title: Separation of "glycosphingolipid signaling domain" from caveolin-containing membrane fraction in mouse melanoma B16 cells and its role in cell adhesion coupled with signaling.
abstract: 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".
authors.affiliation: Pacific Northwest Research Institute, Seattle, Washington 98122 and the Departments of Pathobiology and Microbiology, University of Washington, Seattle, Washington 98195, USA.
authors.forename: K
authors.initials: K
authors.lastname: Iwabuchi
authors.forename: K
authors.initials: K
authors.lastname: Handa
authors.forename: S
authors.initials: S
authors.lastname: Hakomori
mesh_terms: D000818:Animals; D051242:Caveolin 1; D022461:Caveolins; D002448:Cell Adhesion / Q000187:drug effects; D002462:Cell Membrane / Q000378:metabolism; D004285:Dogs; D005372:Filipin / Q000494:pharmacology; D006028:Glycosphingolipids / Q000378:metabolism*; D008024:Ligands; D008546:Melanoma, Experimental / Q000378:metabolism* / Q000473:pathology; D008563:Membrane Lipids / Q000302:isolation & purification; D008565:Membrane Proteins / Q000378:metabolism*; D051379:Mice; D009761:Nystatin / Q000494:pharmacology; D010766:Phosphorylation; D016392:Proto-Oncogene Proteins pp60(c-src) / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects; D014407:Tumor Cells, Cultured; D018631:ras Proteins / Q000378:metabolism
chemical_list: C495541:Cav1 protein, mouse; D051242:Caveolin 1; D022461:Caveolins; D006028:Glycosphingolipids; D008024:Ligands; D008563:Membrane Lipids; D008565:Membrane Proteins; D009761:Nystatin; D005372:Filipin; D016392:Proto-Oncogene Proteins pp60(c-src); D018631:ras Proteins