Glycosphingolipids (GSLs) at the cell surface membrane are associated or complexed with signal transducers (Src family kinases and small G-proteins), tetraspanins, growth factor receptors, and integrins. Such organizational framework, defining GSL-modulated or -dependent cell adhesion, motility, and growth, is termed "glycosynapse" (Hakomori, S., and Handa, K. (2002) FEBS Lett. 531, 88-92; Hakomori, S. (2004) Ann. Braz. Acad. Sci. 76, 553-572). We describe here the functional organization of the glycosynaptic microdomain, and the mechanisms for control of cell motility and invasiveness, in normal bladder epithelial HCV29 cells versus highly invasive bladder cancer YTS1 cells, both derived from transitional epithelia. (i) Ganglioside GM2, but not GM3 or globoside, interacted specifically with tetraspanin CD82, and such a complex inhibited hepatocyte growth factor (HGF)-induced activation of Met tyrosine kinase in a dose-dependent manner. (ii) Depletion of GM2 in HCV29 cells by treatment with D-threo-1-phenyl-2-palmitoylamino-3-pyrrolidino-1-propanol (P4), or reduction of CD82 expression by RNA interference, significantly enhanced HGF-induced Met tyrosine kinase and cell motility. (iii) In contrast, YTS1 cells, lacking CD82, displayed HGF-independent activation of Met tyrosine kinase and high cell motility. Transfection of the CD82 gene to YTS1 inhibited HGF dose-dependent Met tyrosine kinase activity and cell motility, due to formation of the GM2-CD82 complex. (iv) Adhesion of YTS1 or YTS1/CD82 cells to laminin-5-coated plates, as compared with noncoated plates, strongly enhanced Met activation, and the degree of activation was further increased in association with GSL depletion by P4. Laminin-5-dependent Met activation was minimal in HCV29 cells. These findings indicate that GSL, particularly GM2, forms a complex with CD82, and that such complex interacts with Met and thereby inhibits HGF-induced Met tyrosine kinase activity, as well as integrin to Met cross-talk.