Profiling of the Tetraspanin Web of Human Colon Cancer Cells

Profiling of the Tetraspanin Web of HumanColon Cancer Cells*□S

Francois Le Naour‡§, Magali Andre‡¶, Celine Greco‡, Martine Billard‡,Bernard Sordat�, Jean-Francois Emile‡**, Francois Lanza‡‡, Claude Boucheix‡,and Eric Rubinstein‡

Tetraspanins are integral membrane proteins involved in avariety of physiological and pathological processes. Incancer, clinical and experimental studies have reported alink between tetraspanin expression levels and metasta-sis. Tetraspanins play a role as organizers of multimolecu-lar complexes in the plasma membrane. Indeed each tet-raspanin associates specifically with one or a few othermembrane proteins forming primary complexes. Thus,tetraspanin-tetraspanin associations lead to a molecularnetwork of interactions, the “tetraspanin web.” We per-formed a proteomic characterization of the tetraspaninweb using a model of human colon cancer consisting ofthree cell lines derived from the primary tumor and twometastases (hepatic and peritoneal) from the same pa-tient. The tetraspanin complexes were isolated after im-munoaffinity purification using monoclonal antibodies di-rected against the tetraspanin CD9, and the associatedproteins were separated by SDS-PAGE and identified bymass spectrometry using LC-MS/MS. This allowed theidentification of 32 proteins including adhesion molecules(integrins, proteins with Ig domains, CD44, and epithelialcell adhesion molecule) (EpCAM), membrane proteases(ADAM10, TADG-15, and CD26/dipeptidyl peptidase IV),and signaling proteins (heterotrimeric G proteins). Impor-tantly some components were differentially detected inthe tetraspanin web of the three cell lines: the lamininreceptor Lutheran/B-cell adhesion molecule (Lu/B-CAM)was expressed only on the primary tumor cells, whereasCD26/dipeptidyl peptidase IV and tetraspanin Co-029were observed only on metastatic cells. Concerning Co-029, immunohistofluorescence showed a high expressionof Co-029 on epithelial cells in normal colon and a lowerexpression in tumors, whereas heterogeneity in terms ofexpression level was observed on metastasis. Finally wedemonstrated that epithelial cell adhesion molecule andCD9 form a new primary complex in the tetraspanin web.Molecular & Cellular Proteomics 5:845–857, 2006.

Tetraspanins are integral membrane proteins characterizedby the presence of four transmembrane domains delimitingthree short intracellular domains and two extracellular regionsof unequal size. They exhibit significant sequence identity aswell as specific structural features in the larger of the twoextracellular domains (1–3). All cell types studied so far ex-press several tetraspanins, often to a high level. These mole-cules have been implicated in a large variety of physiologicalprocesses such as immune cell activation, cell migration,cell-cell fusion (including fertilization), and various aspects ofcellular differentiation. These molecules have also beenshown to play a role in infectious diseases (e.g. malaria,hepatitis C virus, and human immunodeficiency virus), andseveral genetic diseases are linked to mutations in certain ofthese molecules (e.g. X-linked mental retardation, retinal de-generation, and incorrect assembly of human basementmembranes in kidney and skin) (1–8).

In cancer, clinical studies have reported a link betweentetraspanin expression levels and prognosis and/or metasta-sis. Indeed a high level of the tetraspanins CD9 and CD82/KAI-1 on tumor cells is associated with a favorable prognosisin breast, lung, colon, prostate, and pancreas cancers. Addi-tionally a decreased expression level of these molecules iscorrelated with metastasis in these cancers (for a review, seeRefs. 1 and 4). In contrast, overexpression of CD151 in lung,colon, and prostate cancers was correlated with poor prog-nosis (9–11). Furthermore using in vitro and in vivo experi-mental models, CD9 and CD82 have been shown to act as“metastasis suppressors,” whereas CD151 was shown to in-crease the metastatic potential (1, 4, 12–14).

The function of tetraspanins is still not precisely known. Wehave demonstrated that several tetraspanins associate withone or a few specific molecular partners, forming small pri-mary complexes. Thus the tetraspanin CD151 associates di-rectly with the integrins �3�1 and �6�1 (3, 15), whereas CD9and CD81 have been shown to associate with two moleculeswith immunoglobulin domains, CD9P-1 and EWI-2 (3, 16–18).Furthermore tetraspanins have been shown to associate witheach other by a mechanism involving palmitoylation and cho-lesterol (3, 19–22). Under lysis conditions preserving tet-raspanin to tetraspanin interactions, these molecules havebeen shown to partition into the low density fractions of asucrose gradient, indicating association with detergent-resis-tant domains in the membrane (3, 22). Thus, tetraspanins mayorganize particular microdomains on the plasma membraneto which they target their partner proteins. These microdo-mains appear to be different from the so-called lipid rafts. The

From the ‡INSERM U602, Institut Andre Lwoff, Universite Paris XI,Hopital Paul Brousse, 94807 Villejuif Cedex, France, �Institut Suissede Recherches Experimentales sur le Cancer, 1066 Epalinges, Swit-zerland, **Hopital Ambroise Pare, 92100 Boulogne-Billancourt,France, and ‡‡INSERM U311, Etablissement Francais du Sang-Al-sace, 67065 Strasbourg, France

Received, October 6, 2005, and in revised form, January 10, 2006Published, MCP Papers in Press, February 7, 2006, DOI 10.1074/

mcp.M500330-MCP200

Research

© 2006 by The American Society for Biochemistry and Molecular Biology, Inc. Molecular & Cellular Proteomics 5.5 845This paper is available on line at http://www.mcponline.org

entire set of interactions involving tetraspanins is called the“tetraspanin web” (1, 3, 23).

A better description of the composition and the organiza-tion of the tetraspanin web appears essential to understandthe function of these molecules. Moreover a comparison ofthe tetraspanin web in primary and metastatic cancer cellsmay provide new clues for understanding the role of tet-raspanins in metastasis. In this study, we performed a pro-teomic analysis of CD9-containing complexes by mass spec-trometry using several cancer cell lines from the same patient.

EXPERIMENTAL PROCEDURES

Cell Culture—The human Isreco1, Isreco2 and Isreco3 cell lineswere described previously (24). HeLa and colon carcinoma cell linesCaco-2, SW48, HT29, SW480, Lovo, Colo205, and SW620 wereobtained from ATCC. All cell lines were cultured in Dulbecco’s mod-ified Eagle’s medium supplemented with 10% FCS, 2 mM glutamine,and antibiotics (all from Invitrogen). The cells were maintained in a37 °C humidified incubator in the presence of 5% CO2.

Monoclonal Antibodies—Anti-tetraspanin mAbs1 used in this studywere ALB-6 (CD9) (25), TS9 (CD9), TS53 (CD53), TS63 (CD63), TS81(CD81), TS82 (CD82), TS151 (CD151) (16), Z81 (CD81), and AZM22(Co-029) (26). Anti-integrin mAbs used were �1-vjf (integrin �1) (16);HP2B6 (integrin �1) (Immunotech, Marseille, France); AK7 (integrin�2) (Diaclone, Besancon, France); M-KID-2 (integrin �3) (27); v4-vjf(integrin �4), v5-vjf (integrin �5), and 4F10 (integrin �6) (Serotec,Oxford, UK); and 450-9D and 450-11A (CD104/integrin �4) (BD Bio-sciences). Other mAbs were 1F11 (CD9P-1) (16), 8A12 (EWI-2) (18),12A12 (CD55) (28), HEA125 (epithelial cell adhesion molecule (Ep-CAM)) (Progen Biotechnik, Heidelberg, Germany), VIM15 (CDw92)(Research Diagnostics, Flanders, NJ), M-A261 (CD26) (Serotec,Cergy Saint-Christophe, France), and AC-74 (�-actin) (Sigma). TheF241 mAb (Lutheran/B-cell adhesion molecule (Lu/B-CAM)) was a giftfrom Dr. Wassim El Nemer.

Flow Cytometry Analysis—Cells were detached using a non-enzy-matic solution (Invitrogen), washed, and stained with saturating con-centrations of primary mAb. After washing three times with medium,cells were incubated with 10 �g/ml FITC-labeled goat anti-mouseantibody. After washing, cells were fixed with 1% formaldehyde inPBS. All incubations were performed for 30 min at 4 °C. The analysisof cell surface staining was performed using a FACSCalibur (BDBiosciences).

Immunoisolation of CD9-containing Complexes and In-gel TrypticDigestion—For identification of CD9-associated molecules, 5 � 108

cells were lysed in 40 ml of lysis buffer containing 10 mM Tris, pH 7.4,150 mM NaCl, 1 mM CaCl2, 1 mM MgCl2, 1% Brij97 in the presence ofprotease inhibitors. Insoluble material was removed by centrifugationat 12,000 � g for 15 min, and the lysates were precleared three timessuccessively with Sepharose 4B beads (Amersham Biosciences) cou-pled to BSA, to goat serum (Sigma), and then to an isotype-matchedmAb. Isolation of CD9-containing complexes was performed usingbeads coupled to mAb ALB-6. The beads were washed five timeswith lysis buffer, and the proteins were eluted using 1% Triton X-100and then acetone-precipitated. The proteins were separated by5–15% SDS-polyacrylamide gel electrophoresis under non-reducingconditions. For profiling of the tetraspanin complexes, gels were

silver-stained as described previously (18). For mass spectrometryanalysis, the gels were stained with colloidal Coomassie Blue (Bio-Rad). The proteins were excised and destained. The gel pieces wereincubated in 100% acetonitrile for 10 min, dried, and incubated in 100mM ammonium bicarbonate containing 10 mM DTT for 30 min at56 °C. After cooling to room temperature, the DTT solution wasreplaced with 55 mM iodoacetamide in 100 mM ammonium bicarbon-ate for 20 min at room temperature in the dark. The gel pieces werewashed in 100 mM ammonium bicarbonate for 20 min, dehydrated in100% acetonitrile, and dried. The gel pieces were swollen in a diges-tion buffer containing 25 mM ammonium bicarbonate and 100 ng oftrypsin (Roche Applied Science). Following enzymatic digestion over-night at 37 °C, the resulting peptides were extracted with 50 �l of 5%formic acid for 15 min at 37 °C followed by addition of 100 �l of 100%acetonitrile for another 15 min at 37 °C. The peptides were then driedand rehydrated in 1% formic acid.

LC-ESI-MS/MS on Ion Trap and Data Analysis—LC-MS/MS anal-yses were performed using an ESI ion trap mass spectrometer (LCQDeca XP, ThermoElectron, San Jose, CA) coupled on line with acapillary nano-HPLC system (LC Packings) (Dionex, Amsterdam, ND)for liquid chromatography. The capillary column used in this studywas a PepMap C18 reverse phase (75-�m inner diameter, 15 cm) (LCPackings). A linear 20-min gradient (flow rate, 170 nl/min) from 5 to50% acetonitrile in 0.1% (v/v) aqueous formic acid was performed. Alldata were collected in centroid mode using data-dependent acquisi-tion mode. After the acquisition of a full MS scan (m/z 400–2000 Da)in the first scan event, the three most intense ions present above athreshold of 105 counts were subsequently isolated for fragmentation(MS/MS scan). The collision energy for the MS/MS scan events waspreset at a value of 35%. The sequences of the MS/MS spectra wereidentified by correlation with the peptide sequences from humanproteins present in the non-redundant protein sequence database (nrfrom the National Center for Biotechnology Information (NCBI)) usingthe SEQUEST algorithm incorporated into the Finnigan BIOWORKS3.1 software. The SEQUEST search results were initially assessed byexamination of the Xcorr (cross-correlation) and the �Cn (delta nor-malized correlation) scores. As a general rule, an Xcorr value ofgreater than 1.5, 2.0, and 2.5, respectively, for 1�, 2�, and 3�charged peptides and a �Cn greater than 0.1 were accepted as apositive identification (29).

Cell Surface Biotinylation, Chemical Cross-linking, Immunoprecipi-tation, and Western Blot—Surface labeling of cells with EZ-Link sulfo-NHS-LC-biotin (Pierce) was performed as described previously (16,18). Briefly cells were washed three times in Hank’s buffered salineand incubated for 30 min in 10 mM Hepes, pH 7.3, 150 mM NaCl, 0.2mM CaCl2, 0.2 mM MgCl2 containing 0.5 mg/ml EZ-Link sulfo-NHS-LC-biotin. For cross-linking, the cells were incubated for 30 min at4 °C, in the culture flask, with 100 or 500 �M DSP (Pierce) in the samebuffer. After cell surface biotinylation or chemical cross-linking, cellswere washed three times in 20 mM Tris, pH 7.4, 150 mM NaCl, 0.2 mM

CaCl2, 0.2 mM MgCl2 before lysis.Cells were lysed directly in the flask in the lysis buffer (10 mM Tris,

pH 7.4, 150 mM NaCl, 1 mM CaCl2, 1 mM MgCl2, 0.02% NaN3)containing 1% of the appropriate detergent (Brij97, Triton X-100, ordigitonin) (Sigma; Calbiochem) and protease inhibitors. Digitonin wasfirst dissolved in methanol at the concentration of 10% (w/v) and thendiluted in lysis buffer without CaCl2 and MgCl2 as described previ-ously (15). After 30 min at 4 °C, insoluble material was removed bycentrifugation at 10,000 � g, and cell lysate was precleared for 2 h byaddition of 1⁄1000 volume of heat-inactivated goat serum and 20 �l ofprotein G-Sepharose beads (Amersham Biosciences). Proteins werethen immunoprecipitated by adding 2 �g of specific antibody and 10�l of protein G-Sepharose beads to 200–400 �l of lysate. After 2 h ofincubation at 4 °C under constant agitation, beads were washed five

1 The abbreviations used are: mAb, monoclonal antibody; NHS,N-hydroxysuccinimide; DSP, dithiobis(succinimidyl)propionate; Lu/B-CAM, Lutheran/B-cell adhesion molecule; EpCAM, epithelial cell ad-hesion molecule; ADAM, a disintegrin and metalloprotease; TADG-15,tumor-associated differentially expressed gene 15; GPCR, G protein-coupled receptor; DAPI, 4�,6-diamidino-2-phenylindole.

Profiling of the Tetraspanin Web

846 Molecular & Cellular Proteomics 5.5

times in lysis buffer containing 1% of the appropriate detergent. Theimmunoprecipitates were separated by 5–15% SDS-polyacrylamidegel electrophoresis under non-reducing conditions and transferred toa PVDF membrane (Amersham Biosciences). Western blotting onimmunoprecipitates was performed using specific mAbs. Proteinswere revealed by enhanced chemiluminescence (PerkinElmer LifeSciences) after incubation with a streptavidin-biotinylated horseradishperoxidase complex (Amersham Biosciences) when the first antibodywas coupled with biotin; otherwise a secondary goat anti-mouseantibody coupled with horseradish peroxidase (Amersham Bio-sciences) was used.

Immunofluorescence Staining and Confocal Microscopy of FrozenSections—Four micrometer-thick serial sections of frozen human co-lon were fixed for 20 min in acetone at �20 °C. After drying at roomtemperature, the sections were incubated for 10 min in PBS contain-ing 10% heat-inactivated goat serum and then with 5 �g/ml mAb inthe same buffer for 20 min at room temperature in a moist chamber,washed in PBS, and further incubated for 20 min with goat anti-mouse-FITC and DAPI. After three washes the sections weremounted in Mowiol and examined with a Leica DMR fluorescencemicroscope. For confocal microscopy, the sections were incubatedwith 5 �g/ml TS9b (CD9, IgG2b) and HEA125 (EpCAM, IgG1) mAbsand then with a combination of goat anti-mouse IgG2b and IgG1antibodies labeled, respectively, with Alexa Fluor 488 and Alexa Fluor568 (Molecular Probes). Appropriate controls were performed to as-sess the specificity of the labeling. Analysis was performed with aTCS SP2 confocal microscope (Leica, Wetzlar, Germany).

RESULTS

Proteomic Analysis of CD9-containing Complexes of IsrecoCell Lines Using LC-MS/MS—To better define the role oftetraspanins, we investigated the composition of CD9-con-taining complexes in three colon carcinoma cell lines. TheIsreco1 cell line was established from a primary colon cancer,whereas Isreco2 and Isreco3 were established, respectively,from liver and peritoneal metastases from the same patient(24). The two metastatic cell lines were shown to express a

lower level of CD9 than did the primary tumor using PCRdisplay and Western blot analysis (24). However, flow cyto-metric analysis indicated that the metastatic cell lines stillexpressed a high level of CD9 as compared with other surfaceantigens (see below).

The profiling of CD9 complexes of the three cell lines wasperformed by immunoprecipitation experiments. After biotinlabeling of cell surface proteins, cells were lysed with the milddetergent Brij97. We have shown previously that under theseconditions, tetraspanin-tetraspanin interactions are pre-served, and tetraspanin complexes can be immunoprecipi-tated by mAbs directed against any tetraspanin (23). CD9-containing complexes were isolated by immunoprecipitation,and the CD9-associated proteins were eluted using the morestringent detergent Triton X-100, which dissociates tet-raspanin-tetraspanin associations (15, 23). After SDS-PAGE,the proteins were either transferred to a PVDF membrane andrevealed by enhanced chemiluminescence or silver-stained tovisualize the pattern of tetraspanin-associated molecules. Thepattern of proteins co-immunoprecipitated with CD9 from theprimary tumor cell line exhibited several differences com-pared with the pattern obtained from the two metastatic celllines (Fig. 1). One of the major differences was an intense30-kDa band observed in the pattern of the metastatic celllines and absent in the immunoprecipitates collected from theprimary tumor cell line.

The components of CD9 complexes in the Isreco cell lineswere identified by mass spectrometry. The CD9-associatedproteins were isolated from �5 � 108 cells using anti-CD9-coated beads, separated by SDS-PAGE, and stained usingsilver or colloidal Coomassie Blue. Each lane was systemati-cally cut (in about 20 slices), and the proteins were in-gel

FIG. 1. Profiling of CD9-containing complexes. The CD9-containing complexes were solubilized using the mild detergent Brij97 andisolated by immunoprecipitation experiments using specific CD9 mAbs. The associated proteins were eluted using the more stringentdetergent Triton X-100 and separated by SDS-PAGE. A, the proteins were labeled with biotin before lysis and transferred to a PVDF membraneafter SDS-PAGE. The proteins were revealed by streptavidin-peroxidase and chemiluminescence. B, the proteins were revealed by silverstaining. IP, immunoprecipitation; cont., control.


Molecular & Cellular Proteomics 5.5 847

digested with trypsin. To eliminate background proteins fromanalysis, the IgG1-coated beads that were used in the lastpreclearing step were treated identically to anti-CD9 beads.

Any protein identified in both samples was not considered asa tetraspanin-associated protein. The resulting peptides wereanalyzed using LC-MS/MS, which allows separation, isola-

FIG. 2. Protein identification using LC-MS/MS. After in-gel trypsin digestion of the proteins, the resulting peptides were analyzed usingLC-MS/MS. The peptides were separated by nano-HPLC. Total ion count was measured and visualized on a chromatogram (upper panel). Ata precise time (e.g. 27.74 min, dotted straight line), the mass spectrum obtained is shown (middle panel) in which a parent ion can be selected(e.g. m/z � 699.7, black arrow). The fragmentation of that parent ion led to MS/MS spectrum generation containing b and y ions and thussequence information of the parent ion (lower panel). The amino acid sequence can be deduced after search in the NCBI database using theprogram SEQUEST. The putative sequence of the peptide is shown with associated Xcorr and �Cn. THis peptide sequence led to theidentification of the protein CTL2.



tion, and fragmentation of peptides from a complex mixtureand thus protein identification from a single peptide (Fig. 2).The proteins were identified with one to eight peptides (seesupplemental data).

The proteomic analysis led to the identification of 32 differ-ent proteins (Table I). Among them, eight were tetraspanins(CD9, CD81, CD151, Tspan1/NET1, Tspan14/DC-TM4F2, Ts-pan9/NET5, Tspan15/NET7, and Co-029). This analysis re-vealed the presence in the tetraspanin web of several cate-gories of proteins including adhesion molecules andmolecules with Ig domains (integrins �3�1, �6�1, �6�4,CD44, EpCAM, Lu/B-CAM, CD9P-1, and EWI-2), membraneproteases (ADAM10, TADG-15, and CD26), putative cholinereceptors that are poorly characterized (CTL1/CDw92 andCTL2), and signaling molecules (heterotrimeric G protein sub-units) as well as a protein involved in membrane fusion (syn-taxin-3). Of 24 CD9-associated non-tetraspanin proteins iden-tified, 13 were reported previously to associate with at least

one tetraspanin (1, 3, 18, 30, 31). Finally 11 proteins of the 24identified have never been described in the tetraspanin web.These molecules are CD26, TADG-15, Lu/B-CAM, CTL1/CDw92, CTL2, G�2, G�3, G�13, G�2, G�3, and syntaxin-3.

A comparative analysis of the three Isreco cell lines showedthat some proteins were identified in all cells (such as EpCAM,ADAM10, CTL1/CDw92, and CTL2) whereas others were dif-ferentially detected (Table I). The proteins Lu/B-CAM, TADG-15, syntaxin-3, some G proteins, and most tetraspanins weredetected only in the primary tumor cell line Isreco1. By con-trast, the tetraspanin Co-029 was detected only in the meta-static cell lines, and CD26 was detected only in Isreco3.

FIG. 3. Expression of some tetraspanins and associated mole-cules on Isreco cells. The expression levels of the indicated mole-cules at the cell surface were determined by indirect immunofluores-cence and flow cytometry analysis. Int., integrin.

TABLE IList of identified proteins

Proteins are listed according to their position on the gel. IS, Isreco;DPP IV, dipeptidyl peptidase IV; MHC, major histocompatibilitycomplex.

Protein nameNCBI

accessionno.

Theoreticalmolecular

mass

No. ofpeptides

IS1 IS2 IS3

kDa

Integrin �6 5726563 120 0 4 2Integrin �3 11467963 119 3 1 0Integrin �4 2119645 208a 0 1 1Integrin �1 19743821 91 1 4 0CD9P-1 28201801 98 5 3 1DPP IV/CD26 18765694 88 0 0 1Lu/B-CAM 1708887 67 3 0 0CTL2 34784988 80 7 5 1CTL1/CDw92 16945323 73 4 3 4ADAM10 4557251 84 8 6 1TADG-15 10257390 95 1 0 0IgSF8/EWI-2 16445029 65 1 0 0CD46 41019474 44 1 1 0CD44 10835163 40 0 1 0EpCAM 4505059 35 4 2 1G�13 2494886 44 4 3 0G�q 7441570 41 1 1 1G�2 30585131 40 1 0 0G�3 37539140 38 1 0 0G�2 13937391 37 3 1 0G�3 4504053 37 1 0 0Syntaxin-3 6175045 35 1 0 0�-GT 1/CD224 37563581 26 2 2 0MHC class I 15553694 21 2 0 0Co-029 4759238 26 0 5 2NET-7 6912530 33 3 0 0NET-5 5729941 27 2 0 0DC-TM4F2 22266722 31 2 1 0NET-1 11277023 26 1 0 0CD151 21237748 29 1 1 0CD9 4502693 25 4 4 1CD81 4757944 26 1 1 0

a Integrin �4 exhibited a 125-kDa molecular mass because of aproteolytic cleavage as described previously (61).



Cell Surface Expression and Association with CD9 of Se-lected Proteins—The differences observed in the CD9 com-plexes collected from the three cell lines may be the conse-quence of different levels of expression of these molecules.We thus examined the expression levels of tetraspanins andsome of their associated proteins at the cell surface by indi-rect immunofluorescence and flow cytometry when antibod-ies were available (Fig. 3).

There was a reasonable relationship between the expres-sion of the different molecules tested and their associationwith CD9. Thus, CD9P-1, CDw92, and EpCAM, which areexpressed by all three cell lines, were detected in the CD9immunoprecipitates collected from the three cell lines. Addi-tionally the differential detection of CD26 in Isreco3 and ofCo-029 in Isreco2 and Isreco3 reflects differences in expres-sion. On the other hand, Lu/B-CAM was expressed only byIsreco1, consistent with the detection of Lu/B-CAM pep-tides only in this cell line. It should be noted that EWI-2 andintegrins �6 and �4 were differentially detected by massspectrometry despite a similar expression at the surface ofall three cell lines. This apparent discrepancy may suggesta differential targeting of these proteins to the tetraspaninweb. According to this hypothesis, we observed a higheramount of integrin �6�4 co-immunoprecipitated with CD9 in

Isreco2 and Isreco3 as compared with Isreco1 (data notshown).

To further validate the association with CD9 of some novelidentified molecules, we examined whether the interactionscould be observed by reverse co-immunoprecipitation (Fig.4). After cell surface biotinylation and lysis using Brij97, im-munoprecipitation experiments were performed using mAbsdirected against Co-029, EpCAM, CTL1/CDw92, Lu/B-CAM,and CD26. The pattern of proteins co-immunoprecipitatedwith Co-029 in the metastatic cell lines was identical to that ofCD9 (Fig. 4A). This suggests that when the tetraspanin Co-029 is expressed it is included in the tetraspanin web, and itassociates with the same proteins as CD9. Co-029 is a 30-kDa molecule, and immunoprecipitation after Triton X-100lysis demonstrated that the intense 30-kDa molecule presentin the CD9 or Co-029 immunoprecipitates collected from themetastatic cell lines is indeed Co-029 (data not shown). Themajor proteins immunoprecipitated with anti-CD26, -Lu/B-CAM, and -EpCAM mAbs exhibited a molecular mass of 110,80, and 40 kDa, respectively. This is consistent with thepreviously described molecular masses for these molecules.CDw92 was poorly labeled with biotin and was not visible atthe exposure shown in Fig. 4. Each of these molecules co-immunoprecipitated from Isreco1 cell lysates a band co-mi-

FIG. 4. Association of novel identified proteins with tetraspanins. A, after cell surface biotinylation, cells were lysed using Brij97.Immunoprecipitation experiments were performed using mAbs directed against tetraspanins CD9 and Co-029 or novel identified proteins. AfterSDS-PAGE, the proteins were transferred to a PVDF membrane and revealed by streptavidin-peroxidase and chemiluminescence. B, cells werelysed using Brij97 followed by immunoprecipitation and Western blotting with biotin-labeled CD9 mAbs. IP, immunoprecipitation; Int., integrin.



grating with CD9. This band, as well as a band co-migratingwith Co-029, was present in EpCAM and CDw92 immunopre-cipitates collected from Isreco2 and Isreco3 cell lysates (Fig.4A). The identity of this band as CD9 was demonstrated byWestern blotting using CD9 mAbs (Fig. 4B).

EpCAM Is a CD9 Molecular Partner—Inside the tetraspaninweb, certain tetraspanins have been shown to specifically anddirectly interact with a limited number of proteins, their mo-lecular partners (forming primary complexes). These specificassociations can be observed using detergents that disrupttetraspanin-tetraspanin interactions or precipitate tetraspan-ins that interact with each other as in the case of digitonin (1,15, 16, 18). To identify a tetraspanin partner for EpCAM,CDw92, and Lu/B-CAM, immunoprecipitation experimentswere performed after cell lysis using digitonin. Under theseconditions, EpCAM but not CDw92 clearly immunoprecipi-tated a molecule co-migrating with CD9 (Fig. 5A). This bandwas identified as CD9 by Western blotting (Fig. 5B). Impor-tantly EpCAM co-immunoprecipitated a higher amount ofCD9 than did a well established CD9 partner, CD9P-1. Inaddition, no CD81 was detected in the EpCAM immunopre-cipitate. An additional band of �20 kDa was co-immunopre-cipitated with EpCAM (Fig. 5A). This protein might correspondto Claudin 7, which was identified recently as an EpCAM-associated protein (32). No band co-migrating with EpCAMwas observed in the CD9 immunoprecipitate after digitoninlysis. This suggests that the CD9 mAb may dissociate theCD9�EpCAM complex or that EpCAM interaction prevents thebinding of the CD9 mAb. This is not unprecedented as CD81mAbs fail to co-immunoprecipitate CD19 (a well characterizedpartner) (33), and certain CD151 mAbs fail to co-immunopre-cipitate the integrins �3�1 and �6�1 (15). It should be notedthat a weak band co-migrating with CD9 was also observed inthe Lu/B-CAM immunoprecipitate after digitonin lysis (Fig.5A). However, we believe that it would be premature to con-sider Lu/B-CAM as a CD9 partner because we could notobserve Lu/B-CAM�CD9 complexes after Western blotting orin cross-linking experiments.

Cross-linking experiments were performed to determinewhether CD9 interacts directly with EpCAM. Intact Isreco1cells were first pretreated with DSP as a cross-linking reagent.Then cells were lysed under stringent conditions to disrupt thenon-covalent associations, and immunoprecipitation experi-ments were performed with a specific anti-EpCAM mAb. Theimmunoprecipitates were run under non-reducing conditions,and the complexes containing EpCAM were first visualized byWestern blot using the anti-EpCAM mAb. This approach re-vealed the existence at the cell surface of two complexescontaining EpCAM with molecular masses of �60 and 80kDa. The �60-kDa complex was recognized by the CD9 mAbafter Western blotting. Importantly the molecular mass of thiscomplex is consistent with a complex containing only CD9 (24kDa) and EpCAM (40 kDa). The �80-kDa complex may cor-respond to EpCAM dimers (34) (Fig. 5C).

FIG. 5. CD9 and EpCAM form a novel primary complex. A, aftercell surface biotinylation of Isreco1 cells, immunoprecipitations wereperformed as indicated at the top of each lane after lysis with digito-nin. The proteins were transferred to a PVDF membrane and revealedby streptavidin-peroxidase and chemiluminescence. B, Isreco1 cellswere lysed using digitonin followed by immunoprecipitations andWestern blotting with biotin-labeled CD9, CD81, or B-CAM mAbs. C,in situ cross-linking experiments were performed on living Isreco1cells using DSP. Then cells were lysed using Triton X-100 beforeimmunoprecipitation of EpCAM. The immunoprecipitates were ana-lyzed by Western blot using EpCAM or CD9 mAbs. A band observedonly after cross-linking and revealed in both EpCAM and CD9 West-ern blot is labeled with *. IP, immunoprecipitation; WB, Western blot;Int., integrin.



To gain further information about the potential relevance ofCD9�EpCAM complexes, the distributions of these moleculesin normal and colon cancer were compared by confocal mi-croscopy. There was a substantial colocalization of these twomolecules in the normal colon and a lower level of colocal-ization in primary tumor and metastasis (Fig. 6).

Expression of Components of the Tetraspanin Web in Nor-mal Tissue and Colon Cancer—The most striking differencesbetween the primary cell line and the metastatic cell lines are

the expression of Lu/B-CAM in cells from the primary tumorand the high expression of Co-029 by the metastatic cell lines.To examine the in vivo relevance of these observations, wefirst investigated the expression of Co-029 and Lu/B-CAM byWestern blot experiments in comparison with CD9 and Ep-CAM (Fig. 7). Both molecules were expressed in primary coloncancer and adjacent “normal” colon tissue. A clear reductionof B-CAM expression was observed in all tumor biopsies,whereas a reduction of Co-029 expression was observed inabout half of the eight samples tested (Fig. 7 and data notshown). In addition, the molecular weight of Co-029 waslower in tumor samples than in normal tissues. This suggeststhat modifications of the protein Co-029 that remain to becharacterized may occur in certain tumors (Fig. 7). The mo-lecular weight of Co-029 in the panel of cell lines describedbelow was identical to that observed in normal adjacent tis-sues (data not shown).

We then examined the expression of these molecules inprimary tumors, normal adjacent colon, and metastasis fromthe same patients by immunolabeling of frozen sections. Onnormal colon tissue, an intense labeling of the lateral surfaceof epithelial cells was observed with the CD9 and Co-029mAbs. The expression of Co-029 was restricted to epithelialcells, whereas CD9 was also expressed by mesenchymalcells. The overall expression of CD9 and Co-029 was lower onprimary and metastatic tumors with Co-029 labeling beingheterogeneous (Fig. 8). Similar results were obtained with twoadditional patients.

The high expression of Co-029 observed on Isreco2 andIsreco3 contrasted with the low expression of this moleculeobserved in the metastasis of the three patients analyzed.

FIG. 7. Western blotting analysis on normal colon mucosa andtumors. Biopsies from normal colon mucosa (N) and primary tumors(T) from the same patients were used as the source of protein extractsfor Western blotting experiments. Whole cell extracts from Isreco celllines were also used as control. Expression of tetraspanins CD9 andCo-029 as well as Lu/B-CAM and EpCAM was checked. The amountof protein loaded on SDS-PAGE was normalized to actin. Threerepresentative patients of eight are shown.

FIG. 6. Colocalization of CD9 and EpCAM in normal and cancer colon. Cryostat sections of normal human colon, colon cancer, and colonmetastasis in the liver (all from the same patient) were stained with mAb TS9b (IgG2b) against CD9 and HEA125 (IgG1) against EpCAM andthen with a combination of goat anti-IgG2b and IgG1 antibodies labeled with Alexa Fluor 488 and Alexa Fluor 568, respectively. The sampleswere analyzed by confocal microscopy using a 63� objective. Composite images were generated by superimposition of the green (CD9) andred (EpCAM) signals with areas of overlap appearing as yellow (upper panel). The image of the normal colon is the combination of three differentoverlapping acquisitions. Arrowheads indicate the limits of the different acquisitions. On the lower panel is shown a superimposition with theDAPI staining (blue) that gives an estimate of the number of cells. Bar, 40 �m (identical in all panels).



This suggested a possible variability of Co-029 expression inmetastasis. To challenge this hypothesis, we first tested theexpression of Co-029 in a panel of cell lines derived fromprimary tumors and metastasis. As shown in Fig. 9A, the twocell lines derived from low grade colon cancers (HT29 andCaco-2) had a higher Co-029 expression than the other pri-mary tumor-derived cell lines. The Co-029 expression levelwas highly variable in the metastatic cell lines as comparedwith the cells derived from primary tumors. Variability in thelevel of expression of Co-029 was also observed when ana-lyzing a small series of liver metastasis by immunohistochem-istry. Both Co-029-positive and -negative metastasis wereobserved, and this heterogeneity was confirmed by Westernblot (Fig. 9B).

DISCUSSION

In this study, we investigated the composition of CD9-containing complexes in colon carcinoma cells using pro-teomics. This analysis led to the identification of 32 proteinsand showed that CD9 associates with a so far unsuspectedvariety of membrane proteins. Most of these molecules canbe classified as adhesion molecules and/or molecules with Igdomains, membrane proteases, signaling molecules, and fi-nally tetraspanins.

Several categories of adhesion molecules in the tetraspanin

web were identified including integrins (�3�1, �6�1, and�6�4), molecules with Ig domains (CD9P-1, EWI-2, and Lu/B-CAM), and EpCAM. The protein Lu/B-CAM exhibits fiveIg-like domains and is a laminin receptor with a restrictivespecificity to laminin �5 chain (35). Lu/B-CAM was detectedat the surface of the Isreco1 cell line but not on the metastaticcell lines. Furthermore Lu/B-CAM expression was decreasedin tumor samples compared with normal adjacent tissue asdetermined by Western blot. However, Lu/B-CAM was mainlydetected in some stroma cells in tissue sections (data notshown), and thus further work will have to determine whetherthe loss of expression of this molecule on metastatic cells is ageneral feature of colon cancer. Another new molecule in thetetraspanin web is EpCAM. EpCAM is a molecule with twoepidermal growth factor-like domains that functions as a ho-mophilic cell-cell adhesion molecule. EpCAM is expressed inmany human epithelial tissues (36) and is overexpressed inthe majority of epithelial carcinomas (for a review, see Ref.37). For this reason, it has attracted major attention as a targetfor mAb-based immunotherapy of carcinomas (38). There isalso evidence that EpCAM regulates cell proliferation of car-cinoma cells (39, 40). During the course of this study, Claas etal. (31) demonstrated an association of EpCAM with CD9 andCo-029 in rat cells. This association could be observed onlywhen using weak detergents. In this study we demonstrated

FIG. 8. Expression of CD9 and Co-029 in normal and cancer colon. Cryostat sections of normal human colon, colon cancer, and colonmetastasis in the liver (all from the same patient) were stained with mAb TS9 to CD9 (A) or AZM22 to Co-029 (B) and then with a FITC-labeledgoat anti-mouse antibody. The acquisition time was 2 s except for the staining of Co-029 in the primary tumor and the liver metastasis for whichthere was a 4-s acquisition. On the right is shown a superimposition with the DAPI staining (blue) that gives an estimate of the number of cells.Bar, 100 �m (identical in all panels).



that the interaction of EpCAM with CD9 can be visualizedusing digitonin (and thus is observed under conditions wheretetraspanin to tetraspanin interactions are not observed or arestrongly diminished) and stabilized by chemical cross-linking.Therefore, CD9�EpCAM constitutes a new primary complex inthe tetraspanin web. Recently knock-down of EpCAM by RNAinterference was shown to strongly diminish migration andinvasion of a breast cancer cell line in vitro (39). It will be ofspecial interest to determine whether the effects of EpCAMand tetraspanins on cell migration and invasion are function-ally linked.

An exciting finding of this study is the presence of severaltransmembrane proteases in the tetraspanin web. Thesecomponents may shed new light on the function of tet-raspanin complexes, which may regulate proteolytic activitiesat the cell surface. Indeed transmembrane proteases partici-pate in extracellular proteolysis such as degradation of extra-cellular matrix components, regulation of chemokine activity,

and release of membrane-anchored growth factors, recep-tors, and adhesion molecules that influence cell growth andmotility (41). Our data suggest that ADAM10 is a componentof the tetraspanin web in colon carcinoma cells. ADAM (adisintegrin and metalloprotease) proteins are membrane-an-chored metalloproteases that process and shed ectodomainsof membrane-anchored growth factors, cytokines, and recep-tors. ADAMs also have essential roles in cell-cell and cell-matrix interactions and therefore in a variety of physiologicaland pathological processes including angiogenesis and can-cer (42). ADAM10 may play a role in cancer through its abilityto cleave transmembrane precursors of epidermal growthfactor receptor ligands, including heparin-binding epidermalgrowth factor (43), which has long been known to associatewith tetraspanins (44, 45). CD26/dipeptidyl peptidase IV is a110-kDa glycoprotein that belongs to the prolyl-oligopepti-dase family. It selectively removes the N-terminal dipeptidefrom peptides with proline or alanine in the second position.Thus CD26 truncates many bioactive molecules includinggrowth factors, chemokines, neuropeptides, and vasoactivepeptides and is responsible for the inactivation of many bio-active peptides (46, 47). It is expressed on a variety of tissuesincluding T lymphocytes and endothelial and epithelial cells.CD26 plays an important role in immune regulation, signaltransduction, and apoptosis as well as in tumor progression(46, 47). In colorectal cancer, although CD26 is not present onnormal human colon epithelium, it is sometimes aberrantlyexpressed in colon tumors. TADG-15 (tumor-associated dif-ferentially expressed gene 15)/matriptase is a transmembranetrypsin-like serine protease. TADG-15 expression was foundin all types of epithelia. TADG-15 has been shown to cleaveand activate several proteins that may play a role in thegrowth and invasion of cancer cells during tumor progressionsuch as hepatocyte growth factor/scatter factor, urokinaseplasminogen activator, and protease-activated receptor-2(48–50). TADG-15 was identified in the CD9 complex withonly one peptide. Further work will be necessary to confirmthis interaction

Other findings that may shed new light on the function oftetraspanins are the presence of signaling molecules in thecomplexes. Only a few signaling molecules were demon-strated to associate with tetraspanins. Indeed the associationof tetraspanins with phosphatidylinositol 4-kinase and withseveral protein kinase isoforms has been reported previously(3). However, the interaction of phosphatidylinositol 4-kinasewith tetraspanins was only observed using detergents milderthan Brij97, and that of protein kinase C was only observedafter activation with phorbol esters. Recently Little et al. (30)showed that a fraction of heterotrimeric G proteins, G�q andG�11 subunits, specifically associates with CD9 and CD81 aswell as an unidentified G� subunit. Our data suggest thatadditional G proteins could be associated with tetraspanins.Heterotrimeric G proteins are coupled with seven-transmem-brane domain receptors also called G protein-coupled recep-

FIG. 9. Co-029 is heterogeneously expressed on colon metas-tasis. A, the expression level of Co-029 at the cell surface of cell linesoriginated from colon tumor (T) or metastasis (M) was determined byflow cytometry. B, biopsies from colon metastasis in the liver wereused as the source of protein extracts for Western blotting experi-ments. Expression of tetraspanins Co-029 and CD9 was checked.Extracts from HeLa (uterine cervical cancer) and Lovo (colon metas-tasis) cell lines as well as from a biopsy of normal liver were also used.



tors (GPCRs) (51). However, no GPCR was identified in ouranalysis of CD9-containing complexes. This may be related toa difficulty in obtaining peptides from largely hydrophobicproteins. Alternatively tetraspanins may associate with G pro-teins in the absence of GPCR.

Mass spectrometry and flow cytometry analysis of CD9-containing complexes allowed the detection of 10 tetraspan-ins in the Isreco1 cell line. Among them, Tspan14/DC-TM4F2,Tspan-1/NET-1, Tspan9/NET-5, and Tspan15/NET-7 wereobserved only by mass spectrometry as there are no availablereagents to these molecules. Tspan1/NET-1, Tspan9/NET-5,and Tspan15/NET-7 were not observed on the metastatic celllines. This may indicate a lower expression level of thesetetraspanins. In this regard, two tetraspanins (CD9 and CD82)of five studied by flow cytometry were down-regulated at thesurface of the metastatic cell lines. By contrast, the tet-raspanin Co-029 was detected by mass spectrometry only inCD9 complexes collected from the metastatic cell lines Is-reco2 and Isreco3. Flow cytometry and immunoprecipitationsshowed the expression of Co-029 only in Isreco2 and Isreco3cell lines. Co-029 was originally identified by the generation ofmAbs against a human colon carcinoma cell line (52, 53) andwas reported to be expressed in a variety of carcinomas. Itwas later identified in the rat as a molecule expressed on ametastatic subclone of a pancreatic adenocarcinoma cell linebut not on a low metastasizing subclone of the same cell line(54). Co-029 was shown to be overexpressed in cirrhosis (55)as well as in hepatocarcinoma in particular with intrahepaticspreading (56). Altogether these results prompted us to ex-amine the expression of Co-029 in the colon in comparisonwith CD9 whose expression in the normal colon has beenreported (57). Surprisingly a high expression of Co-029 wasobserved in the normal colon that was confirmed by Westernblot analysis. In contrast to CD9, the expression of Co-029was restricted to epithelial cells. Both molecules were onlyexpressed at lateral surfaces. For three patients, we couldcompare the normal adjacent colon with the primary tumorand the liver metastasis. In these patients, the expression ofCo-029 was low both in the primary tumor and in metastasisin apparent contradiction with the analysis of Isreco cell lines.This discrepancy led us to investigate a possible heterogene-ity of Co-029 expression in colon metastatic cells. Such het-erogeneity is shown through the analysis of different cell linesand different biopsies of colon metastases in the liver. It willbe necessary to determine in further studies with larger seriesof patients whether the level of Co-029 expression in metas-tasizing cells could influence their behavior and the clinicaloutcome. In this regard, it has been suggested that Co-029may promote metastasis using in vitro and in vivo experimen-tal models (58–60). Indeed transfection of a low metastasizingrat pancreatic cell line with Co-029 resulted in increased meta-static potential with massive bleeding around the metastases. Inaddition, transfection of Co-029 in HCC cells promoted devel-opment of intrahepatic metastatic lesions. Further work will

have to determine whether Co-029 plays an active role in themetastatic process during colon cancer progression.

In conclusion, we largely extended the list of moleculesassociating with CD9 and therefore to the tetraspanin web.Clinical studies have previously shown a link between theexpression level of tetraspanins and tumor progression andmetastasis in many cancers. We made the observation usingtumor cell lines and a few patient samples that several com-ponents of the tetraspanin web appear to be differentiallyexpressed during tumor progression. That may change tumorcell properties and contribute to invasion and metastasis. Theclinical and functional relevance of the association in theplasma membrane of the various components of the tet-raspanin web remain to be addressed in further studies.

Acknowledgments—We are grateful to Zohar Mishal for assistancein mass spectrometry and to Pierre Eid for helpful discussions.

* This work was supported by an Action Concertee Incitative ofMinistere de la Recherche, Gefluc, Institut du Cancer etd’Immunogenetique, Ligue nationale contre le cancer, Associationpour la Recherche contre le Cancer, and Nouvelles Recherches Bio-medicales-Vaincre le Cancer (NRB). The costs of publication of thisarticle were defrayed in part by the payment of page charges. Thisarticle must therefore be hereby marked “advertisement” in accord-ance with 18 U.S.C. Section 1734 solely to indicate this fact.

□S The on-line version of this article (available at http://www.mcponline.org) contains supplemental material.

§ To whom correspondence should be addressed: INSERM U602,Hopital Paul Brousse, 94807 Villejuif Cedex, France. Tel.: 33-1-45-59-53-13; Fax: 33-1-45-59-53-29; E-mail: [email protected].

¶ Recipient of a grant from NRB.

REFERENCES

1. Boucheix, C., and Rubinstein, E. (2001) Tetraspanins. Cell. Mol. Life Sci. 58,1189–1205

2. Levy, S., and Shoham, T. (2005) The tetraspanin web modulates immune-signaling complexes. Nat. Rev. Immunol. 5, 136–148

3. Hemler, M. E. (2005) Tetraspanin functions and associated microdomains.Nat. Rev. Mol. Cell. Biol. 10, 801–811

4. Boucheix, C., Duc, G. H., Jasmin, C., and Rubinstein, E. (2001) Tetraspan-ins and malignancy. Expert Rev. Mol. Med. 31, 1–17

5. Silvie, O., Rubinstein, E., Franetich, J. F., Prenant, M., Belnoue, E., Renia,L., Hannoun, L., Eling, W., Levy, S., Boucheix, C., and Mazier, D. (2003)Hepatocyte CD81 is required for Plasmodium falciparum and Plasmo-dium yoelii sporozoite infectivity. Nat. Med. 9, 93–96

6. von Lindern, J. J., Rojo, D., Grovit-Ferbas, K., Yeramian, C., Deng, C.,Herbein, G., Ferguson, M. R., Pappas, T. C., Decker, J. M., Singh, A.,Collman, R. G., and O’Brien, W. A. (2003) Potential role for CD63 inCCR5-mediated human immunodeficiency virus type 1 infection ofmacrophages. J. Virol. 77, 3624–3633

7. Karamatic Crew, V., Burton, N., Kagan, A., Green, C. A., Levene, C., Flinter,F., Brady, R. L., Daniels, G., and Anstee, D. J. (2004) CD151, the firstmember of the tetraspanin (TM4) superfamily detected on erythrocytes,is essential for the correct assembly of human basement membranes inkidney and skin. Blood 104, 2217–2223

8. Garcia, E., Pion, M., Pelchen-Matthews, A., Collinson, L., Arrighi, J. F., Blot,G., Leuba, F., Escola, J. M., Demaurex, N., Marsh, M., and Piguet, V.(2005) HIV-1 trafficking to the dendritic cell-T-cell infectious synapseuses a pathway of tetraspanin sorting to the immunological synapse.Traffic 6, 488–501

9. Tokuhara, T., Hasegawa, H., Hattori, N., Ishida, H., Taki, T., Tachibana, S.,Sasaki, S., and Miyake, M. (2001) Clinical significance of CD151 geneexpression in non-small cell lung cancer. Clin. Cancer Res. 7,4109–4114



10. Hashida, H., Takabayashi, A., Tokuhara, T., Hattori, N., Taki, T., Hasegawa,H., Satoh, S., Kobayashi, N., Yamaoka, Y., and Miyake, M. (2003) Clinicalsignificance of transmembrane 4 superfamily in colon cancer. Br. J.Cancer 89, 158–167

11. Ang, J., Lijovic, M., Ashman, L. K., Kan, K., and Frauman, A. G. (2004)CD151 protein expression predicts the clinical outcome of low-gradeprimary prostate cancer better than histologic grading: a new prognosticindicator? Cancer Epidemiol. Biomark. Prev. 13, 1717–1721; Correction(2005) Cancer Epidemiol. Biomark. Prev. 14, 553

12. Dong, J. T., Lamb, P. W., Rinker-Schaeffer, C. W., Vukanovic, J., Ichikawa,T., Isaacs, J. T., and Barrett, J. C. (1995) KAI1, a metastasis suppressorgene for prostate cancer on human chromosome 11p11.2. Science 268,884–886

13. Testa, J. E., Brooks, P. C., Lin, J. M., and Quigley, J. P. (1999) Eukaryoticexpression cloning with an antimetastatic monoclonal antibody identifiesa tetraspanin (PETA-3/CD151) as an effector of human tumor cell migra-tion and metastasis. Cancer Res. 59, 3812–3820

14. Kohno, M., Hasegawa, H., Miyake, M., Yamamoto, T., and Fujita, S. (2002)CD151 enhances cell motility and metastasis of cancer cells in thepresence of focal adhesion kinase. Int. J. Cancer 97, 336–343

15. Serru, V., Le Naour, F., Billard, M., Azorsa, D. O., Lanza, F., Boucheix, C.,and Rubinstein, E. (1999) Selective tetraspan-integrin complexes (CD81/�4�1, CD151/�3�1, CD151/�6�1) under conditions disrupting tetraspaninteractions. Biochem. J. 340, 103–111

16. Charrin, S., Le Naour, F., Oualid, M., Billard, M., Faure, G., Hanash, S. M.,Boucheix, C., and Rubinstein, E. (2001) The major CD9 and CD81 mo-lecular partner. Identification and characterization of the complexes.J. Biol. Chem. 276, 14329–14337

17. Clark, K. L., Zeng, Z., Langford, A. L., Bowen, S. M., and Todd, S. C. (2001)PGRL is a major CD81-associated protein on lymphocytes and distin-guishes a new family of cell surface proteins. J. Immunol. 167,5115–5121

18. Charrin, S., Le Naour, F., Billard, M., Labas, V., Le Caer, J. P., Emile, J. F.,Petit, M. A., Boucheix, C., and Rubinstein, E. (2003) EWI-2 is a newcomponent of the tetraspanin web in hepatocytes and lymphoid cells.Biochem. J. 373, 409–421

19. Berditchevski, F., Odintsova, E., Sawada, S., and Gilbert, E. (2002) Expres-sion of the palmitoylation-deficient CD151 weakens the association of�3�1 integrin with the tetraspanin-enriched microdomains and affectsintegrin-dependent signaling. J. Biol. Chem. 277, 36991–37000

20. Charrin, S., Manie, S., Oualid, M., Billard, M., Boucheix, C., and Rubinstein,E. (2002) Differential stability of tetraspanin/tetraspanin interactions: roleof palmitoylation. FEBS Lett. 516, 139–144

21. Charrin, S., Manie, S., Thiele, C., Billard, M., Gerlier, D., Boucheix, C., andRubinstein, E. (2003) A physical and functional link between cholesteroland tetraspanins. Eur. J. Immunol. 33, 2479–2489

22. Charrin, S., Manie, S., Billard, M., Ashman, L., Gerlier, D., Boucheix, C., andRubinstein, E. (2003) Multiple levels of interactions within the tetraspaninweb. Biochem. Biophys. Res. Commun. 304, 107–112

23. Rubinstein, E., Le Naour, F., Lagaudriere-Gesbert, C., Billard, M., Con-jeaud, H., and Boucheix, C. (1996) CD9, CD63, CD81 and CD82 arecomponents of a surface tetraspan network connected to HLA-DR andVLA integrins. Eur. J. Immunol. 26, 2657–2665

24. Cajot, J. F., Sordat, I., Silvestre, T., and Sordat, B. (1997) Differential displaycloning identifies motility-related protein (MRP1/CD9) as highly ex-pressed in primary compared to metastatic human colon carcinomacells. Cancer Res. 57, 2593–2597

25. Boucheix, C., Perrot, J. Y., Mirshahi, M., Giannoni, F., Billard, M., Bernadou,A., and Rosenfeld, C. (1985) A new set of monoclonal antibodies againstacute lymphoblastic leukemia. Leuk. Res. 9, 597–604

26. Azorsa, D. O., Moog, S., Cazenave, J. P., and Lanza, F. (1999) A generalapproach to the generation of monoclonal antibodies against membersof the tetraspanin superfamily using recombinant GST fusion proteins.J. Immunol. Methods 229, 35–48

27. Bartolazzi, A., Fraioli, R., Tarone, G., and Natali, P. G. (1991) Generation andcharacterization of the murine monoclonal antibody M-KID 2 to VLA-3integrin. Hybridoma 10, 707–720

28. Lozahic, S., Christiansen, D., Manie, S., Gerlier, D., Billard, M., Boucheix,C., and Rubinstein, E. (2000) CD46 (membrane cofactor protein) asso-ciates with multiple �1 integrins and tetraspans. Eur. J. Immunol. 30,900–907

29. Ducret, A., Van Oostveen, I., Eng, J. K., Yates, J. R., III, and Aebersold, R.(1998) High throughput protein characterization by automated reverse-phase chromatography/electrospray tandem mass spectrometry. Pro-tein Sci. 7, 706–719

30. Little, K. D., Hemler, M. E., and Stipp, C. S. (2004) Dynamic regulation of aGPCR-tetraspanin-G protein complex on intact cells: central role ofCD81 in facilitating GPR56-G�q/11 association. Mol. Biol. Cell 15,2375–2387

31. Claas, C., Wahl, J., Orlicky, D. J., Karaduman, H., Schnolzer, M., Kempf, T.,and Zoller, M. (2005) The tetraspanin D6.1A and its molecular partners onrat carcinoma cells. Biochem. J. 389, 99–110

32. Ladwein, M., Pape, U. F., Schmidt, D. S., Schnolzer, M., Fiedler, S., Lang-bein, L., Franke, W. W., Moldenhauer, G., and Zoller, M. (2005) Thecell-cell adhesion molecule EpCAM interacts directly with the tight junc-tion protein claudin-7. Exp. Cell Res. 309, 345–357

33. Horvath, G., Serru, V., Clay, D., Billard, M., Boucheix, C., and Rubinstein, E.(1998) CD19 is linked to the integrin-associated tetraspans CD9, CD81,and CD82. J. Biol. Chem. 273, 30537–30543

34. Trebak, M., Begg, G. E., Chong, J. M., Kanazireva, E. V., Herlyn, D., andSpeicher, D. W. (2001) Oligomeric state of the colon carcinoma-associ-ated glycoprotein GA733-2 (Ep-CAM/EGP40) and its role in GA733-mediated homotypic cell-cell adhesion. J. Biol. Chem. 276, 2299–2309

35. El Nemer, W., Gane, P., Colin, Y., D’Ambrosio, A. M., Callebaut, I., Cartron,J. P., and Le Van Kim, C. (2001) Characterization of the laminin bindingdomains of the Lutheran blood group glycoprotein. J. Biol. Chem. 276,23757–23762

36. De Boer, C. J., van Krieken, J. H., Janssen-van Rhijn, C. M., and Litvinov,S. V. Expression of Ep-CAM in normal, regenerating, metaplastic, andneoplastic liver. J. Pathol. 188, 201–206

37. Armstrong, A., and Eck, S. L. (2003) EpCAM: a new therapeutic target for anold cancer antigen. Cancer Biol. Ther. 2, 320–326

38. Veronese, M. L., and O’Dwyer, P. J. (2004) Monoclonal antibodies in thetreatment of colorectal cancer. Eur. J. Cancer 40, 1292–1301

39. Osta, W. A., Chen, Y., Mikhitarian, K., Mitas, M., Salem, M., Hannun, Y. A.,Cole, D. J., and Gillanders, W. E. (2004) EpCAM is overexpressed inbreast cancer and is a potential target for breast cancer gene therapy.Cancer Res. 64, 5818–5824

40. Munz, M., Kieu, C., Mack, B., Schmitt, B., Zeidler, R., and Gires, O. (2004)The carcinoma-associated antigen EpCAM upregulates c-myc and in-duces cell proliferation. Oncogene 23, 5748–5758

41. Bauvois, B. (2004) Transmembrane proteases in cell growth and invasion:new contributors to angiogenesis? Oncogene 23, 317–329

42. Blobel, C. P. (2005) ADAMs: key components in EGFR signaling anddevelopment. Nat. Rev. 6, 32–43

43. Yan, Y., Shirakabe, K., and Werb, Z. (2002) The metalloprotease Kuzbanian(ADAM10) mediates the transactivation of EGF receptor by G protein-coupled receptors. J. Cell Biol. 158, 221–226

44. Iwamoto, R., Higashiyama, S., Mitamura, T., Taniguchi, N., Klagsbrun, M.,and Mekada, E. (2004) Heparin-binding EGF-like growth factor, whichacts as the diphtheria toxin receptor, forms a complex with membraneprotein DRAP27/CD9, which up-regulates functional receptors and diph-theria toxin sensitivity. EMBO J. 13, 2322–2330

45. Lagaudriere-Gesbert, C., Le Naour, F., Lebel-Binay, S., Billard, M.,Lemichez, E., Boquet, P., Boucheix, C., Conjeaud, H., and Rubinstein, E.(1997) Functional analysis of four tetraspans, CD9, CD53, CD81, andCD82, suggests a common role in costimulation, cell adhesion, andmigration: only CD9 upregulates HB-EGF activity. Cell. Immunol. 182,105–112

46. Lambeir, A. M., Durinx, C., Scharpe, S., and De Meester, I. (2003) Dipep-tidyl-peptidase IV from bench to bedside: an update on structural prop-erties, functions, and clinical aspects of the enzyme DPP IV. Crit. Rev.Clin. Lab. Sci. 40, 209–294

47. Pro, B., and Dang, N. H. (2004) CD26/dipeptidyl peptidase IV and its role incancer. Histol. Histopathol. 19, 1345–1351

48. Lee, S. L., Dickson, R. B., and Lin, C. Y. (2000) Activation of hepatocytegrowth factor and urokinase/plasminogen activator by matriptase, anepithelial membrane serine protease. J. Biol. Chem. 275, 36720–36725

49. Oberst, M., Anders, J., Xie, B., Singh, B., Ossandon, M., Johnson, M.,Dickson R. B., and Lin, C. Y. (2001) Matriptase and HAI-1 are expressedby normal and malignant epithelial cells in vitro and in vivo. Am. J. Pathol.158, 1301–1311



50. Oberst, M. D., Singh, B., Ozdemirli, M., Dickson, R. B., Johnson, M. D., andLin, C. Y. (2003) Characterization of matriptase expression in normalhuman tissues. J. Histochem. Cytochem. 51, 1017–1025

51. Pierce, K. L., Premont, R. T., and Lefkowitz, R. J. (2002) Seven-transmem-brane receptors. Nat. Rev. Mol. Cell. Biol. 3, 639–650

52. Sela, B. A., Steplewski, Z., and Koprowski, H. (1989) Colon carcinoma-associated glycoproteins recognized by monoclonal antibodies CO-029and GA22-2. Hybridoma 8, 481–491

53. Szala, S., Kasai, Y., Steplewski, Z., Rodeck, U., Koprowski, H., and Lin-nenbach, A. J. (1990) Molecular cloning of cDNA for the human tumor-associated antigen CO-029 and identification of related transmembraneantigens. Proc. Natl. Acad. Sci. U. S. A. 87, 6833–6837

54. Claas, C., Herrmann, K., Matzku, S., Moller, P., and Zoller, M. (1996)Developmentally regulated expression of metastasis-associated anti-gens in the rat. Cell Growth Differ. 7, 663–678

55. Shackel, N. A., McGuinness, P. H., Abbott, C. A., Gorrell, M. D., andMcCaughan, G. W. (2003) Novel differential gene expression in humancirrhosis detected by suppression subtractive hybridization. Hepatology38, 577–588

56. Kanetaka, K., Sakamoto, M., Yamamoto, Y., Yamasaki, S., Lanza, F.,Kanematsu, T., and Hirohashi, S. (2001) Overexpression of tetraspaninCO-029 in hepatocellular carcinoma. J. Hepatol. 35, 637–642

57. Okochi, H., Mine, T., Nashiro, K., Suzuki, J., Fujita, T., and Furue, M. (1999)Expression of tetraspans transmembrane family in the epithelium of thegastrointestinal tract. J. Clin. Gastroenterol. 29, 63–67

58. Claas, C., Seiter, S., Claas, A., Savelyeva, L., Schwab, M., and Zoller, M.(1998) Association between the rat homologue of CO-029, a metastasis-associated tetraspanin molecule and consumption coagulopathy. J. CellBiol. 141, 267–280

59. Herlevsen, M., Schmidt, D. S., Miyazaki, K., and Zoller, M. (2003) Theassociation of the tetraspanin D6.1A with the �6�4 integrin supports cellmotility and liver metastasis formation. J. Cell Sci. 116, 4373–43790

60. Kanetaka, K., Sakamoto, M., Yamamoto, Y., Takamura, M., Kanematsu, T.,and Hirohashi, S. (2003) Possible involvement of tetraspanin CO-029 inhematogenous intrahepatic metastasis of liver cancer cells. J. Gastro-enterol. Hepatol. 18, 1309–1314

61. Potts, A. J., Croall, D. E., and Hemler, M. E. (1994) Proteolytic cleavage ofthe integrin �4 subunit. Exp. Cell Res. 212, 2–9



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Profiling of the Tetraspanin Web of Human Colon Cancer Cells