Proc. Natl. Acad. Sci. USAVol. 93, pp. 7069-7074, July 1996Cell Biology

Matrix metalloproteinase 2 releases active soluble ectodomain offibroblast growth factor receptor 1

(fibroblast growth factor receptor cleavage/fibroblast growth factor receptor crosslinking/gelatinase A)

EHUD LEVI*t, RAFAEL FRIDMANtt, HUA-QUAN MIAO*, YONG-SHENG MA§, AVNER YAYON§,AND ISRAEL VLODAVSKY*¶*Department of Oncology, Hadassah-Hebrew University Hospital, Jerusalem 91120, Israel; tDepartment of Pathology, Wayne State University School ofMedicine, Detroit, MI 48201; and §Department of Molecular Cell Biology, Weizmann Institute of Science, Rehovot 76100, Israel

Communicated by Leo Sachs, Weizmann Institute of Science, Rehovot, Israel, April 5, 1996 (received for review February 25, 1996)

ABSTRACT Recent studies have demonstrated the exis-tence of a soluble fibroblast growth factor (FGF) receptor type1 (FGFR1) extracellular domain in the circulation and invascular basement membranes. However, the process ofFGFR1 ectodomain release from the plasma membrane is notknown. Here we report that the 72-kDa gelatinase A (matrixmetalloproteinase type 2, MMP2) can hydrolyze the Val368-Met369 peptide bond of the FGFR1 ectodomain, eight aminoacids upstream of the transmembrane domain, thus releasingthe entire extracellular domain. Similar results were obtainedregardless of whether FGF was first bound to the receptor ornot. The action of MMP2 abolished binding of FGF to animmobilized recombinant FGFR1 ectodomain fusion proteinand to Chinese hamster ovary cells overexpressing FGFR1.The released recombinant FGFR1 ectodomain was able tobind FGF after MMP2 cleavage, suggesting that the cleavedsoluble receptor maintained its FGF binding capacity. Theactivity of MMP2 could not be reproduced by the 92-kDagelatinase B (MMP9) and was inhibited by tissue inhibitor ofmetalloproteinase type 2. These studies demonstrate thatFGFR1 may be a specific target for MMP2 on the cell surface,yielding a soluble FGF receptor that may modulate themitogenic and angiogenic activities of FGF.

The fibroblast growth factors (FGFs) constitute a family ofnine structurally related polypeptides characterized by highaffinity to heparin. The FGFs participate in a wide array ofbiological activities, including the induction of cellular prolif-eration, tissue regeneration, neurite outgrowth, angiogenesis,and embryonic mesoderm induction (1-3). This gene familyincludes the prototypes acidic FGF (aFGF) and basic FGF(bFGF) that, unlike most other polypeptide growth factors, areprimarily cell associated proteins, consistent with a lack of aconventional signal sequence for secretion (1-3). FGFs elicittheir biological responses by binding to cell surface tyrosinekinase receptors. Four distinct but highly related FGF recep-tors (FGFRs) have been identified (4-6). The prototypeFGFR contains three IgG-like domains in its extracellularportion, a single transmembrane domain, and a tyrosine kinasedomain that is split into two segments by a short interkinaseregion (5). In addition to the four distinct FGFRs, an alter-native splicing mechanism gives rise to multiple forms ofFGFR type 1 (FGR1), FGFR2, and FGFR3 (4-9). In additionto interacting with their high affinity receptors, FGFs alsointeract with lower affinity binding sites identified as heparansulfate proteoglycans and found on the surface of cells and inthe extracellular matrix (ECM; refs. 1-3). Cells that naturallydo not express heparan sulfate proteoglycans but overexpresshigh affinity receptors for FGFs fail to respond mitogenicallyto aFGF or bFGF, unless heparin is added (10, 11).

The 72-kDa [matrix metalloproteinase type 2 (MMP2)] andthe 92-kDa (MMP9) gelatinases are two members of a largefamily of zinc-dependent endopeptidases, the matrix metal-loproteinases (MMP), known to cleave ECM proteins innormal and pathological conditions (12-16). Like other mem-bers of the MMP family, the gelatinases are secreted in a latentform, requiring activation for proteolytic activity, and areinhibited by the tissue inhibitors of metalloproteinases(TIMPs; refs. 12-14). Recent studies demonstrated that latentproMMP2 is activated by a novel subgroup of MMPs, themembrane-type MMPs, which are bound to the plasma mem-brane of normal and tumor cells by a unique transmembranedomain (17, 18). The localization of active MMP2 and mem-brane-type MMPs on the cell surface may play a role in thedegradation of ECM components in areas of cell-matrixinteractions. It is conceivable that nonmatrix cell surfaceproteins, including cell surface receptors, may also be thetarget of membrane-bound MMPs. It has been recently shownthat MMP2 can hydrolyze ,B-amyloid isolated from brains ofAlzheimer disease patients (19, 20), and galectin-3, a cellsurface lectin involved in cell-cell and cell-matrix interactionsin tumor cell metastasis (21).The biological activity of FGF may be regulated by several

mechanisms, including binding to high and low affinity recep-tors on the cell surface (1-3), release of FGF from ECM byheparanase (22, 23), and as recently proposed, release of theentire ectodomain of the FGFR into the circulation and ECM(24, 25). A soluble truncated FGFR1 was recently detected inthe basement membrane of retinal vascular endothelial cells byuse of an antibody raised against the extracellular domain ofFGFR1 (25). The mechanism by which FGFR may be releasedfrom the cell surface was not elucidated, but it may involve theaction of a protease acting on the cell surface and cleaving theextracellular domain of the high affinity receptor. In our study,we examined the ability of the gelatinases to hydrolyze murineFGFR1 and FGFR2 ectodomains. Here we demonstrate thathuman recombinant MMP2, but not MMP9, cleaves a recom-binant FGFR1 fusion protein, releasing the entire ectodomain.MMP2 hydrolysis was achieved with both free and FGF-occupied receptor. The MMP2-hydrolyzed ectodomain re-tained its ability to bind FGF. We also show that MMP2treatment of cells overexpressing FGFR1 markedly reduces

Abbreviations: FGF, fibroblast growth factor; FGFR, FGF receptor;aFGF, acidic FGF; bFGF, basic FGF; FGFR1, FGF receptor type 1;ECM, extracellular matrix; MMP, matrix metalloproteinase; MMP2,MMP type 2; TIMP2, tissue inhibitor of metalloproteinase type 2;proMMP, latent MMP; AP, alkaline phosphatase; APMA, p-aminophenylmercuric acetate. FRAP, FGFR1 ectodomain-AP fusionprotein; BAP, mouse Bek ectodomain-AP fusion protein; DSS,disuccinimidylsuberate; CHO, Chinese hamster ovary.tThe first two authors contributed equally to this work.ITo whom reprint requests should be addressed at: Department ofOncology, Hadassah Hospital, P.O. Box 12000, Jerusalem 91120,Israel.

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

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binding of FGF. The results suggest a novel mechanism of FGFregulation modulated by the action of MMP2.

MATERIALS AND METHODS

Materials. Sodium heparin from porcine intestinal mucosawas obtained from Hepar (Franklin, OH). Recombinant hu-man bFGF, kindly provided by Takeda (Osaka), and aFGF (agift of G. Neufeld, Technion, Haifa, Israel) were iodinated bychloramine T as described (26). The specific activity was-1.7 x 105 cpm/ng of 1251-bFGF and -0.4 x 105 cpm/ng of125I-aFGF, and the labeled preparations were kept for up to 3weeks at -70°C. 1251-Na was purchased from Amersham.Mouse anti-human placental alkaline phosphatase (AP) mAbscoupled to agarose were purchased from Sigma. Rabbit anti-Bek polyclonal antiserum was generated by injecting glutathi-one S-transferase fused with the ectodomain region of mouseBek (9).

Expression and Purification of Recombinant MMPs andTIMPs. Human recombinant proMMP2, proMMP9, andTIMP type 2 (TIMP2) were all expressed in HeLa cells usinga recombinant vaccinia virus expression system (Vac/T7) asdescribed (27, 28). proMMP2 and proMMP9 were purified bygelatin-affinity chromatography from the media (Opti-MEMI, GIBCO/BRL) of HeLa cells coinfected with the appropri-ate recombinant viruses as described (27, 28). TIMP2 waspurified by affinity chromatography using an mAb (CA-101)against human TIMP2, as described (27, 28). The concentra-tions of the purified enzymes and TIMP2 were determined byamino acid analysis (29).Enzyme Activation. The gelatinases, diluted in 5 mM

Tris-HCl, 150 mM NaCl, 5 mM CaCl2, and 0.02% Brij-35buffer, were activated (30 min, 37°C) with a final concentrationof 1 mM p-aminophenylmercuric acetate (APMA). The pres-ence of active forms and the actual activity of the recombinantenzymes were determined by zymography and gelatinaseassays using [3H]gelatin (27, 28), respectively.

Electrophoresis and Immunoblotting. SDS/PAGE was per-formed under reducing conditions, using a 7.5% polyacryl-amide separating gel and a 3.5% stacking gel. The gels werestained with Coomassie blue and/or subjected to autoradiog-raphy. For immunoblot analysis, the separated proteins weretransferred to a nitrocellulose membrane, followed by blocking(3% BSA and 3% nonfat dry milk) and incubation with thecorresponding primary antibodies diluted in 50 mM Tris HCl(pH 7.5), 150 mM NaCl, and 0.1% Tween 20. The immuno-detection of the antigen was performed using the appropriateantibodies and an enhanced chemiluminescence kit (Amer-sham) for development.Preparation of Immobilized FGFR1 Ectodomain-AP

(FRAP) Fusion Protein. A soluble FGF receptor ectodomainwas constructed by cloning the extracellular domain of murineFGFR1 into the APtag expression vector (11, 30). The FRAPfusion protein was immobilized onto either Sepharose beads or96-well plates with use of antibodies to AP. Briefly, proteinA-Sepharose beads (30 ,l; RepliGen) were incubated (2 h,22°C) with 10 gl of affinity-purified rabbit anti-human pla-cental AP antiserum. The beads were washed twice andincubated (2 h, 22°C) with FRAP fusion protein (12 ml ofconditioned medium; -0.5 ,ug of FRAP per ml) followed bythree washes with 0.1% BSA in DMEM containing 25 mMHepes, pH 7.5 (binding buffer). Wells of 96-well plates (F96Maxisorp, Nunc-immuno plate) were incubated (18 h, 4°C)with monoclonal anti-human placental AP antibodies (Sigma)diluted (1:1000) in 0.1 M bicarbonate buffer, pH 8.0 (75 ,ul perwell). The wells were then washed two times with bindingbuffer (250 ,ul per well), incubated (2 h, 220C) with FRAPconditioned medium (-0.5 ,ug of FRAP per ml, 100 ,ul perwell), and washed once with binding buffer.

Treatment of Immobilized FGFR-AP Fusion Protein withMMPs Followed by 125I-bFGF Binding and Crosslinking.Ninety-six-well plates. FGFR-AP fusion protein [FRAP ormouse Bek ectodomain-AP fusion protein (BAP)] immobi-lized onto 96-well plates was incubated (37°C, 5 h, except whenstated otherwise) with recombinant proMMP2 or proMMP9(3 ,ug/ml, except when stated otherwise) in the presence orabsence of 1 mM APMA, 10 mM EDTA, or recombinantTIMP2 (1:1 molar ratio of MMP/TIMP2). The wells were thenwashed three times and incubated (2 h, 22°C) with 5 ng/mliodinated aFGF or bFGF in the absence and presence of 0.1,tg/ml or 0.02 Ag/ml heparin, respectively. The wells werewashed once with binding medium followed by one wash withHepes buffer, pH 7.4, containing 0.5 M NaCl (aFGF) or 2 MNaCl (bFGF), and a final wash with binding buffer. The bound1251-FGF was released with 2 M NaCl in 20 mM sodiumacetate, pH 4.5, and counted in a y counter.

Sepharose beads. The immobilized FGFR-AP (FRAP orBAP) was equilibrated with MMP reaction buffer (50 mMTris-HCl, pH 7.2/5 mM CaCl2/0.02% sodium azide/1 mMAPMA) and incubated (5 h, 37°C) with 3 p,g/ml recombinantproMMP2 in a final volume of 80 ,lI. The beads were washedand suspended in 80 Al of MMP reaction buffer. EDTA (10mM) was then added to both the supernatant and the pellet toinhibit the enzyme activity before bFGF binding and crosslink-ing. The supernatant containing the released FGFR1 ectodo-main and the pellet containing the FRAP fragment remainingbound to the beads were diluted (4x) with Hepes-bufferedsaline, pH 7.5. 125I-bFGF (150 ng/ml) was then incubated (2h, 40C) with both the supernatant and the pellet in the presenceof heparin (0.2 ,ug/ml) and subjected to crosslinking (30 min,220C) with 0.15 mM disuccinimidylsuberate (DSS; dissolved indimethyl sulfoxide and diluted 1:100 in PBS). The crosslinkingreaction was then quenched with 10 mM ethanolamine-HCl,pH 8.0, for 30 min at 24°C. Samples were diluted with SxSDS/PAGE sample buffer and subjected to SDS/PAGE(7.5% gel) under reducing conditions. The gels were dried, andthe 125I-bFGF was visualized by autoradiography on KodakXAR film.

In some experiments, treatment with MMPs was performedafter the crosslinking of 125I-bFGF to FRAP beads. For thispurpose, 125I-bFGF (150 ng/ml) was incubated (2 h, 22°C) withthe FRAP beads in the presence of heparin (0.2 Ag/ml) in 30Al of binding medium. The beads were then washed once with2 M NaCl in 25 mM Hepes, pH 7.4, and once with PBS. Thepellet was suspended in 150 ,lI of DSS (0.15 mM finalconcentration, 30 min, 24°C). The crosslinked 125I-bFGF-FRAP beads were washed four times with PBS, centrifuged,and equilibrated with MMP reaction buffer. RecombinantproMMP2 or proMMP9 (0.2-3 ,ug/ml) was then added to thebeads in the presence of 1 mM APMA, and the reactionmixture (-80 ,ul final volume) was incubated for 5 h at 37°C.At the end of the incubation, the beads were centrifuged, andthe pellets and supernatants were each dissolved in Sx SDS/PAGE sample buffer and subjected to SDS/PAGE analysisand autoradiography as described above.High Affinity Binding of FGF to Chinese Hamster Ovary

(CHO) Cells. proMMP2 was activated with APMA, dialyzedto remove the APMA, and incubated (3 ,ug/ml, 3 h, 370C) withpgsA-745-flg CHO cells (2.5 x 105 cells per 16-mm well) inPBS containing Ca+2 and Mg+2. The culture was washed twice,and receptor binding was performed as described (31-33).Microsequence Analysis. FRAP beads were treated (5 h,

37°C) with MMP2 at an enzyme/substrate ratio of 1:5 (wt/wt).The beads were washed and boiled in sample buffer, and thesolubilized proteins were separated by SDS/PAGE underreducing conditions and transferred to an Immobilon [poly-(vinylidene difluoride)] membrane (Millipore). The trans-ferred proteins were stained with 1% -amido black in 20%isopropanol and 10% acetic acid, and the major -70-kDa band

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was cut and subjected to NH2-terminal sequencing (19 cycles)on an Applied Biosystems 475A gas-phase protein sequencer.

RESULTSEffect of MMP2 on Binding of aFGF and bFGF to FGFR1.

The ability of MMP2 and MMP9 to cleave the FGFR wasdetermined using a FRAP construct attached to anti-AP-coated wells of a 96-well plate. The functionality of the FRAPfusion protein was demonstrated by the binding of 125I-aFGFor 125I-bFGF that was enhanced in the presence of heparin(Fig. 1), in agreement with previous results (11, 33). Treatment(5 h, 37°C) of the immobilized FRAP with 3 ,ug/ml MMP2abolished the binding of 125I-aFGF (Fig. 1) or 125I-bFGF (notshown) to the FRAP construct, regardless of heparin presence.About 50% inhibition of FGF receptor binding was obtainedwhen the immobilized FRAP was treated with 0.3 ,ug/mlMMP2 (not shown). Addition of TIMP2 or EDTA, twoinhibitors of MMPs, to the reaction mixture abrogated theinhibitory effect of MMP2 on FGF-receptor binding. MMP9or proMMP2 had no effect on FGF binding to the FRAPconstruct (Fig. 1). In addition, purified aFGF and bFGF werenot susceptible to cleavage by the MMPs. These results suggestthat MMP2 but not MMP9 can hydrolyze the FRAP fusionprotein, abrogating its capacity to bind FGF. Similar experi-ments performed with a fusion protein formed by AP andFGFR2 (mouse Bek) ectodomain (designated BAP) showed

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FIG. 1. Effect of MMP2 on binding of aFGF to FGFR1 (FRAP).FRAP immobilized onto an anti-AP-conjugated 96-well plate wasincubated (5 h, 37°C) with or without 3 ,g/ml recombinant proMMP2or proMMP9 in the presence of 1 mMAPMA in MMP reaction buffer(110 ,ul per well). Some of the wells were incubated in the presence ofEDTA (10 mM) or recombinant TIMP2 (1:1 molar ratio of MMP/TIMP2) or in the absence ofAPMA (i.e., proMMP2) as indicated. Thewells were washed three times and further incubated (2 h, 22°C) with5 ng/ml 125I-aFGF in the absence and presence of 0.1 ,tg/ml heparinin binding medium. The wells were washed with binding medium andsalt solutions, and the bound 1251-aFGF was released and counted asdescribed in the text. Similar results were obtained with 1251-bFGF.Each point represents the mean ± SD from triplicate wells. Allexperiments were performed at least three times, and the variationbetween different experiments did not exceed ± 10%.

that binding of either aFGF or bFGF to this receptor was notabrogated by pretreatment with MMP2 or MMP9 (not shown).

Identification of the MMP2 Cleavage Site. To determinewhether MMP2 was cleaving within the FGFR1 ectodomain orwithin AP, FRAP attached to Sepharose beads was treatedwith MMP2, and proteins remaining bound to the beads weresolubilized and subjected to immunoblot analysis using anantibody to AP. The results demonstrated the presence of a70-kDa protein, possibly representing the full-length AP.Untreated FRAP or FRAP exposed to MMP2 in the presenceof EDTA showed a molecular mass of 155 kDa (not shown),consistent with the size of the full-length FRAP fusion protein.These studies suggested that the MMP2 cleavage must haveoccurred within the FGFR1 ectodomain or within AP residuesadjacent to FGFR1. Since antibodies to FGFR1 ectodomainwere not available, we analyzed the supernatant fraction,containing the MMP2 cleavage product of FRAP, by SDS/PAGE and Coomassie blue staining. A degradation product(-85 kDa) was identified, consistent with the molecular massof the recombinant ectodomain of FGFR1. Similar studiesperformed with the BAP fusion protein (9), using an antibodyto FGFR2 (anti-Bek), confirmed the resistance of BAP toMMP2 cleavage since only a 155-kDa protein was detected inassociation with the beads (Fig. 2, pellet). These experimentsindicate that of the two FGF receptor ectodomains, onlyFGFR1 may be a substrate susceptible to MMP2 cleavage.To determine the precise cleavage site of MMP2 in the

FRAP fusion protein, the pellet containing the 70-kDa MMP2degradation product was separated by SDS/PAGE, blottedonto poly(vinylidene difluoride) membrane, and subjected toN-terminal sequencing. A sequence of 18 amino acids wasobtained, identical to the known FRAP sequence around thefusion point between FGFR1 ectodomain and AP (Fig. 3). TheN terminus of the 70-kDa protein was found to be within thereceptor sequence, beginning from Met369, eight residues(MTSPLYLE) upstream of the transmembrane domain thatstarts at Arg377 of the murine FGFR1 sequence (5). Thus,MMP2 hydrolyzes the Val368-Met369 bond of the FGFR1ectodomain. Beyond the eight amino acids of the receptor, theother 10 adjacent amino acids (RSSGIIPVEE) that weresequenced were identical to those of AP, starting at position377. Thus, AP, together with eight amino acids of the FGFR1ectodomain, was the immobilized FRAP fragment remainingafter MMP2 cleavage.FGF Binding to FGFR1 Ectodomain Released by MMP2.

The above results demonstrate that MMP2 cleaved the FGF

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FIG. 2. Western blot analysis of BAP fusion protein treated withMMP2. BAP immobilized onto anti-AP conjugated Sepharose beadswas treated (5 h, 37°C) with MMP2 (3 jig/ml) in MMP reaction buffer.The beads were centrifuged, and both the supernatant (S) and thewashed pellet (P) were boiled in SDS/PAGE sample buffer. Solubi-lized proteins were subjected to SDS/PAGE (7.5% gel) followed byelectroblotting and detection with anti-Bek antibodies. Lane 1, super-natant; lane 2, pellet of immobilized BAP that was not exposed toMMP2; lane 3, supernatant; lane 4, pellet of immobilized BAP that wastreated with MMP2. Molecular weight standards are marked on theleft.

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FGFR-1 ectodomain

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FIG. 3. Schematic presentation of the MMP2 cleavage site in theFGFR1 ectodomain. The FGFR ectodomain-AP fusion protein (A) iscleaved at the Val368-Met369 peptide bond (arrow) of the ectodomain.The Arg residue with the asterisk indicates the start of the AP in thefusion protein. For comparison, the natural murine FGFR1 is shownin B. III, immunoglobulin domain III; TM, transmembrane domain; k,kinase domain.

receptor ectodomain immediately next to the transmembranedomain, maintaining the FGF binding domain intact. There-fore, we examined whether the MMP2-cleaved FGFR1ectodomain can bind FGF after being released from the FRAPbeads. Immobilized FRAP was first subjected to MMP2cleavage in the absence and presence ofEDTA. Then, both thebeads (pellet) and the released material (supernatant) werecrosslinked to 125I-bFGF in the presence of heparin. Thecomplex was then analyzed by SDS/PAGE followed by auto-radiography. As demonstrated in Fig. 4 (lane 4), the superna-tant showed a 105-kDa product representing the '25I-bFGFcrosslinked to the solubilized FGFR1 ectodomain. In contrast,the FRAP fragment remaining bound to the beads almost didnot bind 125I-bFGF (lane 3), consistent with the release of theectodomain into the supernatant by MMP2. A lack of 1251-bFGF crosslinking was also observed in the supernatant ofFRAP beads treated with MMP2 in the presence of EDTA(not shown). These results demonstrate that following hydro-lysis of the FGFR1 ectodomain by MMP2, the soluble ectodo-main is still capable of binding bFGF.

Effect of MMP2 on FRAP-bFGF Complex. Since the cleav-age site of MMP2 was found to be immediately upstream of thetransmembrane domain, we examined whether a FRAP fusionprotein bound to FGF was still susceptible to MMP2 cleavage.To this end, immobilized FRAP was crosslinked to 125I-bFGFin the presence of heparin, as described in Materials andMethods. The FRAP-125I-bFGF complex was then treated withMMP2, and both the beads and the supernatant were subjectedto SDS/PAGE analysis followed by autoradiography. Asshown in Fig. 5 (lane 1), the untreated FRAP-125I-bFGFcomplex showed a molecular mass of 175 kDa. Treatment withMMP2 released into the supernatant a 105-kDa protein (lane5) representing the crosslinked 125I-bFGF/FGFR1 ectodo-main complex. In the presence of EDTA, all the radioactivityremained associated with the beads in the form of the 175-kDacomplex (Fig. 5, lane 3). Taken together, the above studiesdemonstrate that an occupied FGFR1 ectodomain is stillsusceptible to hydrolysis by MMP2.

FIG. 4. Binding of 125I-bFGF to MMP2 cleavage products ofFRAP. FRAP immobilized onto anti-AP-conjugated Sepharose beadswas incubated (5 h, 37°C) with MMP2 (3 g/g/ml) in MMP reactionbuffer. The beads were centrifuged, and both the pellet and thesupernatant were incubated (2 h, 4°C) with 125I-bFGF (150 ng/ml) inthe presence of 0.2 /Lg/ml heparin. Binding was followed by crosslink-ing with DSS, boiling in SDS/PAGE sample buffer, electrophoresis,and autoradiography as described in text. Lanes 1 and 2, 125I-bFGFbinding to the pellet (P) and supernatant (S) of control immobilizedFRAP that was not exposed to MMP2; lanes 3 and 4: 125I-bFGFbinding to the pellet and supernatant of immobilized FRAP that wasfirst exposed to MMP2. Molecular weight standards are marked on theleft.

Effect of MMP2 on FGF Binding to CHO Cells. To test theeffect of MMP2 treatment on the ability of cells to bind FGF,we exposed a CHO cell line (pgsA-745-flg) overexpressing themurine FGFR1 (31-33) to recombinant MMP2. The pgsA-745-flg CHO cells were also defective in their metabolism ofheparan sulfate, making them dependent on exogenous hep-

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FIG. 5. Cleavage of '25I-bFGF-FRAP complexes by MMP2. 125I_bFGF (150 ng/ml) was incubated (2 h, 22°C) with FRAP immobilizedonto anti-AP conjugated Sepharose beads in the presence of 0.2 glg/mlheparin in binding medium. The beads were washed with PBS (pH7.4), subjected to crosslinking with DSS, washed three times with PBS,and equilibrated with MMP reaction buffer as described in the text.The beads were then incubated (5 h, 37°C) with MMP2 (3 ALg/ml) inthe absence or presence of 10 mM EDTA, followed by centrifugationand boiling of both the pellet and the supernatant (Sup) fractions inSDS/PAGE sample buffer. Solubilized proteins were subjected toSDS/PAGE and autoradiography. Lanes 1 and 4, pellet and super-natant of control immobilized 125I-bFGF-FRAP complexes that werenot exposed to MMP2, respectively; lanes 2 and 5, pellet and super-natant of immobilized bFGF-FRAP complexes that were exposed toMMP2, respectively; lanes 3 and 6, pellet and supernatant of immo-bilized bFGF-FRAP complexes that were exposed to MMP2 in thepresence of 10 mM EDTA, respectively. The molecular masses ofuntreated (175 kDa, pellet) and MMP2-treated (105 kDa, superna-tant) 125I-bFGF-FRAP are marked on the left.

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arin for binding of FGF (31, 34). The CHO cells were treated(2 h, 37°C) with MMP2 (3 ,ug/ml), washed with PBS, and thenincubated with 125I-aFGF at 4°C for 2 h in the presence ofheparin. High affinity binding of 125I-aFGF was tested asdescribed in Materials and Methods. As shown in Fig. 6, MMP2treatment of the pgsA-745-flg CHO cells caused a markedreduction in the binding of 125I-aFGF to the cells whencompared with the binding to untreated cells. Moreover,FGFR1 ectodomain was detected in the incubation medium ofMMP2 treated CHO cells, and this medium competed withaFGF binding to untreated CHO cells. In other studies,pretreatment of bovine aortic endothelial and smooth musclecells with MMP2 resulted in a significant (approximately 40%and 30%, respectively) loss of aFGF and bFGF binding to highaffinity cell surface receptor sites, indicating that FGFR1 is amajor receptor for aFGF and bFGF in these cells. Thesestudies indicate that FGF receptors expressed on the cellsurface are susceptible to MMP2 hydrolysis, which in turndecreases the binding of FGF.

DISCUSSIONThe present studies demonstrate that human recombinantMMP2, but not MMP9, can hydrolyze the Val368-Met369peptide bond ofFGFR1 located eight residues upstream of thetransmembrane domain. This cleavage resulted in release ofthe entire FGFR1 ectodomain from an immobilized AP-FGFR1 ectodomain fusion protein and inhibited binding of1251-FGF to the remaining immobilized fusion protein. Thecleavage of the FGFR1 ectodomain was MMP2-dependentsince presence of either TIMP2, the natural inhibitor ofMMP2(35), or EDTA, a known chelating inhibitor of MMPs (12, 13),inhibited hydrolysis and subsequent solubilization of theectodomain. It also required proMMP2 activation since withlatent enzyme no hydrolysis occurred. Sequencing data re-vealed that the MMP2 cleavage site was within the FGFR1ectodomain and not within the AP enzyme. No evidence forfurther processing of the FGFR1 ectodomain was observed

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even after a long exposure to MMP2, indicating that thecleavage of the Val368-Met369 peptide bond was unique. Incontrast to FGFR1, BAP was not hydrolyzed by either MMP2or MMP9. This difference is most probably due to a lack ofsequence identity between these two FGF receptors, particu-larly in the area adjacent to the transmembrane domain thatis susceptible to MMP2 hydrolysis in FGFR1. Indeed, align-ment of the sequences of FGFR1 and FGFR2 reveals that theVMTSPLY motif of FGFR1 is replaced by ITASPDY inFGFR2 (5, 6).The hydrolysis of the FGFR1 ectodomain by MMP2 was

observed with both an unoccupied ectodomain, or with anectodomain crosslinked to bFGF, indicating that the MMP2cleavage site is accessible to MMP2 attack regardless of thepresence of FGF. In addition, FGFR1 ectodomain retained itsability to bind FGF after MMP2 hydrolysis. Thus, MMP2cleavage of the receptor yields a functional soluble FGFR1ectodomain capable of FGF binding. The molecular mass ofthe extracellular domain ofFGFR1 cleaved by MMP2 from thefusion protein was approximately 85 kDa, similar to that of thefree FGFR1 ectodomain recently identified in human plasmaand in the ECM of retinal vascular endothelial cells (24, 25).It is tempting to speculate, based on our data, that the presenceof the ectodomain of FGFR1 in plasma and ECM results fromthe effect of MMP2 on FGFR1. We have shown here thattreatment of heparan sulfate-deficient CHO cells overexpress-ing FGFR1 with MMP2 markedly reduced their binding ofFGF. A significant loss of FGFR1 was also observed inMMP2-treated vascular endothelial and smooth muscle cells.Thus, it is likely that a plasma membrane-bound FGFR1 issusceptible to MMP2 hydrolysis. The released ectodomainbinds extracellular FGF, possibly controlling the biologicalavailability and growth promoting activity of FGF.

In a recent study, the ECM produced by bovine endothelialcells was found to contain proMMP2, free of TIMP2 andsusceptible to release by MMP9 and to activation by APMA(36). Once activated, the ECM-resident MMP2 may cleave theFGFR1 of cells that contact the ECM, resulting in ECMsequestration and/or release of the FGFR1 ectodomain.MMP2 expression has been reported in a variety of endothelialcells (37), and MMP2 can be induced by FGF (38, 39). Also,thrombin was recently reported to elicit activation ofMMP2 invascular endothelial cells (40). This may result in a moreefficient cleavage of FGFR1, possibly providing a feedbackcontrol mechanism that will down-regulate cellular responsesto bFGF.A cell surface localization of MMP2 can facilitate the access

of the protease to membrane-bound proteins susceptible toMMP hydrolysis. Studies of human tumors showed that thegelatinases localize on the tumor cells in a pericellular pattern(41-44). In addition, the invadopodia of cultured transformedchicken embryo fibroblasts showed the presence of MMP2(45), and plasma membranes isolated from phorbol ester-treated fibrosarcoma HT1080 cells were found to contain the62-kDa active form of MMP2 (unpublished results). It isconceivable that on the cell surface an appropriate localconcentration and availability of both MMP2 and FGFR1 canbe achieved, so that a physiologically efficient cleavage of thereceptor can take place. A similar situation was proposed forother cell surface (i.e., receptor bound urokinase) (46) and/orECM-bound molecules (i.e., plasminogen and thrombin) (47,48) that are protected and/or better situated for interactionwith effector molecules when immobilized to a solid support,as compared with their behavior in a fluid phase (48). Theeffect of MMP2 on FGFR1 and other cellular proteins such asgalectin-3 (21) and j-amyloid (19, 20) suggests that thisproteinase may also play a role in other biological processbesides ECM degradation and remodeling. This property is notunique to MMP2. MMP1 and MMP3 were-shown to hydrolyzeinsulin growth factor-binding protein type 3 in solution and in

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rat pregnancy serum (49) and MMPs are also involved in theprocessing of tumor necrosis factor a to biologically activetumor necrosis factor a (50). Taken together, these studiesdemonstrate that a spectrum of proteins can be hydrolyzed byMMPs and suggest a novel role for these enzymes in theprocessing of FGFR1 and other regulatory molecules.

We are grateful to Dr. J. D. Esko (University of Birmingham, AL)for providing the pgsA-745 CHO mutant cell line. This work wassupported by grants from the United States-Israel Binational ScienceFoundation, Jerusalem, Israel (to I.V. and A.Y.); the Israel ScienceFoundation administered by the Israel Academy of Sciences andHumanities (to I.V.); and the Israel Cancer Research Fund-CarrierDevelopment Award (A.Y.); and by Grant DAMD17-94-J-4356 fromthe U.S. Department of Defense (to R.F.).

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