| First Author | Iwabuchi K | Year | 1998 |
| Journal | J Biol Chem | Volume | 273 |
| Issue | 50 | Pages | 33766-73 |
| PubMed ID | 9837965 | Mgi Jnum | J:51414 |
| Mgi Id | MGI:1316595 | Doi | 10.1074/jbc.273.50.33766 |
| Citation | Iwabuchi K, et al. (1998) Separation of glycosphingolipid signaling domain from caveolin-containing membrane fraction in mouse melanoma B16 cells and its role in cell adhesion coupled with signaling. J Biol Chem 273(50):33766-73 |
| abstractText | Two membrane subfractions, one enriched in GM3 ganglioside and the other containing caveolin, were separated from low density detergent- insoluble membrane fraction prepared by sucrose density gradient centrifugation of postnuclear fraction of mouse melanoma B16 cells. The GM3-enriched subfraction, separated by anti-GM3 monoclonal antibody DH2, contained sphingomyelin, cholesterol, c-Src, and Rho A but not caveolin. In contrast, the caveolin-containing subfraction, separated by anti-caveolin antibody, contained neither GM3, c-Src, nor Rho A but did contain glucosylceramide, Ras, a very small quantity 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 signal transduction through GM3. 32P-Phosphorylation of c-Src (Mr 60,000) together with two other components (Mr 45,000 and 29,000) was enhanced in the fraction bound to dishes coated with asialo-GM2 (Gg3) or with anti-GM3 monoclonal antibody DH2, detected by incubation with [gamma-32P]ATP at 37 degreesC 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 either filipin or nystatin, which are cholesterol-binding reagents known to abolish caveolae structure and function. B16 melanoma cells incubated with filipin (0.16-0.3 micrograms/ml) or with nystatin (25 micrograms/ml) for 30 min showed depletion of cholesterol in detergent-insoluble membrane fraction but were still capable of binding to Gg3-coated plate and capable of the associated signaling. Thus, the GM3-enriched subfraction, involved in cell adhesion and capable of sending signals through GM3, represents a membrane domain distinguishable from caveolin-containing subfraction or caveolae. This microdomain is hereby termed the glycosphingolipid signaling domain or glycosignaling domain. |
