title: Tetraspanin CD9 is a "proteolipid," and its interaction with alpha 3 integrin in microdomain is promoted by GM3 ganglioside, leading to inhibition of laminin-5-dependent cell motility.
abstract: 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 alpha(3) integrin were concentrated together in the low-density glycolipid-enriched microdomain (GEM) of ldlD/CD9 cells, and the alpha(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 alpha(3) in GEM under +Gal condition, whereby GM3 is present. (iii) Chemical levels of alpha(3) and CD9 in the total extract or membrane fractions from cells grown under +Gal versus -Gal condition were nearly identical, whereas alpha(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 alpha(3) with CD9, which restricts alpha(3) binding to its antibody. A concept of the alpha(3)/CD9 interaction promoted by GM3 was further supported by (i) co-immunoprecipitation of CD9 and alpha(3) under +Gal but not -Gal condition, (ii) enhanced co-immunoprecipitation of CD9 and alpha(3) when GM3 was added exogenously to cells under -Gal condition, and (iii) the co-localization images of CD9 with alpha(3) and of GM3 with CD9 in fluorescence laser scanning confocal microscopy. Based on the promotion of alpha(3)/CD9 interaction by GM3 and the status of laminin-5 as a true ligand for alpha(3), the laminin-5/alpha(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-alpha(3) antibody and is therefore based on interaction between alpha(3), CD9, and GM3 in GEM.
authors.affiliation: Division of Biomembrane Research, Pacific Northwest Research Institute, 720 Broadway, Seattle, WA 98122-4327, USA.
authors.forename: Yasushi
authors.initials: Y
authors.lastname: Kawakami
authors.forename: Kumiko
authors.initials: K
authors.lastname: Kawakami
authors.forename: Wim F A
authors.initials: WF
authors.lastname: Steelant
authors.forename: Masaya
authors.initials: M
authors.lastname: Ono
authors.forename: Rena C
authors.initials: RC
authors.lastname: Baek
authors.forename: Kazuko
authors.initials: K
authors.lastname: Handa
authors.forename: Donald A
authors.initials: DA
authors.lastname: Withers
authors.forename: Senitiroh
authors.initials: S
authors.lastname: Hakomori
mesh_terms: D000818:Animals; D015703:Antigens, CD / Q000032:analysis / Q000737:chemistry*; D016466:CHO Cells; D015815:Cell Adhesion Molecules / Q000502:physiology*; D002465:Cell Movement / Q000502:physiology*; D006224:Cricetinae; D005679:G(M3) Ganglioside / Q000502:physiology*; D006031:Glycosylation; D039422:Integrin alpha3 / Q000032:analysis / Q000737:chemistry*; D008562:Membrane Glycoproteins / Q000032:analysis / Q000737:chemistry*; D011233:Precipitin Tests; D060245:Tetraspanin 29; D000097794:Kalinin
chemical_list: D015703:Antigens, CD; D015815:Cell Adhesion Molecules; D005679:G(M3) Ganglioside; D039422:Integrin alpha3; D008562:Membrane Glycoproteins; D060245:Tetraspanin 29; D000097794:Kalinin