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• W2078162414 abstract "CD9, a tetraspanin protein, makes crucial contributions to sperm egg fusion, other cellular fusions, epidermal growth factor receptor signaling, cell motility, and tumor suppression. Here we characterize a low affinity anti-CD9 antibody, C9BB, which binds preferentially to homoclustered CD9. Using mAb C9BB as a tool, we show that cell surface CD9 homoclustering is promoted by expression of α3β1 and α6β4 integrins and by palmitoylation of the CD9 and β4 proteins. Conversely, CD9 is shifted toward heteroclusters upon expression of CD9 partner proteins (EWI-2 and EWI-F) or other tetraspanins, or upon ablation of CD9 palmitoylation. Furthermore, unpalmitoylated CD9 showed enhanced EWI-2 association, thereby demonstrating a previously unappreciated role for tetraspanin palmitoylation, and underscoring how depalmitoylation and EWI-2 association may collaborate to shift CD9 from homo- to heteroclusters. In conclusion, we have used a novel molecular probe (mAb C9BB) to demonstrate the existence of multiple types of CD9 complex on the cell surface. A shift from homo- to heteroclustered CD9 may be functionally significant because the latter was especially obvious on malignant epithelial tumor cells. Hence, because of its specialized properties, C9BB may be more useful than other anti-CD9 antibodies for monitoring CD9 during tumor progression. CD9, a tetraspanin protein, makes crucial contributions to sperm egg fusion, other cellular fusions, epidermal growth factor receptor signaling, cell motility, and tumor suppression. Here we characterize a low affinity anti-CD9 antibody, C9BB, which binds preferentially to homoclustered CD9. Using mAb C9BB as a tool, we show that cell surface CD9 homoclustering is promoted by expression of α3β1 and α6β4 integrins and by palmitoylation of the CD9 and β4 proteins. Conversely, CD9 is shifted toward heteroclusters upon expression of CD9 partner proteins (EWI-2 and EWI-F) or other tetraspanins, or upon ablation of CD9 palmitoylation. Furthermore, unpalmitoylated CD9 showed enhanced EWI-2 association, thereby demonstrating a previously unappreciated role for tetraspanin palmitoylation, and underscoring how depalmitoylation and EWI-2 association may collaborate to shift CD9 from homo- to heteroclusters. In conclusion, we have used a novel molecular probe (mAb C9BB) to demonstrate the existence of multiple types of CD9 complex on the cell surface. A shift from homo- to heteroclustered CD9 may be functionally significant because the latter was especially obvious on malignant epithelial tumor cells. Hence, because of its specialized properties, C9BB may be more useful than other anti-CD9 antibodies for monitoring CD9 during tumor progression. Tetraspanin protein CD9 has attracted considerable attention for its crucial role on oocytes during sperm egg fusion (1Miyado K. Yamada G. Yamada S. Hasuwa H. Nakamura Y. Ryu F. Suzuki K. Kosai K. Inoue K. Ogura A. Okabe M. Mekada E. Science. 2000; 287: 321-324Crossref PubMed Scopus (553) Google Scholar, 2Le Naour F. Rubinstein E. Jasmin C. Prenant M. Boucheix C. Science. 2000; 287: 319-321Crossref PubMed Scopus (533) Google Scholar, 3Kaji K. Oda S. Shikano T. Ohnuki T. Uematsu Y. Sakagami J. Tada N. Miyazaki S. Kudo A. Nat. Genet. 2000; 24: 279-282Crossref PubMed Scopus (384) Google Scholar). CD9 also contributes to myoblast fusion (4Tachibana I. Hemler M.E. J. Cell Biol. 1999; 146: 893-904Crossref PubMed Scopus (203) Google Scholar) mononuclear phagocyte fusion (5Takeda Y. Tachibana I. Miyado K. Kobayashi M. Miyazaki T. Funakoshi T. Kimura H. Yamane H. Saito Y. Goto H. Yoneda T. Yoshida M. Kumagai T. Osaki T. Hayashi S. Kawase I. Mekada E. J. Cell Biol. 2003; 161: 945-956Crossref PubMed Scopus (142) Google Scholar), virus-induced syncytia formation (6Willett B. Hosie M. Shaw A. Neil J. J. Gen. Virol. 1997; 78: 611-618Crossref PubMed Scopus (39) Google Scholar, 7Fukudome K. Furuse M. Imai T. Nishimura M. Takagi S. Hinuma Y. Yoshie O. J. Virol. 1992; 66: 1394-1401Crossref PubMed Google Scholar), osteoclastogenesis (5Takeda Y. Tachibana I. Miyado K. Kobayashi M. Miyazaki T. Funakoshi T. Kimura H. Yamane H. Saito Y. Goto H. Yoneda T. Yoshida M. Kumagai T. Osaki T. Hayashi S. Kawase I. Mekada E. J. Cell Biol. 2003; 161: 945-956Crossref PubMed Scopus (142) Google Scholar), and paranodal junction formation in the peripheral nervous system (8Ishibashi T. Ding L. Ikenaka K. Inoue Y. Miyado K. Mekada E. Baba H. J. Neurosci. 2004; 24: 96-102Crossref PubMed Scopus (63) Google Scholar). CD9 also promotes juxtacrine signaling by associating with epidermal growth factor (EGF) receptor membrane-bound agonists, pro-TGFα, pro-HB-EGF, and pro-amphiregulin (9Shi W. Fan H. Shum L. Derynck R. J. Cell Biol. 2000; 148: 591-602Crossref PubMed Scopus (144) Google Scholar, 10Higashiyama S. Iwamoto R. Goishi K. Raab G. Taniguchi N. Klagsbrun M. Mekada E. J. Cell Biol. 1995; 128: 929-938Crossref PubMed Scopus (279) Google Scholar, 11Inui S. Higashiyama S. Hashimoto K. Higashiyama M. Yoshikawa K. Taniguchi N. J. Cell. Physiol. 1997; 171: 291-298Crossref PubMed Scopus (68) Google Scholar). CD9 may affect paracrine signaling by either promoting (12Yan Y. Shirakabe K. Werb Z. J. Cell Biol. 2002; 158: 221-226Crossref PubMed Scopus (278) Google Scholar) or inhibiting (9Shi W. Fan H. Shum L. Derynck R. J. Cell Biol. 2000; 148: 591-602Crossref PubMed Scopus (144) Google Scholar) proteolytic production of soluble EGF receptor agonist. The latter result could help to explain CD9 tumor suppressor properties. Indeed, CD9 expression is often markedly reduced in malignant melanoma (13Si Z. Hersey P. Int. J. Cancer. 1993; 54: 37-43Crossref PubMed Scopus (98) Google Scholar), colon (14Mori M. Mimori K. Shiraishi T. Haraguchi M. Ueo H. Barnard G.F. Akiyoshi T. Clin. Cancer Res. 1998; 4: 1507-1510PubMed Google Scholar), bladder (15Mhawech P. Herrmann F. Coassin M. Guillou L. Iselin C.E. Cancer. 2003; 98: 1649-1657Crossref PubMed Scopus (34) Google Scholar), lung (16Higashiyama M. Taki T. Ieki Y. Adachi M. Huang C.-L. Koh T. Kodama K. Doi O. Miyake M. Cancer Res. 1995; 55: 6040-6044PubMed Google Scholar), pancreatic (17Sho M. Adachi M. Taki T. Hashida H. Konishi T. Huang C.L. Ikeda N. Nakajima Y. Kanehiro H. Hisanaga M. Nakano H. Miyake M. Int. J. Cancer. 1998; 79: 509-516Crossref PubMed Scopus (117) Google Scholar), squamous cell (18Erovic B.M. Pammer J. Hollemann D. Woegerbauer M. Geleff S. Fischer M.B. Burian M. Frommlet F. Neuchrist C. Head Neck. 2003; 25: 848-857Crossref PubMed Scopus (37) Google Scholar, 19Mhawech P. Dulguerov P. Tschanz E. Verdan C. Ares C. Allal A.S. Br. J. Cancer. 2004; 90: 471-475Crossref PubMed Scopus (21) Google Scholar), and breast cancers (20Miyake M. Nakano K. Itoi S.I. Koh T. Taki T. Cancer Res. 1996; 56: 1244-1249PubMed Google Scholar, 21Miyake M. Nakano K. Ieki Y. Adachi M. Huang C.-L. Itoi S. Koh T. Taki T. Cancer Res. 1995; 55: 4127-4131PubMed Google Scholar). Furthermore, CD9 signaling can decrease cell proliferation while promoting apoptosis (22Murayama Y. Miyagawa J. Oritani K. Yoshida H. Yamamoto K. Kishida O. Miyazaki T. Tsutsui S. Kiyohara T. Miyazaki Y. Higashiyama S. Matsuzawa Y. Shinomura Y. J. Cell Sci. 2004; 117: 3379-3388Crossref PubMed Scopus (49) Google Scholar), and ectopic CD9 can suppress tumor cell motility and metastasis (23Ikeyama S. Koyama M. Yamaoko M. Sasada R. Miyake M. J. Exp. Med. 1993; 177: 1231-1237Crossref PubMed Scopus (274) Google Scholar, 24Miyake M. Inufusa H. Adachi M. Ishida H. Hashida H. Tokuhara T. Kakehi Y. Oncogene. 2000; 19: 5221-5226Crossref PubMed Scopus (47) Google Scholar), and down-regulate Wnt signaling pathways (25Huang C.L. Liu D. Masuya D. Kameyama K. Nakashima T. Yokomise H. Ueno M. Miyake M. Oncogene. 2004; 23: 7475-7483Crossref PubMed Scopus (62) Google Scholar).Tetraspanins typically assemble into multimolecular membrane complexes. In this regard, CD9 can directly associate with transmembrane Ig superfamily proteins EWI 2The abbreviations used are: EWI proteins, a family of four proteins each containing a conserved Glu-Phe-Ile motif; mAb, monoclonal antibody; FACS, fluorescence activated cell sorter; GFP, green fluorescent protein; MFI, mean fluorescence intensity; Wt, wild type; FITC, fluorescein isothiocyanate. 2The abbreviations used are: EWI proteins, a family of four proteins each containing a conserved Glu-Phe-Ile motif; mAb, monoclonal antibody; FACS, fluorescence activated cell sorter; GFP, green fluorescent protein; MFI, mean fluorescence intensity; Wt, wild type; FITC, fluorescein isothiocyanate.-F (CD9P-1, FPRP) and EWI-2 (26Stipp C.S. Orlicky D. Hemler M.E. J. Biol. Chem. 2001; 276: 4853-4862Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar, 27Charrin 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 (188) Google Scholar, 28Stipp C.S. Kolesnikova T.V. Hemler M.E. J. Biol. Chem. 2001; 276: 40545-40554Abstract Full Text Full Text PDF PubMed Scopus (173) Google Scholar, 29Charrin S. Le Naour F. Labas V. Billard M. Le Caer J.P. Emile J.F. Petit M.A. Boucheix C. Rubinstein E. Biochem. J. 2003; 373: 409-421Crossref PubMed Scopus (118) Google Scholar), which can drive CD9 into filopodia (32Stipp C.S. Kolesnikova T.V. Hemler M.E. J. Cell Biol. 2003; 163: 1167-1177Crossref PubMed Scopus (71) Google Scholar). CD9 also directly associates with itself, suggesting that CD9-CD9 homodimers are building blocks for larger tetraspanin complexes (30Kovalenko O.V. Yang X. Kolesnikova T.V. Hemler M.E. Biochem. J. 2004; 377: 407-417Crossref PubMed Scopus (108) Google Scholar). Additional partners for CD9 include the laminin-binding integrins α3β1, α6β1, and α6β4 (31Berditchevski F. J. Cell Sci. 2001; 114: 4143-4151Crossref PubMed Google Scholar). One consequence of integrin-CD9 association is the recruitment of EWI-2, via CD9, into a functionally important EWI2-CD9-integrin complex, that affects integrin-dependent morphology and motility (32Stipp C.S. Kolesnikova T.V. Hemler M.E. J. Cell Biol. 2003; 163: 1167-1177Crossref PubMed Scopus (71) Google Scholar). The distribution and signaling functions of CD9 also could be affected by its associations with PKC (33Zhang X.A. Bontrager A.L. Hemler M.E. J. Biol. Chem. 2001; 276: 25005-25013Abstract Full Text Full Text PDF PubMed Scopus (297) Google Scholar), type II phosphatidylinositol 4-kinase (34Yauch R.L. Hemler M.E. Biochem. J. 2000; 351: 629-637Crossref PubMed Scopus (150) Google Scholar), gangliosides (35Kawakami Y. Kawakami K. Steelant W.F. Ono M. Baek R.C. Handa K. Withers D.A. Hakomori S. J. Biol. Chem. 2002; 277: 34349-34358Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar), and cholesterol (36Charrin S. Manie S. Thiele C. Billard M. Gerlier D. Boucheix C. Rubinstein E. Eur. J. Immunol. 2003; 33: 2479-2489Crossref PubMed Scopus (181) Google Scholar).Like other tetraspanins, CD9 can undergo palmitoylation on each of its membrane-proximal cysteines (37Charrin S. Manie S. Oualid M. Billard M. Boucheix C. Rubinstein E. FEBS Lett. 2002; 516: 139-144Crossref PubMed Scopus (172) Google Scholar). Although palmitoylation is not required for CD9 homodimer formation (30Kovalenko O.V. Yang X. Kolesnikova T.V. Hemler M.E. Biochem. J. 2004; 377: 407-417Crossref PubMed Scopus (108) Google Scholar), or for CD9 association with EWI proteins (this article), it does support CD9 associations with other tetraspanins, including CD81 and CD53 (37Charrin S. Manie S. Oualid M. Billard M. Boucheix C. Rubinstein E. FEBS Lett. 2002; 516: 139-144Crossref PubMed Scopus (172) Google Scholar). In addition, CD9 association with the α6β4-CD151 protein complex is enhanced by palmitoylation of the integrin β4 (38Yang X. Kovalenko O.V. Tang W. Claas C. Stipp C.S. Hemler M.E. J. Cell Biol. 2004; 167: 1231-1240Crossref PubMed Scopus (165) Google Scholar) and CD151 (39Yang X. Claas C. Kraeft S.K. Chen L.B. Wang Z. Kreidberg J.A. Hemler M.E. Mol. Biol. Cell. 2002; 13: 767-781Crossref PubMed Scopus (189) Google Scholar, 40Berditchevski F. Odintsova E. Sawada S. Gilbert E. J. Biol. Chem. 2002; 277: 36991-37000Abstract Full Text Full Text PDF PubMed Scopus (173) Google Scholar) proteins. CD9 palmitoylation should be functionally important because palmitoylations of other tetraspanins (CD151 and CD82) markedly affect cell morphology and signaling (39Yang X. Claas C. Kraeft S.K. Chen L.B. Wang Z. Kreidberg J.A. Hemler M.E. Mol. Biol. Cell. 2002; 13: 767-781Crossref PubMed Scopus (189) Google Scholar, 40Berditchevski F. Odintsova E. Sawada S. Gilbert E. J. Biol. Chem. 2002; 277: 36991-37000Abstract Full Text Full Text PDF PubMed Scopus (173) Google Scholar, 41Zhou B. Liu L. Reddivari M. Zhang X.A. Cancer Res. 2004; 64: 7455-7463Crossref PubMed Scopus (82) Google Scholar).Monoclonal antibodies that detect dynamic cell surface molecular events have been extremely useful during studies of integrins (42Hughes P.E. Renshaw M.W. Pfaff M. Forsyth J. Keivens V.M. Schwartz M.A. Ginsberg M.H. Cell. 1997; 88: 521-530Abstract Full Text Full Text PDF PubMed Scopus (434) Google Scholar, 43Bazzoni G. Ma L. Blue M.-L. Hemler M.E. J. Biol. Chem. 1998; 273: 6670-6678Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar, 44Diamond M.S. Springer T.A. J. Cell Biol. 1993; 120: 545-556Crossref PubMed Scopus (453) Google Scholar). By comparison, mAb tools that can detect molecular changes in tetraspanins on the surface of live cells have been scarce. Instead, cell surface tetraspanin studies have relied on covalent cross-linking, co-capping, immunofluorescence co-localization, and fluorescence resonance energy transfer (45Levy S. Shoham T. Nat. Rev. Immunol. 2005; 5: 136-148Crossref PubMed Scopus (479) Google Scholar, 46Szollosi J. Horejsi V. Bene L. Angelisova P. Damjanovich S. J. Immunol. 1996; 157: 2939-2946PubMed Google Scholar). Here we describe a new tool, anti-CD9 mAb C9BB, which enables novel insights into CD9 molecular organization. mAb C9BB is distinct from other anti-CD9 antibodies in terms of binding affinity, and preference for clustered CD9. Using C9BB as a probe, we demonstrate the contrasting effects of protein palmitoylation, integrins, tetraspanins, and EWI proteins on CD9 organization, and we show that CD9 palmitoylation markedly affects CD9-EWI2 association. Furthermore, we provide evidence that homoclustered CD9 may diminish even more than total CD9 on malignant tumor cells, thus making C9BB particularly useful for monitoring tumor progression.Antibodies, Cells, and Chimeric Proteins—mAbs to tetraspanins CD9 (ALB6 and DU-ALL-1), CD63 (6H1), CD82 (M104); and to integrins α2 (A2-IIE10), α3 (A3-X8), β1 (TS2/16), and β4 (3E1) were referenced elsewhere (38Yang X. Kovalenko O.V. Tang W. Claas C. Stipp C.S. Hemler M.E. J. Cell Biol. 2004; 167: 1231-1240Crossref PubMed Scopus (165) Google Scholar, 39Yang X. Claas C. Kraeft S.K. Chen L.B. Wang Z. Kreidberg J.A. Hemler M.E. Mol. Biol. Cell. 2002; 13: 767-781Crossref PubMed Scopus (189) Google Scholar). Other anti-CD9 mAbs were C9BB (formerly called 4D5 (47Tachibana I. Bodorova J. Berditchevski F. Zutter M.M. Hemler M.E. J. Biol. Chem. 1997; 272: 29181-29189Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar)), SYB.1 (48Worthington R.E. Carroll R.C. Boucheix C. Br. J. Haematol. 1990; 74: 216-222Crossref PubMed Scopus (110) Google Scholar), PAIN-13 (49Gutierrez-Lopez M.D. Ovalle S. Yanez-Mo M. Sanchez-Sanchez N. Rubinstein E. Olmo N. Lizarbe M.A. Sanchez-Madrid F. Cabanas C. J. Biol. Chem. 2003; 278: 208-218Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar), and MM2/57 (from Research Diagnostics, Flanders, NJ). FITC-ALB6 and FITC-MM2/57 were from Immunotech, and Research Diagnostics, respectively. Cultured human cell lines were purchased from ATCC and grown in Dulbecco's modified Eagle's medium or RPMI 1640 supplemented with 10% fetal calf serum (Invitrogen), 10 mm HEPES plus antibiotics. For studies of endogenous CD9, we mostly used human A431 epidermoid carcinoma cells (which have abundant CD9 levels) and for studies of CD9 mutants, we used RD rhabdomyosarcoma cells, since they express minimal endogenous CD9. Palmitoylation deficient CD9-Pal- and GFP-CD9-Pal- (containing 6 membrane-proximal Cys→Ser mutations) were 100% deficient in palmitate incorporation (30Kovalenko O.V. Yang X. Kolesnikova T.V. Hemler M.E. Biochem. J. 2004; 377: 407-417Crossref PubMed Scopus (108) Google Scholar). GFP-CD9 (30Kovalenko O.V. Yang X. Kolesnikova T.V. Hemler M.E. Biochem. J. 2004; 377: 407-417Crossref PubMed Scopus (108) Google Scholar), GFP-EWI-2 (32Stipp C.S. Kolesnikova T.V. Hemler M.E. J. Cell Biol. 2003; 163: 1167-1177Crossref PubMed Scopus (71) Google Scholar), and CD9 × CD82 chimeras (29Charrin S. Le Naour F. Labas V. Billard M. Le Caer J.P. Emile J.F. Petit M.A. Boucheix C. Rubinstein E. Biochem. J. 2003; 373: 409-421Crossref PubMed Scopus (118) Google Scholar) were prepared as described.Transfection and Immunoprecipitation—The Fugene 6 method (Roche Applied Science) was used for transient or stable expression of CD9 (or CD82, or CD9 plus GFP-EWI-2) proteins in RD or MDA-MB-435 cells, and CD9 (or CD9 plus CD81) in U937 cells. Expression of EWI-2 in A431 cells was described elsewhere (32Stipp C.S. Kolesnikova T.V. Hemler M.E. J. Cell Biol. 2003; 163: 1167-1177Crossref PubMed Scopus (71) Google Scholar). Following cell surface biotin labeling, cells were lysed in 1% Brij 96, and the proteins were immunoprecipitated and immunoblotted as previously described (38Yang X. Kovalenko O.V. Tang W. Claas C. Stipp C.S. Hemler M.E. J. Cell Biol. 2004; 167: 1231-1240Crossref PubMed Scopus (165) Google Scholar, 39Yang X. Claas C. Kraeft S.K. Chen L.B. Wang Z. Kreidberg J.A. Hemler M.E. Mol. Biol. Cell. 2002; 13: 767-781Crossref PubMed Scopus (189) Google Scholar).Chemical cross-linking of oligomerized CD9 was achieved as described previously (30Kovalenko O.V. Yang X. Kolesnikova T.V. Hemler M.E. Biochem. J. 2004; 377: 407-417Crossref PubMed Scopus (108) Google Scholar). Briefly, intact A431 cells were treated for 20-22 h with 50 μm 2-bromopalmitate (to block cysteine palmitoylation), and then cross-linked with a thiol-specific reagent, DTME (Pierce) prior to lysis. Alternatively, RD cells transiently expressing CD9 were treated with DTME 24-h post-transfection, and then lysed. Prior to cell lysis, residual free cysteines were blocked by incubation with 10 mm N-ethyl maleimide.Flow Cytometry and Immunofluorescence Microscopy—For flow cytometry, cells were detached, stained on ice with either control IgG or specific mAbs (at ∼10 μg/ml for 30 min), followed by 20 min with FITC-conjugated secondary antibody (BIOSOURCE, Camarillo, CA), and then analyzed using a FACSCalibur (Becton Dickinson, Bedford, MA). At least 10,000 cells were counted per experiment, unless otherwise indicated. Background staining, obtained using negative control antibodies, was subtracted from all mean fluorescence intensity (MFI) values prior to calculating antibody staining ratios. For confocal microscopy 2-color imaging, cells were cultured overnight on 60-mm dishes with coverglass bottoms (MaTek Corp., Ashland, MA). Cells were then stained on ice with anti-CD9 mAb C9BB, which was detected using ALEX-549-conjugated 2nd antibody (Molecular Probes, Portland, OR). The same cells were then incubated with FITC-conjugated MM2/57 or FITC-ALB6 anti-CD9 mAbs. Slides were then fixed with 2% paraformaldehyde, and images were acquired through the z-axis (at 0.5-1 μm increments) and visualized using a Zeiss LSM 510 laser-scanning confocal microscope, with LSM510 Meta software package.RESULTSCell Type-specific Variations in Recognition of CD9 by mAb C9BB—Compared with other anti-CD9 antibodies, mAb C9BB showed considerable variability in recognition of cell surface CD9. For example, on breast carcinoma HCC1419 cells, staining by C9BB was relatively high compared with anti-CD9 mAb MM2/57 (ratio C9BB/MM2/57, 0.55). In contrast, on MDA-MB-468 cells, the C9BB/MM2/57 ratio was 0.08 (Fig. 1). On human epidermoid carcinoma A431 cells the C9BB/MM2/57 ratio is 0.29 (supplemental Fig. S1). At saturating levels on A431 cells, C9BB staining yielded a MFI of 99, whereas other anti-CD9 antibodies yielded high (MFI = 318, 327) or intermediate (MFI = 198, 243) values (supplemental Fig. S1). Because staining ratios among ALB6, DU-ALL-1, and MM2/57 were relatively stable, these three mAbs were used somewhat interchangeably to assess total CD9 in subsequent experiments. Evaluation of an extensive panel of cells showed consistently variable C9BB staining, relative to that seen using other CD9 antibodies such as DU-ALL-1 and ALB6 (supplemental Table S1). For example, C9BB/ALB-6 ratios ranged from 0.02 to 1.37, and C9BB/DU-ALL-1 ratios ranged from 0.05 to 0.74, mostly because of variations in C9BB staining (supplemental Table S1). Subsequent experiments (Figs. 2, 3, 4, 5, 6) were aimed at determining the molecular basis for widely varying recognition of CD9 by C9BB.FIGURE 2Integrin β4 palmitoylation alters C9BB staining of CD9. Integrin β4 wild type (β4-WT) and β4 lacking all 7 membrane-proximal cysteine palmitoylation sites (β4-7CS) were stably expressed in MDA-MB-435 cells (which lack endogenous β4) as previously described (38Yang X. Kovalenko O.V. Tang W. Claas C. Stipp C.S. Hemler M.E. J. Cell Biol. 2004; 167: 1231-1240Crossref PubMed Scopus (165) Google Scholar). Cell surface staining was measured by flow cytometry counting of ∼10,000 cells, using the indicated antibodies to CD9, integrin α6, and CD81. In each case, single fluorescence peaks were observed, yielding the indicated MFI values. r = C9BB/ALB-6 staining ratio, after background subtraction. Differences between C9BB and ALB-6, and between C9BB (β4-7CS) and C9BB (β4-WT) are highly significant (p < 0.005). Similar results have been seen in multiple experiments.View Large Image Figure ViewerDownload Hi-res image Download (PPT)FIGURE 3CD9 palmitoylation promotes C9BB staining. A, RD cells were transiently transfected to become >90% positive for untagged CD9-Wt or CD9-Pal-. At least 3000 cells were analyzed by flow cytometry in each experiment. r = C9BB/ALB6 and C9BB/MM257 mean fluorescence intensity ratios. Differences between C9BB and other CD9 antibodiesonRD-CD9-Pal- cells,andbetweenC9BB on CD9-Wt and CD9-Pal-cells are highly significant (p < 0.005). Similar results have been seen in multiple experiments. B, RD cells were transiently transfected with GFP-CD9 or GFP-CD9-Pal-. After 48 h, cells were stained with either C9BB or ALB6 mAb, and analyzed by flow cytometry simultaneously for CD9-GFP expression (x-axis) and anti-CD9 mAb staining (using PE-conjugated 2nd Ab,y-axis). Right panels (upper right plus lower right) contain 11-12% of total CD9-Wt cells and 19-20% of total CD9-Pal- cells. C, live MCF-7 cells stably transfected with GFP-tagged CD9-Wt or CD9-Pal- were visualized using a Zeiss Axioskop fluorescence microscope.View Large Image Figure ViewerDownload Hi-res image Download (PPT)FIGURE 4Variable integrin and EWI-2 effects on C9BB staining. A, 501-Mel cells were stably transfected with integrin α3 subunit (dark peaks) or mock-transfected (lighter peaks), and then analyzed by flow cytometry for CD9 (C9BB or ALB-6) and integrin α3 (mAb A3X8) expression. B, A431 cells were stably transfected with EWI-2 (solid line), or mock-transfected (dotted line), and analyzed by flow cytometry, using the indicated anti-CD9 antibodies. C, MDA-MD-231 cells and MCF-7 cells were labeled with biotin, lysed in 1% Brij 96, and then CD9 was immunoprecipitated using mAb MM2/57 or C9BB as indicated. Biotin-labeled proteins are visualized by blotting with Avidin.View Large Image Figure ViewerDownload Hi-res image Download (PPT)FIGURE 5C9BB preference for oligomerized CD9. A, RD cells, transiently transfected to express CD9, were treated with thiol-specific cross-linker, DTME, and then lysed in 1% Brij 96. The presence of constitutively free cysteines made treatment with 2-bromopalmitate unnecessary. Anti-CD9 antibodies ALB6, MM2/57, and C9BB were used for immunoprecipitation. B, A431 cells were incubated in growth medium supplemented with 50 μm 2-bromopalmitate for 20 h (to expose free cysteines) and then treated with DTME. Relative levels of each form of CD9 (right box) were determined from densitometric measurements. Note: although mutation of CD9 palmitoylation sites resulted in diminished C9BB staining of cell surface CD9 (Fig. 3), inhibition of CD9 palmitoylation by 2-bromopalmitate does not decrease C9BB binding in Fig. 5B. Once CD9 oligomers are stabilized by covalent cross-linking, presence or absence of palmitoylation becomes irrelevant. Furthermore, 2-bromopalmitate diminishes CD9 palmitoylation to an extent sufficient to enable covalent cross-linking, but not enough to affect cell surface recognition of CD9 by C9BB (not shown). Note: panels in A and B each are single, contiguous, unsectioned, gel images.View Large Image Figure ViewerDownload Hi-res image Download (PPT)FIGURE 6C9BB affinity, susceptibility to other tetraspanins and immunofluorescence localization. A, A431 cells were incubated with CD9 antibodies (C9BB or ALB6, at 15 μg/ml) for various times (upper panel) or for 30 min at various doses (lower panel), and then MFI values were determined by flow cytometry. B, MDA-MB-435 cells were transfected to express (panel a) vector alone, (panel b) additional CD9 (1.2-fold above endogenous) or (panel c) CD82 proteins. U937 cells were transfected to express CD9 alone (panel d) or CD9 plus CD81 (panel e). Flow cytometry analyses were carried out using the indicated anti-CD9 antibodies. Inset boxes show C9BB/ALB6 and C9BB/DU-ALL-1 ratios. MFI values for CD82 are 8.5, 11, and 105 in panels a, b, and c respectively. MFI values for CD81 are ∼10 and ∼200 in panels d and e respectively. C, for 2-color confocal microscopy analysis, A431 cells were stained with (panel a) C9BB (red), (panel b) MM2/57 (green), or (panel c) an overlay of both red and green.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Protein Palmitoylation Uniquely Affects CD9 Recognition by Monoclonal Antibody C9BB—Removal of palmitoylation sites from the integrin β4 subunit resulted in a substantial decrease in β4-dependent cell spreading and signaling, accompanied by diminished recognition of cell surface CD9 by mAb C9BB (38Yang X. Kovalenko O.V. Tang W. Claas C. Stipp C.S. Hemler M.E. J. Cell Biol. 2004; 167: 1231-1240Crossref PubMed Scopus (165) Google Scholar). In the same experiment, the total amount of cell surface CD9 did not change (38Yang X. Kovalenko O.V. Tang W. Claas C. Stipp C.S. Hemler M.E. J. Cell Biol. 2004; 167: 1231-1240Crossref PubMed Scopus (165) Google Scholar). Here we confirm and extend those results. Upon stable expression of either wild-type β4, or palmitoylation-deficient β4(β4-7CS) in MDA-MB-435 cells, amounts of cell surface CD9 (detected by flow cytometry, using mAb ALB6), integrin α6 (mostly associated with β4), and tetraspanin CD81 were relatively similar in both cell types (Fig. 2). In sharp contrast, staining by mAb C9BB was markedly diminished in cells expressing β4-7CS, as the C9BB/ALB6 staining ratio decreased from 0.58 to 0.085. We showed previously that physical association of CD9 with β4 is substantially diminished when β4 palmitoylation sites are removed (38Yang X. Kovalenko O.V. Tang W. Claas C. Stipp C.S. Hemler M.E. J. Cell Biol. 2004; 167: 1231-1240Crossref PubMed Scopus (165) Google Scholar). Hence, absence of palmitoylation sites in β4-7CS is not causing β4 to shield the C9BB binding epitope.We then determined the effects of removing palmitoylation sites from CD9 itself. Untagged CD9-Wt and CD9-Pal- (lacking 6 membrane-proximal cysteines) were expressed at similar levels on the surface of RD cells, as detected by anti-CD9 mAbs ALB6 and MM2/57 (Fig. 3A). However, the palmitoylation-deficient protein again showed a decrease (>50%) in C9BB staining (Fig. 3A). Consequently there was a substantial decrease in C9BB/ALB6 (1.20 → 0.43) and C9BB/MM2/57 (0.64 → 0.27) ratios (Fig. 3A). As shown previously, C9BB immunoblotting of CD9 was undiminished upon removal of all 6 CD9 palmitoylation sites (Ref. 30Kovalenko O.V. Yang X. Kolesnikova T.V. Hemler M.E. Biochem. J. 2004; 377: 407-417Crossref PubMed Scopus (108) Google Scholar and confirmed in Fig. 6B), indicating that the C9BB binding epitope on CD9 is not directly affected by the presence or absence of palmitoylation sites. In a separate experiment, we also analyzed GFP-tagged CD9 expression in RD cells. Again, both CD9-Wt and CD9-Pal- showed ample staining of CD9 with mAb ALB6 (Fig. 3B, right panels). However, among cells that were GFP-positive (right quadrants) CD9-Pal- cells showed only 0.3% staining for C9BB (Fig. 2B, bottom middle panel, upper right quadrant), whereas wild-type GFP-CD9 yielded 17.5% staining with C9BB (Fig. 3B, upper middle panel, upper right quadrant). By immunofluorescence microscopy, wild-type GFP-tagged CD9 appe" @default.
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• W2078162414 title "Contrasting Effects of EWI Proteins, Integrins, and Protein Palmitoylation on Cell Surface CD9 Organization" @default.
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