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 GM3.32 P-Phosphorylation of c-Src (M r 60,000) together 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 monoclonal antibody DH2, detected by incubation with [␥-32 P]ATP at 37°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 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 g/ml) or with nystatin (25 g/ml) for 30 min showed depletion of cholesterol in detergentinsoluble 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".

The glycosynapse

“…Well studied examples are signal transduction events resulting from GM3-dependent adhesion of B16 cells (42) and Gb4-dependent adhesion of 2102 cells (54). GM3-enriched membranes in B16 cells are separable from caveolar membranes by anti-GM3 or anti-caveolin Abs (20). Contrasting properties of GM3-enriched signaling domain vs. caveolar membranes are summarized in Fig.…”

“…Caveolar membranes showed a composition similar to that of other low-density membranes regardless of the presence or absence of caveolin (1,16). Various terms such as caveolae (1), caveola-like (16), DIM (10), detergent-resistant membrane (2), GEM (17)(18)(19)(20), and most frequently ''raft'' (5, 6) were used to refer to similar mixtures of membranes with low density.…”

“…Examples are (i) modulation of growth factor receptors with intrinsic tyrosine kinases (48-51; for review, see ref. 31); (ii) modulation of integrin function (52) through their complex with tetraspanin CD9 (53); and (iii) interaction with and activation of cytoplasmic signal transducers such as Src family kinases and small G proteins present in microdomain (17)(18)(19)(20).…”

“…We have studied the composition and functional roles of GSL glycosynapses in mouse B16 melanoma (19,20), human teratocarcinoma 2102 (54), mouse neuroblastoma Neuro2a (55), and human renal cell carcinoma TOS-1 cell lines (56). In each case, the major GSL or ganglioside in the assembly mediates cell adhesion or displays susceptibility to exogenous gangliosides to activate cytoplasmic signal transducers.…”

The differential protein and lipid compositions of noncaveolar lipid microdomains and caveolae

“…Caveolae are not as lipid-enriched as lipid rafts. In fact, sphingolipidenriched, but caveolin-depleted, membrane microdomains can be isolated from mouse melanoma B16 cells by immunoprecipitation with an anti-GM3 ganglioside antibody [44]. Caveolae are also different membrane fractions from sphingolipid-enriched membrane microdomains in MDCK II cells [45].…”