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

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 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. 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. NeuAcα3Galβ4GlcCer glycolipid-enriched microdomain laminin-5 monoclonal antibody phosphate-buffered saline first precipitate post-nuclear fraction bovine serum albumin fluorescein isothiocyanate co-immunoprecipitation Tris-buffered saline Integrins and their functions in control of cell adhesion, motility, and signaling have been studied extensively (1Hynes R.O. Cell. 1992; 69: 11-25Abstract Full Text PDF PubMed Scopus (9139) Google Scholar). The effects of integrin N-glycosylation (2Akiyama S.K. Yamada K.M. J. Biol. Chem. 1987; 262: 17536-17542Abstract Full Text PDF PubMed Google Scholar, 3Zheng M. Fang H. Hakomori S. J. Biol. Chem. 1994; 269: 12325-12331Abstract Full Text PDF PubMed Google Scholar), surrounding gangliosides (4Zheng M. Fang H. Tsuruoka T. Tsuji T. Sasaki T. Hakomori S. J. Biol. Chem. 1993; 268: 2217-2222Abstract Full Text PDF PubMed Google Scholar, 5Hyuga S. Yamagata S. Tai T. Yamagata T. Biochem. Biophys. Res. Commun. 1997; 231: 340-343Crossref PubMed Scopus (25) Google Scholar), 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.) (6Mannion B.A. Berditchevski F. Kraeft S.-K. Chen L.B. Hemler M.E. J. Immunol. 1996; 157: 2039-2047PubMed Google Scholar), collectively termed “tetraspanin” (7Maecker H.T. Todd S.C. Levy S. FASEB J. 1997; 11: 428-442Crossref PubMed Scopus (820) Google Scholar) based on the presence of four transmembrane regions, are associated with various types of integrin receptors (Ref. 6Mannion B.A. Berditchevski F. Kraeft S.-K. Chen L.B. Hemler M.E. J. Immunol. 1996; 157: 2039-2047PubMed Google Scholar; for review, see Refs. 8Hemler M.E. Curr. Opin. Cell Biol. 1998; 10: 578-585Crossref PubMed Scopus (323) Google Scholar, 9Hemler M.E. J. Cell Biol. 2001; 155: 1103-1107Crossref PubMed Scopus (335) Google Scholar, 10Berditchevski F. J. Cell Sci. 2001; 114: 4143-4151Crossref PubMed Google Scholar). Tetraspanins interact with many membrane proteins to form a “tetraspanin web” (11Charrin S., Le Naour F. Oualid M. Billard M. Faure G. Hanash S.M. Boucheix C. Rubinstein E. J. Biol. Chem. 2001; 276: 14329-14337Abstract Full Text Full Text PDF PubMed Scopus (192) Google Scholar, 12Horvath G. Serru V. Clay D. Billard M. Boucheix C. Rubinstein E. J. Biol. Chem. 1998; 273: 30537-30543Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar), although the functional significance of this web is unclear. During a search for antibodies that inhibit cell motility (13Miyake M. Hakomori S. Biochemistry. 1991; 30: 3328-3334Crossref PubMed Scopus (96) Google Scholar), a membrane protein with sequence identical to that of the previously known CD9 was identified as a motility regulatory factor (14Miyake M. Koyama M. Seno M. Ikeyama S. J. Exp. Med. 1991; 174: 1347-1354Crossref PubMed Scopus (194) Google Scholar). CD9 expression in metastatic deposits is much lower than in normal tissues or non-metastatic tumors, and transfection of the CD9 in cells results in of and motility M. Hakomori S. Res. Google Scholar). have that of GM3 and CD9 is for motility of various and cell lines and of the cell in M. S. H. Hakomori S. Biochemistry. 2001; PubMed Scopus Google Scholar). cells are for the effect of GM3 and or status of membrane proteins a in to the presence or of in the M. Cell. Full Text PDF PubMed Scopus Google Scholar). GM3 was identified as a of CD9 for of cell motility, but the of this effect has been unclear. CD9 expressed in cells by its transfection is not to or are and their are by N-glycosylation M. Fang H. Hakomori S. J. Biol. Chem. 1994; 269: 12325-12331Abstract Full Text PDF PubMed Google Scholar). is on (i) the interaction of α3 with CD9 and the effect of GM3 ganglioside on this interaction in low-density glycolipid-enriched microdomain (GEM) fractions and (ii) the inhibitory effect of this interaction on laminin-5 cell The of the interaction in GEM is of the total of α3 and CD9, but on the status of the cells, which GEM for the first that CD9 is a based on its in and its interaction with The cell in M. Cell. Full Text PDF PubMed Scopus Google Scholar), was by of of cells CD9 its transfection were as previously M. Hakomori S. Res. Google Scholar). cells with the based on were and as control cells CD9 was in the cells with monoclonal or cells were with the These were in with The was by the with and M. D. M. Cell Biol. PubMed Scopus Google Scholar), termed with or on and in this for GM3 was the in ldlD/moc and ldlD/CD9 cells grown in with but was in cells grown gangliosides were was the in cell lines under −Gal condition, as observed previously M. Hakomori S. Res. Google Scholar). The of was higher under +Gal with −Gal condition, that N-glycosylation of α3 is under the condition, as observed previously for M. Hakomori S. Biochem. Biophys. Res. Commun. PubMed Scopus Google Scholar). of effect on the of α3 and not of gangliosides than The of CD9 was of or GM3 was in this T. G. Hakomori S. Res. Google Scholar). M. T. M. S. M. J. Biol. Chem. Full Text PDF PubMed Google Scholar) was from to CD9 was from antibodies to and the of CD9 were from to α3 integrin and integrin were from These antibodies were with their from and with of is based on to was from to and were from were at the by the A protein was for of protein The of was in which were grown by G. J. Cell Biol. 1996; PubMed Scopus Google Scholar). ldlD/CD9 cells were in serum in and with and with or was were at with for was with and and of and was were in a for The cell was to a by a and at and of the were to and for of α3 and CD9 as previously M. Hakomori S. Res. Google M. Hakomori S. Biochem. Biophys. Res. Commun. PubMed Scopus Google Scholar). for α3 and CD9 were with that for (see of α3 and CD9 by ldlD/CD9 cells were in with were with the of cells, and The was and cells were with with was added to was added to the The were at with for were by and and from cells grown under +Gal and −Gal were in and of and The was for at and with a to the and of the total were for of α3 and CD9 levels based on protein and the control (see the to The total was at for to termed the first precipitate from the post-nuclear fraction The cell was at as previously J. Cell Biol. 1996; PubMed Scopus Google Scholar, S. Hakomori S. Biochem. Biophys. Res. Commun. 1997; PubMed Scopus Google Scholar, S. Hakomori S. J. Biol. Chem. 1998; 273: Full Text Full Text PDF PubMed Scopus Google Scholar). cell and a of Hakomori S. J. Biol. Chem. Full Text PDF PubMed Google were in by for membrane that the membrane in GEM (see The protein of the total and was the and of the total of the total and were for α3 and CD9 by and antibodies as (see to of α3 and CD9 in ldlD/CD9 are α3 and CD9 levels in the total and from cells grown under +Gal condition. The total and fractions were as under and of the total with protein were for of α3 and CD9 with as a under −Gal versus +Gal condition. the value for α3 or CD9 divided by that for from the as for for α3 and for CD9 of with protein were for of and CD9 with as a under −Gal versus +Gal are as for for α3 and for CD9 from of total was in of at for were for of α3 and CD9 with as a under −Gal versus +Gal condition. are as for for α3 and for CD9 from cells was to as under and from fraction the from to was by and with antibody to α3 or on the of that the of α3 and CD9 were in low-density GEM fractions of +Gal or −Gal condition. protein from fraction the soluble fraction or from ldlD/CD9 or ldlD/moc cells was by and that levels of α3 and CD9 from ldlD/CD9 cells were higher under +Gal than −Gal condition whereas difference was for ldlD/moc cells is the of α3 expression for ldlD/CD9 cells as by The ratio value under +Gal condition divided by the value under −Gal condition) was as for and for for of protein from fraction as is the of α3 expression by the ratio for α3 in fraction or in GEM in or as α3 in fraction from ldlD/moc α3 in fraction from ldlD/CD9 α3 in GEM fractions from ldlD/moc α3 in GEM from ldlD/CD9 cells. significance of in α3 is by at CD9 was between +Gal versus −Gal condition in ldlD/CD9 cells. ratio for CD9 in fraction ratio for CD9 in for α3 and CD9 were with that for as the control was for α3 and CD9 by from cells grown under +Gal and −Gal were by the were and The was with in and Biochem. 1987; PubMed Scopus Google Scholar) at for with and with antibody and antibody for The for from the for of α3 and CD9 under +Gal and −Gal were the of were and The by by were from cells was to at to the low-density membrane fractions were and to to as in S. Hakomori S. Biochem. Biophys. Res. Commun. 1997; PubMed Scopus Google Scholar, S. Hakomori S. J. Biol. Chem. 1998; 273: Full Text Full Text PDF PubMed Scopus Google Scholar). The low-density was in fraction and at a much lower in fractions and The protein of fraction was the The of the proteins in were found in soluble fractions were found in low-density fractions and or protein of fraction were by and antibodies as were from the with and and in of cells as in were with of or antibody to α3 or CD9 on with and with or and in in were a flow were with by and at for was by and the cell was of versus The was with of with and with by for and at for of the extract was under the was in of and to this was added of for the J. S. J. Biol. Chem. Full Text PDF PubMed Google Scholar). the and lower were and lower were to in and to by with antibody. ldlD/CD9 cells grown under +Gal or −Gal condition were by with in of for at with a and at for The was for fraction by was for The of as was with of protein and by for at was with of protein and in the The was at for and the was with antibody. between α3 and CD9 was by addition of of antibody. was by addition of of antibody. the was at protein were and the was for at and at for The was with of and and at for was added to the which was to by between α3 and CD9, was with antibody. was with antibody. was in of a of serum was and the was with of this and for at in of this were added to and for The was to or and cells were for The in was with with and in in for at on the were with and in in a cells were in in and for with and with in for was cells were with the of of CD9 and α3, cells on were at for with from a of of and of anti-α3antibody and with on were further in a with a of antibodies of and of with of at for and with of CD9 and cells on were for with from of of and of with and in a with a of antibodies as were first with antibodies in of for were with with in of for and with cells were with a of of fluorescence were observed by laser scanning confocal at of for antibodies and for or The of antibodies in this on a are as by the that J. Cell Biol. 1996; PubMed Scopus Google Scholar). of in was added to the of a membrane and at to the of the membrane with the The membrane was with and of in was added for at in and for to binding to the The membrane was with and of was added to the and for The was with and for was the antibody at the of the membrane with and the membrane was with grown under −Gal or +Gal condition as were with and by of with and were in in or was in the of the in or was added to the lower and for at that to the lower of the membrane were with and with and the cell was as previously M. Hakomori S. Res. Google Scholar). The α3 and CD9 levels in types of extract extract at and at and in types of membrane fraction and from cells grown under +Gal or −Gal condition, were and by by control (see and for α3 and for CD9 under +Gal condition were the as under −Gal condition for the extract for α3 and CD9 under +Gal versus −Gal condition were the for the total and the between the from −Gal condition were as by the value as a of The levels of CD9 at the ldlD/CD9 cell surface, probed by antibody binding in flow cytometry, were for cells grown under −Gal condition. The α3 probed by binding to its antibody was much higher under −Gal than +Gal condition the that levels of α3 and CD9 in the total cell extract or membrane fractions were nearly identical, as The α3 probed by its antibody binding in control ldlD/moc cells was lower than that in ldlD/CD9 cells although the of binding to on antibody not The CD9 at the cell was lower under +Gal condition when GM3 was These the concept that α3 and CD9 levels at the cell are by their which is promoted by GM3 and under +Gal condition (see of α3 and CD9, based on from fraction by of ldlD/CD9 cells grown under −Gal and +Gal are in and that α3 and CD9 were concentrated in fractions The of α3 based on protein of the low-density fraction was based on the value as by for +Gal condition divided by the value for −Gal condition. The ratio was for ldlD/CD9 versus ldlD/moc cells. The of α3 in fraction was higher under +Gal than −Gal condition for ldlD/CD9 cells but difference was for ldlD/moc cells The of α3 based on the in and is in The in and are for the protein from The of α3 based on the was in to in and the or protein The results are in and are in the The expression of CD9 in fraction and fractions (GEM) of ldlD/CD9 cells grown under +Gal versus −Gal condition is in GEM of ldlD/CD9 cells, the ratio was for fraction the ratio was between and ldlD/moc cells, the were for GEM and for fraction fraction and the difference between ldlD/moc and ldlD/CD9 cells was as by the lines in These that the presence of CD9 of α3 and CD9 levels in low-density membrane The of α3 in low-density fractions was not with the of in soluble fractions and low-density were not or to by of cells with of −Gal condition. a was to soluble fractions of cells with a much higher of not that the is and not on a protein and of difference in of fractions under +Gal versus −Gal condition not CD9 was in or by was by with CD9 was found in the lower not is to that of known (see and is a of the J. J. Biol. 157: PubMed Scopus Google Scholar) between CD9 and a membrane protein as previously M. Sci. S. PubMed Scopus Google Scholar). The interaction of α3 with CD9, promoted by was further by from cells grown under +Gal or −Gal condition were to with antibody by with antibody. The CD9 was with antibody in the of cells grown under +Gal condition, but not under −Gal condition a low-density membrane fraction with by with enhanced α3 was observed in the of cells grown under +Gal condition, and a with a lower was observed in cells grown under −Gal condition were the effect of GM3 on the interaction of CD9 with α3 or a effect of GM3 added to ldlD/CD9 cells grown under −Gal condition, whereby GM3 was were grown under −Gal condition for and on the with GM3 of or of the with antibody by with or is in of α3 or were observed with of GM3 added exogenously to cells, with The of CD9 with α3 and of GM3 with CD9 was further by laser scanning confocal microscopy. A co-localization was observed between CD9 and α3 in ldlD/CD9 cells when grown under +Gal condition but was in cells grown under −Gal condition a co-localization was between GM3 and CD9 in cells grown under +Gal condition but was in cells grown under −Gal condition A co-localization in a of cells between CD9 and α3 or between GM3 and CD9 was observed on the than of the cells. These that a of CD9 with α3, or GM3 with CD9, when cells are grown under +Gal condition, whereby GM3 and N-glycosylation of α3 in the GEM at the The motility of ldlD/CD9 and their control cells CD9 membrane is in The motility was for ldlD/CD9 cells under −Gal condition and was strongly inhibited under +Gal condition to the as the control cells +Gal condition GM3 the of motility under this condition is based on +Gal condition in α3, this interaction (see of antibody reversed the inhibitory effect on cell motility, enhanced motility These that GM3 the interaction of α3 with CD9, to of the interaction inhibits to be studied (see on the status of α3 integrin with tetraspanin CD9 in glycolipid-enriched microdomain (GEM) in low-density membrane and the effect of on the interaction of α3 with CD9 and on their cell motility was studied as a of the α3/CD9 which is by The was based on the of cells M. Cell. Full Text PDF PubMed Scopus Google Scholar) CD9 by its transfection and cells CD9 as a The of ldlD/CD9 or ldlD/moc cells under +Gal condition the of to GM3 and expression of GM3 as the ganglioside in GEM. grown under −Gal condition have as the M. Hakomori S. Res. Google Scholar). α3, in cells higher under +Gal than −Gal condition, is under +Gal condition M. Hakomori S. Biochem. Biophys. Res. Commun. PubMed Scopus Google Scholar). that the interaction of α3 with CD9 in ldlD/CD9 cells is promoted by a in GM3 and that α3 in which are in low-density membrane inhibit cell for of the α3/CD9 interaction is as (i) of α3 with CD9 and of CD9 with α3 were greatly enhanced under +Gal with −Gal condition (ii) addition of GM3 to ldlD/CD9 cells grown under −Gal condition GM3 is enhanced of α3 and with CD9 (iii) The co-localization of α3 and CD9 or that of GM3 and CD9 was by laser scanning confocal α3 be to fraction or GEM in the presence of CD9 when GM3 under +Gal condition in ldlD/CD9 cells. is by the higher ratio of α3 in fraction or as in and and the of ldlD/CD9 cells with control ldlD/moc cells The difference in the of α3 expression in fraction or GEM of ldlD/CD9 cells with ldlD/moc cells was in the of The CD9 in fraction or on protein was higher in cells grown under +Gal with −Gal condition These and effects by are based on GM3 under +Gal condition. the status of α3 and CD9 in GEM is strongly by the status in ldlD/CD9 cells as by the various in the in and the levels of α3 and CD9 in the total cell extract or membrane fractions were identical in ldlD/CD9 cells grown under +Gal versus −Gal condition and These status versus total are not in the the status of the interaction of under whereas the the of in the of the interaction this versus flow are of The antibody binding of α3 at the cell was much higher under −Gal with +Gal condition, although the of α3 was the under The for this difference to be a is that α3 strongly with CD9 in the presence of GM3 is synthesized under +Gal the antibody binding status to α3 be is with various in and GM3 is −Gal the α3/CD9 interaction is and binding of α3 to its antibody be and therefore CD9 was found to be and in the lower of M. 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