were sti ll capable of binding to Gg3-coated plate and capable of the associated sig naling. Thus, the GM3-enriched subfraction, involved in cell adhesion and c apable of sending signals through GM3, represents a membrane domain disting uishable from caveolin-containing subfraction or caveolae. This microdomain is hereby termed the "glycosphingolipid signaling domain" or "glycosignali ng domain".