Proc. Nati. Acad. Sci. USAVol. 86, pp. 9906-9910, December 1989Cell Biology

The human integrin VLA-2 is a collagen receptor on some cells anda collagen/laminin receptor on othersMARIANO J. ELICES AND MARTIN E. HEMLER*Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA 02115

Communicated by Elizabeth D. Hay, September 5, 1989

ABSTRACT The integrin heterodimer VLA-2, previouslyknown as a collagen receptor, is now shown also to be a lamininreceptor. Adhesion of the human melanoma cell line LOX tolaminin was inhibited by anti-VLA a2 antibodies. BecauseVLA-2-mediated LOX cell attachment to laminin was notinhibited by digestion with collagenase, collagen contaminationof laminin was not a factor. In addition, VLA-2 from LOX cellsbound to immobilized laminin, and binding was disrupted byEDTA but not by Arg-Gly-Asp (RGD) peptides. VLA-3 alsobound to lammnin-Sepharose, although less avidly than VLA-2.Thus, at least four separate members of the integrin .,8subfamily serve as laminin receptors-i.e., VLA-2 and VLA-3(this study) together with VLA-1 and VLA-6 (other reports).Whereas LOX and other cell lines used VLA-2 as both alaminin and collagen receptor, fibroblast VLA-2 mediatedcollagen but not laminin binding. Likewise, VLA-2 fromplatelets did not interact with laminin. Despite this functionaldiscordancy, VLA-2 from laminin-binding and nonbindingsources was indistinguishable by all immunochemical andbiochemical criteria examined. Thus, functional differences inVLA-2 may be due to cell type-specific modulation.

Laminin, a major constituent of basement membranes, pro-motes adhesion, growth, migration, and differentiation ofcells (1). The laminin glycoprotein (Mr 900,000) consists ofthree subunits arranged in the shape of a cross with one longand three short arms (1). Distinct functions of laminin, suchas promotion of neurite outgrowth, interaction with basementmembrane components, and attachment and spreading ofcells, each reside within separate structural domains (1). Inparticular, cell adhesion appears to be mediated by at leastthree different regions in laminin (2-5), and a peptide derivedfrom one of the cell-binding sites has been shown to inhibitexperimental metastasis (6).Thus far, specific cell-adhesion receptors for laminin in-

clude a 68-kDa protein (7-9), as well as other surface mole-cules that belong to the integrin family. Integrins are a groupof af3 heterodimers involved in cell-cell and cell-extracellu-lar matrix interactions, some of which occur via recognitionofan Arg-Gly-Asp (RGD) tripeptide sequence present in theirligands (10, 11). Members of the integrin family implicated aslaminin receptors include a rat cell complex that resemblesVLA-3 (12), human VLA-6 from platelets (13), and a rat cellcomplex that may be the homolog of human VLA-1 (14).VLA-2, another member of the integrin ,1 subfamily, has

been shown to act as a cell-surface receptor for collagen inplatelets (15-18), fibroblasts (19, 20), and melanoma cells(21). However, in the platelet system VLA-2 was clearlyunable to mediate cell binding to laminin (13, 18). In thepresent report, a role for VLA-2 as a laminin receptor inhuman LOX melanoma cells, as well as other cell lines, isunequivocally demonstrated, despite finding that neither

platelet nor fibroblast VLA-2 appears to interact with lami-nin.

METHODSAntibodies and Cells. The monoclonal antibodies (mAbs)

A-lA5 (anti-f81), TS2/7 (anti-a'), 12F1 (anti-a2), J143 (anti-a3), and the control mAbs P3 and J-2A2 were obtained asdescribed (22). The mAbs PlH5 and PlB5 that recognize theVLA a2 and a3 subunits, respectively (19, 20), were from E.Wayner (University of Washington, Seattle). Anti-VLA-2mAbs 5E8 (23) and Gil4 (24) were from R. Bankert (RoswellPark Memorial Institute, Buffalo, NY) and S. Santoso (Gies-sen, F.R.G.), respectively. The mAbs BlE5 and BlEllrecognize epitopes on VLA aS and f1, respectively, and wereprovided by C. Damsky (University of California, San Fran-cisco). GoH3, a mAb specific for VLA a6 (25), was obtainedfrom A. Sonnenberg (The Netherlands Cancer Institute,Amsterdam). Finally, a rabbit heteroantiserum to a syntheticpeptide from the COOH-terminal intracytoplasmic portion ofVLA a2 (26) was prepared in this laboratory.The human melanoma cell line LOX was obtained from

Lan Bo Chen (Dana-Farber Cancer Institute). All the celllines described in Table 1 were grown in RPMI 1640 mediumcontaining 10o fetal bovine serum.Matrix Proteins. Laminin from the Engelbreth-Holm-

Swarm (EHS) murine tumor (27) was a gift from H. Kleinman(National Institute of Dental Research, Bethesda, MD).Fibronectin was purified from human plasma (28), and col-lagens type I and IV were purchased from Telios Pharma-ceuticals (La Jolla, CA).

Cell Attachment and mAb Inhibition Assays. To assess cellattachment to extracellular matrix proteins, cells were incu-bated with 51Cr (0.5 ,Ci; 1 Ci = 37 GBq) overnight, washedthree times, and resuspended in RPMI 1640 supplementedwith 0.5% fetal bovine serum and 0.1 mM MnC12. Then 1.5x 103 cells per well were plated in triplicate for 20-30 min at37°C on 96-well microtiter dishes (0.8-cm diameter per well)that were previously incubated with laminin, collagen, orfibronectin (1 ,g/well). After unbound cells were aspirated,and the plates were washed twice with RPMI 1640, 51Crpresent in 0.1% SDS cell lysates was measured by using a ycounter. For mAb inhibition experiments, labeled cells wereincubated with mAbs for 30 min at 4°C, washed, and platedon matrix ligand as described above. Specific attachment wascalculated by subtracting the radioactivity bound to bovineserum albumin-coated controls.Laminin-Sepharose Affinity Chromatography. Human

LOX melanoma cells were detached from tissue cultureflasks with 0.03% EDTA in phosphate-buffered saline (PBS),washed twice with PBS, and iodinated with 2 ,uCi of Na1251(DuPont/NEN) and lactoperoxidase at 0.2 mg/ml (Sigma) for2-5 x 107 cells. lodinated cells were lysed with 0.1 M octyl

Abbreviations: mAb, monoclonal antibody; PBS, phosphate-buffered saline.*To whom reprint requests should be addressed.

9906

The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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glucoside (Sigma) and 0.1 M octyl thioglucoside (Calbio-chem) in PBS containing 0.1 mM MnCI2, aprotinin at 10,ug/ml, 10 juM leupeptin, and 1 mM phenylmethylsulfonylfluoride for 4 hr at 40C. Detergent lysates were clarified bycentrifugation at 12,000 x g, supplemented with bovineserum albumin (0.5 mg/ml), and loaded onto 2 ml of laminin-Sepharose columns (3.5 mg of laminin per ml of packedbeads) preequilibrated with 25 mM each of octyl glucosideand octyl thioglucoside in the same buffer as above (bufferA). After equilibration for 2 hr, columns were washed withbuffer A until eluted radioactivity decreased to backgroundlevels (-20 column volumes). Stepwise elution was con-ducted with 0.5 M NaCi, 10 mM EDTA, and 4 M urea, eachin buffer A. Fractions (0.8 ml each) were collected andanalyzed for radioactivity with a y counter. Immunoprecip-itation of labeled proteins was done as described (22).NH2-Terminal Sequencing of a2 Subunit. VLA-2 was pu-

rified from LOX (1 x 109 cells) by using lectin affinitychromatography, followed by Gil4-Sepharose chromatogra-phy with described methods (29). The mAb Gil4 boundquantitatively to all VLA-2, whether from laminin binding ornonbinding cells. After purification, VLA-2 subunits wereseparated by SDS/PAGE and then transferred to polyvinyl-idene difluoride membrane (Immobilon, Millipore), as de-scribed (30). The a2 polypeptide chain was identified bystaining with Coomassie blue, cut from the membrane, andthe NH2-terminal amino acid sequence was determined at theHarvard Microsequencing Facility, Cambridge, MA.

RESULTSInhibition of LOX Cell Attachment to Laminin. The LOX

human melanoma cell line was found to attach efficiently to

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laminin-coated plastic surfaces, with half-maximal binding at0.5 ,ug oflaminin per well (data not shown). To investigate thecontribution of VLA heterodimers to melanoma-cell adhe-sion on laminin, a panel of mAbs was tested for inhibition ofcell binding (Fig. 1A). Cell attachment to laminin could becompletely abolished by incubation with the mAb BlEll(anti-VLA p1), thus indicating that f1 integrins had a majorrole in LOX cell adhesion to laminin. Ofthe mAbs against theVLA proteins expressed on the surface ofLOX cells (VLA-1,-2, -3, -5, and -6) only anti-VLA a2 mAb 5E8 (Fig. 1A) andP1H5 (data not shown) substantially inhibited melanoma-celladhesion to laminin. In contrast, neither mAbs PlB5 (anti-VLA a 3) and BlE5 (anti-VLA a5) (Fig. 1A) nor TS2/7(anti-VLA a') and GoH3 (anti-VLA a 6) were inhibitory (datanot shown). The prototype anti-a2 mAb 12F1 (31) did notblock cell binding to either laminin or collagen type I,although material immunoreactive with either 5E8 or 12F1was reciprocally precleared by incubation with the othermAb (Fig. 1B). Thus, 12F1 and 5E8 both bound to VLA-2 butmight differ in epitope recognition. Upon titration with in-creasing concentrations of mAb 5E8 (Fig. 1C), a maximalinhibition of 70% was obtained when laminin was saturatingfor cell adhesion. At lower levels oflaminin, LOX cells boundto laminin-coated plates with less avidity, and thus inhibitionby mAb 5E8 approached 100% (data not shown).Comparison of Matrix Adhesion for LOX and Other Cells.

To determine whether various human cell lines may useVLA-2 to adhere to laminin, their binding to laminin-coatedplates was analyzed. Table 1 shows that the interaction of theadherent cell lines EJ, LOX, and SK-N-SH with laminin wasblocked by mAb 5E8, whereas cell lines of hematopoieticorigin such as HL-60, HPB-ALL, and K-562, not expressingVLA-2, did not bind to laminin. RD cells bound to laminin

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FIG. 1. Inhibition of melanoma-cell adhesion to laminin by anti-VLA-2 antibodies. (A) Inhibition of LOX-cell binding to laminin was doneby using the following mAbs, each at -10 ,ug/ml: J-2A2 (control), 5E8 (anti-a2), PlB5 (anti-a3), BlE5 (anti-a5), and BlEll (anti-PD). (B)Immunoprecipitation of VLA-2 by mAb 5E8. Radiolabeled material from LOX-cell extracts either was not (lanes a and b) or was preclearedwith mAbs 12F1 (lanes c and d) and 5E8 (lanes e and f), respectively, before reprecipitating with either mAb 12F1 (lanes a, c, and e) or 5E8(lanes b, d, and f). (C) Titration of LOX-cell attachment to laminin with increasing concentrations of mAbs J-2A2 (o), 5E8 (0), and BlEll (A).Cell adhesion assays were conducted as described.

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Table 1. Binding of cell lines to extracellular matrix proteins and inhibition with mAbsCollagen type I Fibronectin Laminin

Inhibited Inhibited InhibitedCell line Binding, % by 5E8, % Binding, % by BlE5, % Binding, % by 5E8, %MRC-5 45 65 20 80 <1EJ 88 74 79 82 69 67LOX 87 66 76 81 65 70RD 13 0 82 86 38 0SK-N-SH 80 61 55 84 72 73HL-60 <1 16 96 <1HPB-ALL <1 - 10 89 <1K-562 <1 - 12 98 <1MRC-5 (normal fibroblast), EJ (bladder carcinoma), LOX (melanoma), SK-N-SH (neuroblastoma), HL-60 (promyelo-

cytic leukemia), HPB-ALL (T lymphoblastoma), and K-562 (erythroleukemia) cells were all tested for attachment toligand-coated plates as described. The mAbs used for inhibition of cell attachment were J-2A2 (control), 5E8 (anti-a 2), PlB5(anti-a3), and BlE5 (anti-a5).

even though they did not express marked levels of VLA-2,and as expected this binding was not inhibited by anti-VLA-2mAb. For the MRC-5 fibroblast line, attachment to collagentype I was mediated by VLA-2 because mAb 5E8 blockedbinding; yet these cells did not adhere to laminin. Thisobservation suggested that VLA-2 may function as a receptorexclusively for collagen in some cell types but may see bothcollagen and laminin in others. For all cell types analyzed,binding to fibronectin was inhibited (80-98%) by the anti-VLA-5 mAb BlE5, regardless of whether laminin and/orcollagen binding was observed. Also, attachment of all celllines listed in Table 1 to collagen, laminin, or fibronectin wasblocked by the anti-,31 mAb BlEll (data not shown).Laminin Binding Not Influenced by Collagen Contamina-

tion. When iodinated laminin was subjected to SDS/PAGEand autoradiography, the corresponding autoradiogram (Fig.2A) showed laminin subunits A, B1, and B2, as well as 10%of entactin/nidogen (32). However, the possibility remainedthat laminin contamination by collagen type IV could infu-ence the results described above. Thus, the effect of bacterialcollagenase upon LOX binding to laminin and collagen typeIV was determined. Fig. 2B shows that melanoma-cell at-tachment to collagen type IV was abrogated after digestionwith collagenase, whereas treatment of laminin resulted in no

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loss of LOX-cell adhesion. In similar fashion, when LOXcells in suspension were incubated with collagenase, cellbinding to laminin did not diminish in subsequent attachmentassays. Thus, laminin binding was not an indirect phenom-enon mediated by a collagen bridge.

Direct Demonstration of VLA Proteins Binding to Laminin.To directly confirm that VLA-2 interacts with laminin, assuggested by the inhibition results obtained above, affinity-chromatography separations were conducted. Fig. 3 shows arepresentative elution profile of an octyl glucoside LOX-celllysate fractionated on a laminin-Sepharose column in thepresence of MnCl2 (33, 34). Although most label passedunretarded through the column, sequential elution with buff-ers containing 0.5 M NaCl, 10 mM EDTA, and 4 M urea,respectively, resulted in selective enrichment for discretepolypeptide species. Notably, elution with EDTA yieldedthree distinct bands of 40, 120, and 150 kDa, the last two withmobilities resembling VLA / and a subunits. Immunoprecip-itation of column eluates (Fig. 4) showed that VLA-2 was themajor component in the EDTA eluate, together with a lesseramount of VLA-3, whereas the NaCI fraction containedmostly VLA-3. When the same laminin affinity-column ex-periments were done using platelets, no detectable VLAproteins were obtained (data not shown). In addition, neither

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FMG. 2. Properties of laminin used for cell-attachment studies. (A)SDS/PAGE analysis of iodinated laminin used in this study. En/Nd,entactin/nidogen. (B) Effect of collagenase treatment of laminin onLOX-cell attachment. Binding ofLOX cells to laminin (o, *) and collagentype IV (o, *) was analyzed with (open symbols) or without (closedsymbols) prior digestion with bacterial collagenase (type VII, Sigma) at 50milliunits/ml in 50 mM Tris, pH 7.5/2.5 mM CaC12/0.02% NaN3 for 16 hrat 15°C. Incubations were terminated by adding EDTA, and then bothtreated and untreated samples were used to coat microtiter plates.LOX-cell attachment was done as described.

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FIG. 3. Affinity chromatography ofLOX-cell extracts on laminin-Sepharose.A radiolabeled extract from human LOXmelanoma cells was passed over laminin-Sepharose as described. Fractions (0.8ml each) were collected and analyzed forradioactivity by using a y counter. Allfour peak fractions and a sample of the

a0 \ column wash (arrows) were analyzed byS1* SDS/PAGE under reducing conditions,

and labeled bands were visualized byF- \ ^autoradiography (between brackets).

Peaks A, B, and C resulted from stepwiseelution with 0.5 M NaCl, then 10 mM

___________________ EDTA, and finally 4 M urea, respec-40 50 60 tively. Myo, myoglobin; 83G, P galacto-sidase; BSA, bovine serum albumin;

Ova, ovalbumin.

VLA-2 nor VLA-3 could be eluted from laminin-Sepharosecolumns using RGD peptides (data not shown).

Structural Similarity in VLA-2 from Funtionally DistinctSources. Because VLA-2 from LOX cells did bind to lamininand VLA-2 from fibroblasts and platelets did not, it wasimportant to determine whether there were any obviousstructural differences in VLA-2 isolated from various cellsources. Thus, VLA-2 preparations from LOX cells, plate-lets, and/or fibroblasts were compared. NH2-terminal aminoacid sequencing was carried out for purified a subunit fromLOX VLA-2 (see Methods), and the unequivocal result

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FIG. 4. Immunoprecipitation ofVLA heterodimers from lamininand collagen type IV columns. The material that passed unretardedthrough the laminin-Sepharose column, as well as the correspondingNaCl and EDTA eluates, were immunoprecipitated with the follow-ing mAbs: control J-2A2 (-), 5E8 (anti-VLA a2), J143 (anti-VLA a3),B1E5 (anti-VLA a5), and A-lA5 [anti-VLA ,81, total VLA proteins(VLA-T)].

through 15 positions was Tyr-Asn-Val-Gly-Leu-Pro-Glu-Ala-Lys-Ile-Phe-Ser-Gly-Pro-Ser. This sequence was 100% iden-tical to that reported for platelet or placenta a2 subunit at theNH2-terminal 15 positions (26). In addition, this NH2-terminal sequence has been consistently obtained by usingthree different anti-a 2 antibodies for purification (data notshown), and also it exactly matched the amino acid sequencederived from a2 cDNA from fibroblast and endothelial celllibraries (26).

In immunoprecipitation and/or flow cytometry experi-ments, VLA-2 from LOX cells, platelets, and fibroblasts wasrecognized by three distinct mAbs (5E8, P1H5, Gil4) and byrabbit anti-intact 2 antiserum (data not shown). In addition,VLA-2 protein immunoprecipitates from each cell sourcewere identical in size (both reduced and nonreduced) with noapparent differences in posttranslational modification. Fi-nally, a2 mRNA from fibroblasts, LOX cells, and other cellswas the same size (-8 kb) when probed with full-length a2cDNA probe (data not shown).

DISCUSSIONVLA-2 as a Laminin Receptor. The present study expands

the range of VLA-2 activities to include a laminin receptorfunction because (i) anti-VLA-2 mAbs inhibit cell attachmentto laminin and (ii) VLA-2 binds directly to laminin-Sepharosecolumns. The laminin receptor described herein is clearlydistinct from other laminin-binding integrins, such as VLA-6(13) and the VLA-3-like integrin from Rugli glioblastoma (12).Whereas the a subunits of those integrins drop upon reduc-tion due to cleavage of a disulfide-linked C-terminal peptide,VLA a2 subunit is not cleaved. In the cases of the laminin-binding integrins from the chicken CSAT complex (35) andthe rat PC-12 pheochromocytoma cell line (36, 37), multipleintegrin complexes appear to be involved, and their relation-ship, if any, to human VLA-2 remains unclear. The VLA-1-like laminin receptor from rat PC-12 cells (14) differs fromVLA-2 in that it has a larger a subunit.VLA-3 as a Laminin Receptor. Although no inhibition of

LOX-cell binding to laminin was seen with the anti-VLA-3mAb P1B5 (Fig. 1A), VLA-3 bound and was subsequentlyeluted from laminin affinity columns (Fig. 3). Previously,mAbs to VLA-3 have been suggested to block cell adhesionto laminin (19, 20). Therefore, it is possible that anti-VLA-3

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blocking of LOX-cell binding to laminin was not seen in thisstudy because either (i) the mAb recognized an inappropriatea3 epitope or (ii) the role of VLA-3 may be overshadowed bythe contribution of VLA-2. In support of this, elution ofVLA-3, but not VLA-2, from laminin columns was accom-plished with 0.5 M NaCl, suggesting (but not proving) thatVLA-3 may display less avidity than VLA-2 toward laminin.

Functional Diversity for VLA-2. For LOX and other celllines, adhesion to laminin appeared chiefly mediated byVLA-2. However, VLA-2 was clearly not a laminin receptorin all cell types in which it was present. For example,antibody-blocking studies showed that fibroblasts used VLA-2 to bind collagen but not laminin. Also, VLA-2 from plateletsdid not appear to be a laminin receptor because it did notinteract with laminin-Sepharose columns. This observationwas consistent with previous results by other investigators(13, 18). In one study, even though VLA-2 was present andfully functional as a collagen receptor, antibodies to VLA-2did not block platelet attachment to laminin (13). In anotherreport, platelet VLA-2 incorporated into liposomes bound tocollagen but not to laminin (18).

Despite functional discordancy among various VLA-2-expressing cell lines, VLA-2 samples from differentsources did not exhibit any obvious physical or immuno-chemical differences. In particular, NH2-terminal a2 se-quences were identical regardless of source, whereas for the10 other integrin a subunits, the NH2-terminal sequences ofwhich are known, there are 8-14 differences between thoseand the a2 subunit in the first 15 positions (38). Also, all a2subunits tested were similarly reactive with anti-a2 COOH-terminal peptide sera, whereas the COOH termini currentlyknown for eight different integrin a subunits (39) plus a3subunit (Y. Takada, M. H., unpublished work) display littleor no sequence homology.Because there was no obvious VLA-2 structural diversity,

other factors such as cellular signaling or activation eventsmay contribute to modify VLA-2 function in a cell type-specific manner. For example, lIb/IIIa, another integrinheterodimer, was expressed on unactivated platelets but wasdependent on platelet activation for function (40). Similarly,triggering of T cells through the T-cell receptor or withphorbol ester activated the integrin LFA-1 without changingits surface expression levels (41). Additional factors thatmerit consideration as possible means of modulating VLA-2activity include variations in divalent cations (33, 34), asso-ciation with glycolipids (42), and changes in phosphorylation(43). However, regardless of the mechanistic details, thisreport suggests that the dual laminin/collagen surface recep-tor functions of VLA-2 can be dissociated, and cells may usethis property to diversify their adhesive capability.The presence of multiple receptors for laminin within the

integrin family (VLA-1, VLA-2, VLA-3, and VLA-6) resem-bles the situation encountered for another extracellular ma-trix component, fibronectin, which is recognized by at leastfive different integrins (44). In future studies, it will beimportant to correlate the multiple cell-binding regions inlaminin (2-5) with its specific integrin receptors.

Note Added in Proof. Results similar to those reported in this paperhave recently been obtained by Languino et al. (45).

The authors gratefully acknowledge Drs. R. Bankert, C. Damsky,R. Kantor, H. Kleinman, L. Old, S. Santoso, A. Sonnenberg, E.Wayner, and V. Woods for providing reagents used in this study.M.J.E. is a fellow of the Cancer Research Institute, Inc., New York.This work was supported by a grant from the National Institutes ofHealth (GM38903).

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