GM3 ganglioside inhibits tetraspanin CD9-facilitated cell motility in various cell lines (Ono, M., Handa, K., Sonnino, S., Withers, D. A., Nagai, H., and Hakomori, S. (2001) Biochemistry 40, 6414 -6421). We now report the following: (i) CD9 has the novel feature of being soluble in chloroform/methanol, and classifiable as "proteolipid"; (ii) CD9 and ␣ 3 integrin were concentrated together in the lowdensity glycolipid-enriched microdomain (GEM) of ldlD/ CD9 cells, and the ␣ 3 expression ratio (value for cells grown under ؉Gal condition divided by the value for cells grown under ؊Gal condition) in GEM of ldlD/CD9 cells was higher than that in control ldlD/moc cells, suggesting that CD9 recruits ␣ 3 in GEM under ؉Gal condition, whereby GM3 is present. (iii) Chemical levels of ␣ 3 and CD9 in the total extract or membrane fractions from cells grown under ؉Gal versus ؊Gal condition were nearly identical, whereas ␣ 3 expressed at the cell surface, probed by antibody binding in flow cytometry, was higher under ؊Gal than ؉Gal condition. These results suggest that GM3 synthesized under ؉Gal condition promotes interaction of ␣ 3 with CD9, which restricts ␣ 3 binding to its antibody. A concept of the ␣ 3 /CD9 interaction promoted by GM3 was further supported by (i) co-immunoprecipitation of CD9 and ␣ 3 under ؉Gal but not ؊Gal condition, (ii) enhanced co-immunoprecipitation of CD9 and ␣ 3 when GM3 was added exogenously to cells under ؊Gal condition, and (iii) the co-localization images of CD9 with ␣ 3 and of GM3 with CD9 in fluorescence laser scanning confocal microscopy. Based on the promotion of ␣ 3 /CD9 interaction by GM3 and the status of laminin-5 as a true ligand for ␣ 3 , the laminin-5/␣ 3 -dependent motility of ldlD/CD9 cells was found to be greatly enhanced under ؊Gal condition, but strongly inhibited under ؉Gal condition. Such a motility difference under ؉Gal versus ؊Gal condition was not observed for ldlD/moc cells. The inhibitory effect observed in ldlD/CD9 cells under ؉Gal condition was reversed upon addition of anti-␣ 3 antibody and is therefore based on interaction between ␣ 3 , CD9, and GM3 in GEM.Integrins and their functions in control of cell adhesion, motility, and signaling have been studied extensively (1). The effects of integrin N-glycosylation (2, 3), surrounding gangliosides (4, 5), and associated tetraspanins (see below) on their functions are increasingly clear, but remain to be studied extensively. A series of membrane proteins (CD9, CD53, CD63, CD81, CD82, etc.) (6), collectively termed "tetraspanin" (7) based on the presence of four transmembrane regions, are associated with various types of integrin receptors (Ref. 6; for review, see Refs. 8 -10). Tetraspanins interact with many membrane proteins to form a "tetraspanin web" (11,12), although the functional significance of this web is unclear. During a search for antibodies that inhibit cell motility (13), a membrane protein with sequence identical to that of the previously known CD9 was identified as a motility regulatory factor (14). CD9 expr...