GM3-enriched Microdomain Involved in Cell Adhesion and Signal Transduction through Carbohydrate-Carbohydrate Interaction in Mouse Melanoma B16 Cells

Abstract

Mouse melanoma B16 cells are characterized by the predominant presence of ganglioside GM3 and adhere to lactosylceramide-or Gg3-coated plates through interaction of GM3 with lactosylceramide or Gg3, whereby not only adhesion but also spreading and enhancement of cell motility occur (Kojima, N., Hakomori, S. (1991) J. Biol. Chem. 266, 17552-17558). We now report that the adhesion process is based essentially on a glycosphingolipid-enriched microdomain (GEM) at the B16 cell surface, since >90% of GM3 present in the original cells is found in GEM, and GEM is also enriched in several signal transducer molecules, e.g. c-Src, Ras, Rho, and focal adhesion kinase (FAK). GEM was isolated as a low density membranous fraction by homogenization of B16 cells in lysis buffer under two different conditions (i.e. buffer containing 1% Triton X-100, or hypertonic sodium carbonate without detergent), followed by sucrose density gradient centrifugation. A close association of GM3 with c-Src, Rho, and FAK was indicated by co-immunoprecipitation of GM3 present in GEM by anti-GM3 monoclonal antibody DH2, followed by Western blotting with antibodies directed to these transducer molecules. The following data indicate that GEM is a structural and functional unit for initiation of GM3-dependent cell adhesion coupled with signal transduction. 1) Tyrosine phosphorylation in FAK was greatly enhanced in B16 cells adhered to Gg3-coated plates but was minimal in cells adhered to GM3-coated, GlcCer-coated, or noncoated plates. 2) GTP loading on Ras and Rho increased significantly when cells were adhered to Gg3-coated plates, compared with GM3-coated, GlcCer-coated, or noncoated plates. Since Ras and Rho are closely associated with GM3 in GEM, cell adhesion/stimulation through GM3 in GEM may induce activation of Ras and Rho through enhanced GTP binding.

The glycosynapse

“…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.…”

Effect of lipid mimetics of GM3 and lyso-GM3 dimer on EGF receptor tyrosine kinase and EGF-induced signal transduction

“…Glycosphingolipids, including gangliosides, have the novel capability to interact with many functional membrane proteins, such as cytoplasmic signal transducers (Src family kinases, small G-proteins) [13,14], tetraspanins (CD9, CD81, CD82) [15][16][17][18], adhesion receptors (integrins) [19,20], and growth factor receptors [1,[21][22][23]. Studies on modulatory effects, initially observed with GM3 on EGFR tyrosine kinase [1], have been extended to effects of GM1, GD1a, GD1b, and GT1b on platelet-derived growth factor receptor [21,24], GM1 and plasmalopsychosine on nerve growth factor receptor [23,25], and GM3 and sialyl-nLc4 on insulin receptors [22,26].…”

Src Kinase Mediates the Regulation of Phospholipase C-γ Activity by Glycosphingolipids

“…Several studies have now implicated the association of most every non-receptor tyrosine kinase with glycosphingolipids. These include c-Src (19), Lyk, Fyn (20), Lyn (21,22), Yes (23), Cbl (24), FAK (19), and Csk (25). These studies have either localized these tyrosine kinases in raft fractions by density gradient separations or have coimmunoprecipitated these kinases with anti-glycosphingolipid antibodies.…”