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Originally published In Press as doi:10.1074/jbc.M510617200 on March 13, 2006

J. Biol. Chem., Vol. 281, Issue 18, 12976-12985, May 5, 2006
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Contrasting Effects of EWI Proteins, Integrins, and Protein Palmitoylation on Cell Surface CD9 Organization*Formula

Xiuwei H. Yang{ddagger}, Oleg V. Kovalenko{ddagger}, Tatiana V. Kolesnikova{ddagger}, Milena M. Andzelm§, Eric Rubinstein, Jack L. Strominger§, and Martin E. Hemler{ddagger}1

From the {ddagger}Dana-Farber Cancer Institute and Department of Pathology, Harvard Medical School, Boston, Massachusetts 02115, the §Department of Molecular and Cellular Biology, Harvard University, Cambridge, Massachusetts 02138, and Inserm U268, Institut Andre Lwoff, Universite Paris XI, 94807 Villejuif, Cedex, France

Received for publication, September 28, 2005 , and in revised form, March 10, 2006.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 REFERENCES
 
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 {alpha}3beta1 and {alpha}6beta4 integrins and by palmitoylation of the CD9 and beta4 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.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 REFERENCES
 
Tetraspanin protein CD9 has attracted considerable attention for its crucial role on oocytes during sperm egg fusion (1-3). CD9 also contributes to myoblast fusion (4) mononuclear phagocyte fusion (5), virus-induced syncytia formation (6, 7), osteoclastogenesis (5), and paranodal junction formation in the peripheral nervous system (8). CD9 also promotes juxtacrine signaling by associating with epidermal growth factor (EGF) receptor membrane-bound agonists, pro-TGF{alpha}, pro-HB-EGF, and pro-amphiregulin (9-11). CD9 may affect paracrine signaling by either promoting (12) or inhibiting (9) 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 (13), colon (14), bladder (15), lung (16), pancreatic (17), squamous cell (18, 19), and breast cancers (20, 21). Furthermore, CD9 signaling can decrease cell proliferation while promoting apoptosis (22), and ectopic CD9 can suppress tumor cell motility and metastasis (23, 24), and down-regulate Wnt signaling pathways (25).

Tetraspanins typically assemble into multimolecular membrane complexes. In this regard, CD9 can directly associate with transmembrane Ig superfamily proteins EWI2-F (CD9P-1, FPRP) and EWI-2 (26-29), which can drive CD9 into filopodia (32). CD9 also directly associates with itself, suggesting that CD9-CD9 homodimers are building blocks for larger tetraspanin complexes (30). Additional partners for CD9 include the laminin-binding integrins {alpha}3beta1, {alpha}6beta1, and {alpha}6beta4 (31). 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 (32). The distribution and signaling functions of CD9 also could be affected by its associations with PKC (33), type II phosphatidylinositol 4-kinase (34), gangliosides (35), and cholesterol (36).

Like other tetraspanins, CD9 can undergo palmitoylation on each of its membrane-proximal cysteines (37). Although palmitoylation is not required for CD9 homodimer formation (30), or for CD9 association with EWI proteins (this article), it does support CD9 associations with other tetraspanins, including CD81 and CD53 (37). In addition, CD9 association with the {alpha}6beta4-CD151 protein complex is enhanced by palmitoylation of the integrin beta4 (38) and CD151 (39, 40) proteins. CD9 palmitoylation should be functionally important because palmitoylations of other tetraspanins (CD151 and CD82) markedly affect cell morphology and signaling (39-41).

Monoclonal antibodies that detect dynamic cell surface molecular events have been extremely useful during studies of integrins (42-44). 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 (45, 46). 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 {alpha}2 (A2-IIE10), {alpha}3 (A3-X8), beta1 (TS2/16), and beta4 (3E1) were referenced elsewhere (38, 39). Other anti-CD9 mAbs were C9BB (formerly called 4D5 (47)), SYB.1 (48), PAIN-13 (49), 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 (30). GFP-CD9 (30), GFP-EWI-2 (32), and CD9 x CD82 chimeras (29) 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 (32). Following cell surface biotin labeling, cells were lysed in 1% Brij 96, and the proteins were immunoprecipitated and immunoblotted as previously described (38, 39).

Chemical cross-linking of oligomerized CD9 was achieved as described previously (30). 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.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 REFERENCES
 
Cell 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 1
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  		FIGURE 1.
Cell type-specific variations in C9BB binding to CD9. Breast carcinoma cell lines HCC1419 and MDA-MB-468 were stained for total cell surface CD9 (mAb MM2/57, dark peaks) and for a variable subset of CD9 (mAb C9BB, lighter peaks), as assessed by flow cytometry. Negative control staining is also shown (lightest peaks). r = C9BB/MM257 staining ratio, based on MFI units, after background subtraction.

 


Figure 2
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  		FIGURE 2.
Integrin beta4 palmitoylation alters C9BB staining of CD9. Integrin beta4 wild type (beta4-WT) and beta4 lacking all 7 membrane-proximal cysteine palmitoylation sites (beta4-7CS) were stably expressed in MDA-MB-435 cells (which lack endogenous beta4) as previously described (38). Cell surface staining was measured by flow cytometry counting of ~10,000 cells, using the indicated antibodies to CD9, integrin {alpha}6, 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 (beta4-7CS) and C9BB (beta4-WT) are highly significant (p < 0.005). Similar results have been seen in multiple experiments.

 
Protein Palmitoylation Uniquely Affects CD9 Recognition by Monoclonal Antibody C9BB—Removal of palmitoylation sites from the integrin beta4 subunit resulted in a substantial decrease in beta4-dependent cell spreading and signaling, accompanied by diminished recognition of cell surface CD9 by mAb C9BB (38). In the same experiment, the total amount of cell surface CD9 did not change (38). Here we confirm and extend those results. Upon stable expression of either wild-type beta4, or palmitoylation-deficient beta4(beta4-7CS) in MDA-MB-435 cells, amounts of cell surface CD9 (detected by flow cytometry, using mAb ALB6), integrin {alpha}6 (mostly associated with beta4), 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 beta4-7CS, as the C9BB/ALB6 staining ratio decreased from 0.58 to 0.085. We showed previously that physical association of CD9 with beta4 is substantially diminished when beta4 palmitoylation sites are removed (38). Hence, absence of palmitoylation sites in beta4-7CS is not causing beta4 to shield the C9BB binding epitope.


Figure 3
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  		FIGURE 3.
CD9 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.

 


Figure 4
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  		FIGURE 4.
Variable integrin and EWI-2 effects on C9BB staining. A, 501-Mel cells were stably transfected with integrin {alpha}3 subunit (dark peaks) or mock-transfected (lighter peaks), and then analyzed by flow cytometry for CD9 (C9BB or ALB-6) and integrin {alpha}3 (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.

 
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. 30 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 appeared to be clustered on the surface of MCF-7 cells and at cell-cell contact sites (Fig. 3C). By contrast, palmitoylation-deficient CD9 (CD9-Pal--GFP) was more diffuse on the surface of MCF-7 cells and diminished at cell-cell contact sites.

Contrasting Effects of CD9 Partner Proteins on CDBB Staining—In a cell line (501-Mel) with minimal endogenous {alpha}3 integrin, {alpha}3 subunit was ectopically expressed (Fig. 4A, bottom panel). The amount of total CD9 in 501-Mel cells, as stained by mAb ALB-6 (Fig. 4A, middle panel) and DU-ALL-1 (not shown), remained essentially unchanged. However, integrin {alpha}3 expression caused a marked increase in C9BB staining (Fig. 4A, top panel), causing the C9BB/ALB6 ratio to increase from 0.12 to 0.34. Ectopic expression of integrin beta4 subunit in MDA-MB-435 cells similarly caused a marked increase (~2-fold) in C9BB/ALB6 ratios (not shown). Selectively elevated C9BB staining was particularly obvious when {alpha}3 or beta4 were expressed in cells that did not already have substantial endogenous levels of those integrin subunits.

EWI proteins (EWI-2 and EWI-F) are major protein partners for CD9 (26-29). Whereas expression of integrin {alpha}3 and beta4 subunits could selectively promote C9BB staining, expression of EWI-2 in HEK-293 cells caused a pronounced diminution of C9BB staining (Fig. 4B, top panel), but had minimal effect on ALB6 or DU-ALL-1 staining of CD9 (Fig. 4B, middle and bottom). EWI-2 was present at a level comparable to tetraspanins CD9 and CD81 in HEK-293 cells, such that ~70% of CD9 was associated with EWI-2 (not shown). In HEK-293 cells (Fig. 4B) and in A431 cells (not shown), ectopic expression of EWI-2 or EWI-F caused C9BB staining to decrease by ~50%, whereas total CD9 was essentially unchanged.


Figure 5
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  		FIGURE 5.
C9BB 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.

 
A possible explanation for results in Fig. 4, A and B is that integrins ({alpha}3, beta4) are exposing the C9BB epitope, whereas EWI-2 is obscuring the C9BB epitope on CD9. Hence, it might be expected that C9BB would selectively immunoprecipitate more CD9-integrin complexes, and less CD9-EWI2 complexes. However, anti-CD9 mAbs C9BB and MM2/57 (Fig. 4C) and other anti-CD9 mAb (not shown) immunoprecipitated comparable proportions of CD9-associated integrins and EWI-2 proteins. For example, in a cell line (MCF-7) in which C9BB cell surface staining is relatively high compared with total CD9, comparable amounts of CD9 and associated proteins were obtained (Fig. 4C, lanes 3 and 4). When C9BB surface staining was low (as in MDA-MB-231 cells), correspondingly lower amounts of CD9 protein complexes were immunoprecipitated by C9BB (Fig. 4C, lanes 1 and 2). Nonetheless, the relative proportions of integrins, EWI-2, and CD9 were maintained.

C9BB Preference for Clustered CD9—The C9BB binding epitope on the CD9 molecule did not appear to be either lost, shielded, or up-regulated upon removal of beta4 or CD9 palmitoylation sites (Figs. 2 and 3), or upon expression of integrins or EWI proteins (Fig. 4). Hence, an alternative explanation is needed for variable C9BB cell staining. A possible clue comes from results in Fig. 3C, where C9BB staining correlated with increased cell surface clustering. Based on this result, we hypothesized: 1) that C9BB is a low affinity antibody that may preferentially stain CD9 when it is clustered on the cell surface, and 2) that manipulations of palmitoylation, integrins and EWI proteins are affecting CD9 clustering, thereby indirectly affecting C9BB staining. As shown for other cell surface molecules (e.g. CD47, CDw78, CD147), cell surface antigen clustering markedly enhances recognition by low affinity antibodies, while having minimal effect on cell surface staining by high affinity antibodies (50-52).

Next we established that C9BB indeed prefers clustered CD9. To achieve this, we first captured cell surface CD9 multimers by chemical cross-linking of cysteines on intact RD and A431 cells as previously described (30). Then we assessed C9BB recognition of CD9 multimers in cell lysates. As indicated in Fig. 5A, C9BB showed a strong preference for CD9 trimer from RD cells. By contrast, CD9 in the cell lysate, and in two other anti-CD9 immunoprecipitations (ALB6 and MM2/57), was much less enriched for the trimeric form. From A431 cells (Fig. 5B), CD9 dimer, trimer, and tetramer were preferentially recognized by mAb C9BB. By contrast, mAb MM2/57 (Fig. 5B) and ALB6 (not shown) recognized mostly monomer (see quantitation in box).

To assess the relative affinity of C9BB for CD9, A431 cells were incubated with C9BB or MM2/57 mAb (at 15 µg/ml) for various times at 4 °C, prior to flow cytometry analysis. MM2/57 binding reached saturation (at ~300 MFI units) within ~20 min (Fig. 6A, panel a). By contrast, C9BB showed less binding and still had not reached saturation after 300 min. Antibody titration curves (Fig. 6A, panel b) showed consistently higher binding by MM2/57, with apparent half-maximal saturation at a 20-fold lower dose compared with mAb C9BB. Hence, C9BB affinity for CD9 is substantially lower compared with MM2/57 (Fig. 6A), and DU-ALL-1 and ALB-6 (not shown).

Next we predicted that if C9BB binding is enhanced by CD9 clustering, then increasing CD9 levels should increase clustering, thereby disproportionately increasing C9BB binding. Indeed, upon overexpression of CD9 in MDA-MB-435 cells, C9BB binding increased 2.4-fold, whereas ALB6 and DU-ALL-1 binding increased by only 1.2-fold. Thus the C9BB/ALB6 and C9BB/DU-ALL-1 ratios each increased by 2-fold (compare Fig. 6B, panels a and b). Overexpression of CD9 in RD cells similarly increased both the C9BB/ALB6 and C9BB/DU-ALL-1 ratios by 2-3-fold (not shown). These results are consistent with C9BB preferentially detecting clustered CD9.

We also analyzed the cellular distribution of CD9 molecules by double immunostaining of A431 cells with C9BB and MM2/57 mAbs. As shown (Fig. 6C, panel b), mAb MM2/57 staining was somewhat concentrated at cell-cell contact sites. C9BB staining of the same cells was perhaps even more concentrated at cell-cell contact sites (Fig. 6C, panel a), and overlapping staining (seen as yellow) was particularly evident at cell-cell contact sites (Fig. 6C, panel c), where clustering appears to be most pronounced.

Whereas elevated CD9 expression caused a selective increase in C9BB staining (Fig. 6B, compare panels a and b), expression of other tetraspanins had the opposite effect. Expression of tetraspanin CD82 decreased C9BB/ALB6 and C9BB/DU-ALL-1 ratios on MDA-MB-435 cells (0.61 -> 0.39; 0.24 -> 0.18; Fig. 6B, compare panels a and c). Like-wise expression of CD81 in CD9-transfected U937 cells caused a consistent small decrease in C9BB/ALB and C9BB/DU-ALL-1 ratios (0.72 -> 0.47; 0.34 -> 0.25; Fig. 6B, compare panels d and e). On the other hand, siRNA-mediated knockdown of tetraspanins CD151 (by 90%) or CD81 (by 90%) in MDA-MB-231 cells caused corresponding elevation in C9BB/MM2/57 ratios (0.27 -> 0.36; 0.27 -> 0.40) respectively (FACS data not shown).


Figure 6
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  		FIGURE 6.
C9BB 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.

 
CD9 Palmitoylation Affects EWI-2 Association—Whereas CD9 palmitoylation appeared to promote homoclustering (CD9-CD9), EWI protein expression decreased CD9-CD9 clustering while increasing heteroclustering (i.e. CD9-EWI-2). Hence we predicted that if CD9 were induced to become less involved in homoclustering (e.g. by removing palmitoylation as seen in Fig. 3), then CD9 would become more available for EWI-2 association (see Fig. 11 below). Indeed, elevated EWI-2 association was seen for CD9-Pal-, compared with CD9-Wt, in RD cells (Fig. 7A). This result was obtained whether CD9 was immunoprecipitated using C9BB (Fig. 7A, compare lanes 2 and 3) or ALB6 (compare lanes 6 and 7). For this experiment, CD9 antibodies were added prelysis, so that only cell surface CD9 complexes would be recovered. C9BB immunoprecipitated >50% less CD9-Pal-, compared with CD9-Wt (Fig. 7B, lanes 2 and 3), likely because of the diminished ability of C9BB to recognize cell surface CD9-Pal- (Fig. 3). If either CD9 or EWI-2-GFP were omitted (Fig. 7A, lanes 1, 4, 5, and 8), no co-precipitation was observed. Only a low level of EWI-2 was expressed in RD cells (Fig. 7) compared with HEK-293 cells (Fig. 4B). Hence, immunoprecipitation of CD9 by C9BB was not notably impaired because of the presence of EWI-2 (Fig. 7B, compare lanes 2 and 4).

CD9 Subdomains Involved in C9BB Binding—Nearly all antitetraspanin monoclonal antibodies recognize epitopes entirely within tetraspanin large extracellular loop (EC2) regions. Here we used chimeric CD9/CD82 molecules (Fig. 8A, top) to localize regions of CD9 needed for C9BB binding. Chimeras were transiently expressed in human RD cells, which have relatively little endogenous CD9 protein. As expected, when the EC2 domain was replaced, neither C9BB nor MM2/57 immunoblotted CD9 (Figs. 8A, chimeras 2 and 3; 7B, lanes 2 and 3). As expected, chimeras containing only the EC2 region from CD9 (chimera 5, lane 5) or CD9 EC2 plus adjacent C-terminal domains (chimera 4, lane 4) were immunoblotted by MM2/57 (and DU-ALL-1, not shown). However, chimeras 4 and 5 were not recognized by C9BB. C9BB immunoblotting was only restored when CD9 EC2 regions plus additional flanking TM2-TM3 regions were present (chimeras 7 and 8; lanes 7 and 8). In control experiments, C9BB failed to immunoblot negative control CD82 (chimera 6, lane 6), but did immunoblot wild-type CD9 (chimera 1, lane 1). Cell surface flow cytometry analysis confirmed that C9BB staining of CD9 was greatly decreased (by 93-100%) when either EC2 or regions adjacent to EC2 were replaced (Fig. 8A). By comparison, staining of intact cells by ALB-6 (Fig. 8A) or DU-ALL-1 or MM2/57 (not shown) was completely eliminated when EC2 was replaced, and was mostly retained when any other region of CD9 was replaced. Anti-CD82 mAb M104 confirmed that wild-type CD82 and chimeras containing CD82-EC2 were well expressed at the cell surface (Fig. 8A).


Figure 7
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  		FIGURE 7.
EWI-2 preference for unpalmitoylated CD9. RD cells transiently expressing CD9-Wt or CD9-Pal-, alone or with GFP-tagged EWI-2, were incubated with mAb C9BB or ALB6 at 4 °C for 1 h. After excess mAb was removed by washing, cells were lysed in 1% Brij 96, and then immune complexes were recovered, resolved by SDS-PAGE, and blotted for EWI-2 (anti-GFP, top panel) or CD9 (C9BB, bottom panel).

 


Figure 8
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  		FIGURE 8.
Atypical C9BB binding epitope on CD9. A, regions of CD9 (dark lines) and CD82 (light lines) are indicated schematically, for chimeric CD9-CD82 molecules. Chimeras, transiently expressed in RD cells, were analyzed by flow cytometry for binding of C9BB, ALB6, and anti-CD82 mAb M104 (right columns). B, RD cells transiently expressing chimeras were lysed in Triton X-100, and then blotted for CD9 using mAb MM2/57 (top panel) or C9BB (bottom panel).

 
Reduced CD9 Clustering on Malignant Epithelial Cancer Cells—Malignant epithelial tumor cells show reduced CD9 levels, consistent with CD9 being a possible tumor suppressor (see Introduction). For example, MDA-MB-231 breast cancer cells showed ~65% reduced CD9 levels (as detected by DU-ALL-1 or ALB6) when compared with other less malignant breast cancer lines, including MCF-7 and MDA-MB-453 cells (Fig. 9A). By contrast, C9BB staining of MDA-MB-231 cells was diminished by over 90% (Fig. 9A). Immunofluorescence confocal microscopy of MCF-7 cells showed preferential staining of cell-cell contact sites by all CD9 antibodies (ALB6, MM2/57, C9BB; Fig. 10, panels a-f). On the other hand, MDA-MB-231 cells (where C9BB staining was particularly low) showed much less CD9 at cell-cell contact sites (Fig. 10, panels g-i). Again C9BB staining (panel g) was more restricted relative to total CD9 staining (panel h).

Comparison of two prostate cell lines (tumorigenic LNCaP, metastatic PC3) revealed only a small change in total CD9 levels in PC3 cells (10-15% decreased), but again more pronounced results were seen with C9BB (~60% decrease; Fig. 9A, lower panel). Immunoprecipitations of CD9 from LNCaP and PC3 cells (Fig. 9B) or from MDA-MB-231 and MCF-7 cells (Fig. 9C) yielded integrins, EWI-2, and a few other unknown proteins. Notably, for the two less malignant cell lines (LNCaP and MCF-7) the ratio of recovered EWI-2/CD9 protein was relatively low, whereas higher EWI-2/CD9 ratios were obtained from the more malignant cell lines (PC3 and MDA-MB-231; Fig. 9, B and C). Hence, increased association with EWI-2 could be at least partly responsible for decreased C9BB staining of MDA-MB-231 and PC3 cells (Fig. 9A).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
REFERENCES
 
Regulation of C9BB Binding—The fraction of CD9 recognized by mAb C9BB, compared with total cell surface CD9, varied widely on different cell types. To help explain this variation, we identified four different molecular manipulations that can selectively affect C9BB recognition of CD9. First, C9BB staining was markedly diminished upon removal of protein palmitoylation sites, either from CD9 itself, or from beta4 integrin, which is known to associate with CD9 (38, 53). Second, expression of some CD9 partner proteins (e.g. {alpha}3 or beta4 integrins) promoted C9BB recognition of CD9. This was especially obvious in cells not already expressing an abundance of those integrins. Third, expression of other CD9 partner proteins (e.g. EWI-2 and EWI-F) down-regulated C9BB staining of CD9. Fourth, other tetraspanins (CD81, CD82, CD151) also negatively influenced C9BB staining of CD9.

None of these molecular manipulations appeared to affect directly the C9BB binding epitope on CD9. For example, C9BB immunoblotted CD9-Wt and CD9-Pal- equally well, despite diminished cell surface recognition of the latter. Also, palmitoylation-deficient beta4 shows decreased association with CD9 (38), and thus is not in position to obscure the C9BB binding epitope. Furthermore, mAb C9BB and other anti-CD9 antibodies immunoprecipitated CD9 partner proteins (integrins, EWI proteins, other tetraspanins) in equal proportion. This would not occur if these partner proteins were selectively exposing or obscuring the C9BB epitope. Hence, we sought an alternative explanation for variable C9BB binding, which did not involve direct effects on epitope exposure.


Figure 9
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  		FIGURE 9.
Diminished C9BB staining on malignant carcinomas. A, indicated breast (top panel) and prostate (bottom panel) cancer cell lines were stained with anti-CD9 mAb and analyzed by flow cytometry. Differences between C9BB and the other CD9 antibodies (ALB6, DU-ALL-1) on MDA-MB-231 and PC3 cells are highly significant (p < 0.005). Similar results have been seen in multiple experiments. B, human prostate PC3 and LNCaP cancer cells and C, breast carcinoma cell lines MDA-MB-231 and MCF-7 were labeled with biotin, lysed in 1% Brij 96, and then CD9 was immunoprecipitated using mAb ALB6. Biotin-labeled proteins are visualized by blotting with Avidin (top panel), and CD9 is blotted with mAb C9BB (bottom panel).

 


Figure 10
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  		FIGURE 10.
Staining of CD9 in breast cancer cells. Human breast cancer cells MCF-7 (panels a-f) and MDA-MB-231 (panels g-i) are stained with C9BB mAb (red; a, d, g), ALB6 (green, b) or MM2/57 (green, e, h). Overlay of red and green is shown in panels c, f, and i.

 
The Role of CD9 Clustering—For antibodies that bind bivalently to cells, there is enormous variation in bivalent binding affinity, dependent on antigen density (54, 55). Antibodies with low monovalent affinity are particularly dependent on bivalent binding, and consequently are particularly sensitive to antigen clustering. Indeed, this phenomenon has been well documented, as low affinity antibodies have been used to detect cell surface clustering of CDw78, CD47, and CD147 (50-52). Our results suggest that C9BB is likewise a low affinity antibody, with cell surface binding strongly dependent on cell surface CD9 clustering. Several results support this low affinity-C9BB-CD9 clustering model. First, as seen from two different cell lines, C9BB, in contrast to other anti-CD9 mAbs, preferentially recognized multimerized CD9. Second, C9BB, compared with other CD9 mAB, indeed has a relatively lower affinity for CD9. Third, C9BB staining was most obvious at cell-cell contact sites, where CD9 accumulates (Figs. 3C, 6C, 10, and Refs. 56 and 57). Fourth, overexpression of CD9, which would be expected to increase clustering, led to disproportionately increased staining by C9BB, relative to other anti-CD9 antibodies. Fifth, C9BB binds very well to CD9 in immunoblots (where CD9 is concentrated/clustered), even when not recognizing the same CD9 on the cell surface. Sixth, removal of CD9 palmitoylation sites (or beta4 sites) decreased C9BB staining. In this regard, loss of palmitoylation also decreases tetraspanin-tetraspanin (37, 39, 40) and CD9-beta4 (38) associations, consistent with CD9 becoming more diffuse on the cell surface.

CD9 Homoclustering and Heteroclustering—We suggest that CD9 can shift between homoclustered and heteroclustered states, as indicated in the model in Fig. 11. As outlined below, the balance between these states may have considerable relevance with respect to CD9 motility regulation and tumor suppression functions. Fig. 11 highlights novel roles for tetraspanin palmitoylation. Whereas it was shown previously that tetraspanin palmitoylation supports various types of heteroclusters (37, 39, 40), this may be the first example of tetraspanin palmitoylation supporting homoclustering. Another previous study showed that CD9 homodimerization (as detected by covalent cross-linking) is unaffected by CD9 palmitoylation (58). However that result does not contradict the current one. Whereas CD9 homodimers arise from direct protein-protein interactions, independent of palmitoylation (58), it is the assembly of those homodimers into expanded homoclusters that appears to be supported by palmitoylation. Although CD9 palmitoylation supports homoclustering, it decreased CD9-EWI-2 heteroclustering (Fig. 7A), possibly because palmitoylation on CD9 partially blocks its accessibility to EWI-2. Alternatively, palmitoylation could shift the balance of CD9 toward homoclusters, thus making CD9 less available for EWI-2 heteroclusters (Fig. 11). This type of negative regulation by palmitoylation was not seen in previous studies of either tetraspanins or EWI-2. In cases where tetraspanin palmitoylation is dynamically regulated (59), the balance between homo- and heteroclustering could therefore be regulated in parallel.

Expression of integrins ({alpha}3beta1, {alpha}6beta4) also promoted CD9 clustering. These integrins are not direct partners of CD9, but rather may promote CD9 clustering through secondary (i.e. indirect) interactions, likely involving CD151 (39, 60). Again, palmitoylation plays a key role as ectopic expression of unpalmitoylated mutant beta4 did not increase CD9 clustering (Ref. 38 and not shown). Furthermore, for CD151-{alpha}3beta1 and CD151-{alpha}6beta4 complexes, removal of palmitoylation sites from either CD151 (39, 40), or from beta4 (38) disrupts secondary interactions of the integrins with other tetraspanins, including CD9. In conclusion, CD9 homoclustering is enhanced within a network of secondary interactions, supported by palmitoylation of CD9, integrin beta4, and possibly also CD151.


Figure 11
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  		FIGURE 11.
Model for CD9 homoclusters and heteroclusters. CD9 homoclusters are favored by CD9 palmitoylation, integrin palmitoylation (38), and by the presence of {alpha}3beta1or {alpha}6beta4 integrins. CD9 heteroclusters are favored by expression of EWI-2 or EWI-F, and by de-palmitoylation of CD9. The presence of other tetraspanins (besides CD9) also diminishes homoclustering (not shown). Also, intermediate mixed complexes may form, containing integrins, tetraspanins, and EWI proteins (32, 61).

 
Whereas palmitoylation and integrin expression increased CD9 clustering, EWI proteins diminished CD9 clustering. Disruption of CD9 clustering could be a key component to the mechanism whereby EWI-2 regulates cell migration, spreading, and signaling (32, 61). Ectopic expression of other tetraspanins (CD81, CD82) also down-modulated CD9 clustering. We suspect that these other tetraspanins, like EWI proteins, can form direct molecular contacts with CD9, thereby disrupting CD9 homo-oligomers. In support of this, a low level of heterotetraspanin dimerization (e.g. CD9-CD81) has been captured covalently (30). In summary, indirectly associated integrins promote palmitoylation-dependent homoclustering of palmitoylated CD9. In sharp contrast, EWI proteins and other tetraspanins can form direct, palmitoylation independent, protein-protein interactions with CD9, thereby disrupting homo-oligomeric clustering, while forming hetero-oligomers. We considered that CD9 associations with cholesterol (36) and gangliosides (62) might also affect CD9 clustering, and therefore C9BB staining. However, treatment of cells with cholesterol depleting agents (e.g. methyl-beta-cyclodextrin) or with sphingomyelinase, alone or together, did not affect C9BB staining (not shown). In summary, we suggest that wide variations in C9BB staining (supplemental Table S1) arise because of variable positive and negative effects of integrins, EWI proteins, palmitoylation, and other tetraspanins, on the organization of cell surface CD9.

CD9 on Tumor Cells—New insights regarding CD9 clustering are also quite relevant to the role of CD9 on tumor cells. Over 50 articles have suggested that CD9 expression levels diminish when various types of tumor cells become more malignant. We show here that CD9 not only decreases expression levels, but also shows less homoclustering. Based on preliminary results with two different malignant cell types, we suggest that C9BB has the potential to be more discriminating, and thus more diagnostically useful than other CD9 antibodies. Decreased CD9 clustering may reflect the tendency of malignant cells to have less organized cell-cell junctions (where tetraspanins are typically known to be clustered). In addition, decreased C9BB binding could involve increased CD9-EWI-2 association on malignant tumor cells, and/or decreased integrin association. For the two pairs of cell lines shown in Fig. 9, B and C, the malignant lines with diminished C9BB staining also had more CD9-EWI-2 association. Hence, EWI-2 association could be play a particularly important role in the diminished CD9 suppressor activity and increased malignancy that accompanies decreased C9BB staining. Conversely, homoclustered CD9 may be more active in tumor suppression. In this regard, CD9 may resemble many other cell surface transmembrane proteins for which homoclustering is typically associated with increased functional activity. In a particularly relevant example, low affinity cluster-specific antibodies to cell surface CD147 protein recognize active subsets of total CD147 (50, 63, 64).

The C9BB Binding Epitope—C9BB clearly binds to an extracellular site, as evidenced by flow cytometry results using intact cells. The large extracellular loop (EC2) is necessary, but not sufficient, whereas the short extracellular loop (EC1) is neither necessary nor sufficient for binding. Studies with chimeric molecules (Fig. 8) point to involvement of transmembrane and/or intracellular regions that flank the EC2 loop. Specific interactions between transmembrane domains TM3 and TM4 (65) could possibly stabilize an extracellular conformation containing the C9BB epitope. By contrast to C9BB, 11/11 other monoclonal antibodies to CD9 (Refs. 49 and 66 and this article), and 22/22 antibodies to other tetraspanins (49, 66, 67), all recognize epitopes entirely within tetraspanin EC2 domains. At present, we have no evidence that the unique properties of the C9BB binding epitope contribute to the wide variations in cell surface CD9 recognition of different cell types. Notably, the native CD9 conformation recognized by C9BB on cell surfaces does not appear to be labile, because C9BB also recognizes CD9 after it has been boiled in SDS and transferred to nitrocellulose (e.g. as shown in Fig. 8). Another anti-CD9 antibody, PAINS-13, has been reported to be regulated by integrin activation (49). In a side-by-side comparison, PAINS-13 showed 90-95% less binding than C9BB to 6 different cell lines, 50-80% less binding to three other cell lines, and no overall correlation to C9BB binding levels. In further contrast to PAINS-13, C9BB binding was not influenced by divalent cations, temperature, or integrin-activating antibodies (not shown).

In conclusion, mAb C9BB represents perhaps the first documented cluster-specific antitetraspanin antibody. Already this antibody has been used to discover contrasting homoclustering effects of palmitoylation and integrin expression and heteroclustering effects of EWI proteins and other tetraspanins. Also C9BB appears to be potentially more useful than other anti-CD9 antibodies in terms of monitoring tumor progression. In future studies, C9BB should continue to be a valuable tool, useful for dissecting the role of CD9 during its many contributions to cell fusion, proliferation, and signaling events. Previously we found that tetraspanins (such as CD9 and CD81), EWI proteins, and integrins can associate together in expanded complexes (32, 61). Now, with our specialized tool, mAb C9BB, we can distinguish between homo-oligomeric complexes (with CD9 mostly clustered with itself) and heterooligomeric complexes (CD9 clustered with other molecules). Furthermore, we can show that whereas integrins promote homo-oligomeric complexes, EWI proteins, and other tetraspanins promote hetero-oligomeric complexes (see Fig. 11). Finally, our C9BB studies establish a precedent that could be applicable to many other tetraspanins. Because tetraspanins are primarily known for their ability to form organized lateral partnerships with many other proteins, other cluster-specific antibodies could again have considerable utility. Such reagents would enable studies of tetraspanin organization on the surface of intact cells in a way that has not previously been possible.


   FOOTNOTES
 
* This work was supported by awards from the DFCI Friends Foundation and a DFCI Claudia Adams Barr grant (to X. H. Y.) and National Institutes of Health Grants GM38903 and CA42368 (to M. E. H.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

Formula The on-line version of this article (available at http://www.jbc.org) contains supplemental figures and tables. Back

1 To whom correspondence should be addressed: Dana-Farber Cancer Inst., Rm D-1430, 44 Binney St., Boston, MA 02115. Tel.: 617-632-3410; Fax: 617-632-2662; E-mail: Martin_Hemler{at}DFCI.Harvard.edu.

2 The 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. Back



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