Tetraspanin CD9 Is a “Proteolipid,” and Its Interaction with α3 Integrin in Microdomain Is Promoted by GM3 Ganglioside, Leading to Inhibition of Laminin-5-dependent Cell Motility*

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 α3 integrin were concentrated together in the low-density glycolipid-enriched microdomain (GEM) of ldlD/CD9 cells, and the α3 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 α3 in GEM under +Gal condition, whereby GM3 is present. (iii) Chemical levels of α3 and CD9 in the total extract or membrane fractions from cells grown under +Gal versus −Gal condition were nearly identical, whereas α3 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 α3 with CD9, which restricts α3 binding to its antibody. A concept of the α3/CD9 interaction promoted by GM3 was further supported by (i) co-immunoprecipitation of CD9 and α3under +Gal but not −Gal condition, (ii) enhanced co-immunoprecipitation of CD9 and α3 when GM3 was added exogenously to cells under −Gal condition, and (iii) the co-localization images of CD9 with α3 and of GM3 with CD9 in fluorescence laser scanning confocal microscopy. Based on the promotion of α3/CD9 interaction by GM3 and the status of laminin-5 as a true ligand for α3, the laminin-5/α3-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-α3antibody and is therefore based on interaction between α3, CD9, and GM3 in GEM.