Urokinase-type Plasminogen Activator Receptor (CD87) Is a Ligand for Integrins and Mediates Cell-Cell Interaction*

Takehiko Tarui, Andrew P. MazarDagger , Douglas B. Cines§, and Yoshikazu Takada

From the Department of Vascular Biology, The Scripps Research Institute, La Jolla, California 92037, Dagger  Attenuon, LLC, San Diego, California 92121, and the § Department of Pathology and Laboratory Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104

Received for publication, September 8, 2000, and in revised form, October 13, 2000



    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Binding of urokinase-type plasminogen activator (uPA) to its receptor (uPAR/CD87) regulates cellular adhesion, migration, and tumor cell invasion. However, it is unclear how glycosyl phosphatidylinositol-anchored uPAR, which lacks a transmembrane structure, mediates signal transduction. It has been proposed that uPAR forms cis-interactions with integrins as an associated protein and thereby transduces proliferative or migratory signals to cells upon binding of uPA. We provide evidence that soluble uPAR (suPAR) specifically binds to integrins alpha 4beta 1, alpha 6beta 1, alpha 9beta 1, and alpha vbeta 3 on Chinese hamster ovary cells in a cation-dependent manner. Anti-integrin and anti-uPAR antibodies effectively block binding of suPAR to these integrins. Binding of suPAR to alpha 4beta 1 and alpha vbeta 3 is blocked by known soluble ligands and by the integrin mutations that inhibit ligand binding. These results suggest that uPAR is an integrin ligand rather than, or in addition to, an integrin-associated protein. In addition, we demonstrate that glycosyl phosphatidylinositol-anchored uPAR on the cell surface specifically binds to integrins on the apposing cells, suggesting that uPAR-integrin interaction may mediate cell-cell interaction (trans-interaction). These previously unrecognized uPAR-integrin interactions may allow uPAR to transduce signals through the engaged integrin without a hypothetical transmembrane adapter and may provide a potential therapeutic target for control of inflammation and cancer.



    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The receptor for urokinase-type plasminogen activator (uPA),1 uPAR/CD87, is a glycoprotein (Mr 35,000-65,000) composed of 283 amino acid residues. It is anchored to the plasma membrane by a glycosyl phosphatidylinositol (GPI) linkage (reviewed in Refs. 1 and 2). uPAR is the cellular receptor for urokinase, a serine protease that is constitutively or inducibly secreted by most uPAR-expressing cells. Receptor-bound uPA can convert plasminogen to plasmin, which mediates pericellular proteolysis of extracellular matrix proteins in the path of cellular invasion. uPAR is expressed by activated leukocytes, endothelial cells, fibroblasts, and different types of cancer cells (reviewed in Refs. 2 and 3 and references therein). Expression of uPAR has been shown to correlate with the prognosis of many human cancers (2). In murine tumor models, expression or administration of uPAR antagonists has a marked inhibitory effect on the metastatic ability of cancer cells (4) and on the growth of the primary tumor (5), and the down-regulation of uPAR leads to dormancy of carcinoma cells in vivo (6, 7). Thus, uPAR expression has been implicated in cancer progression. In addition, it appears that uPAR is up-regulated on cells that are in motion. Migratory/chemotaxis-inducing stimuli (e.g. vascular endothelial growth factor, fibroblast growth factor, platelet-derived growth factor, and interleukins) up-regulate uPAR in endothelial cells, smooth muscle cells, and leukocytes in vitro, whereas unstimulated cells do not have detectable expression of uPAR (reviewed in Refs. 2 and 3 and references therein). Thus, uPAR may play a role in leukocyte recruitment, angiogenesis, and tumor metastasis.

uPAR has been reported to associate with many signaling molecules and to mediate signal transduction (8-10). In recent reports the binding of uPA to uPAR in tumor or endothelial cells has been shown to activate the mitogen-activated protein kinases, extracellular regulated kinase 1 and 2 (11-13). However, a major question is how uPAR mediates cellular signaling, because the molecule has no transmembrane structure. The existence of one or more hypothetical "transmembrane adapter molecules" that connects uPAR and signaling molecules inside cells has been proposed (14).

It has been shown that the beta 1, beta 2, and beta 3 integrin receptor families interact with uPAR using immunocoprecipitation, immunocolocalization, and resonance energy transfer approaches (15-17). The uPAR-integrin interaction may be significant, because many integrin receptors activate intracellular signals coupled to the pathways used by both receptor and nonreceptor tyrosine kinases (18-20). Integrin- and receptor tyrosine kinase-mediated signals may complement each other to fully activate cell survival and proliferation pathways (21, 22). It has been proposed that uPAR forms cis-interactions with integrins on the same cell surface as an integrin-associated protein (reviewed in Ref. 1). However, it has not been established that this is the dominant mode of interaction responsible for signal transduction events.

In the present study, we designed experiments to examine the uPAR-integrin interaction in detail using isolated domains derived from recombinant soluble uPAR (suPAR) and cells expressing recombinant beta 1 or beta 3 integrins. These studies establish that uPAR binds to integrins in a manner that is very similar to that of known integrin ligands (e.g. vascular cellular adhesion molecule-1 (VCAM-1)). Additionally, we have found that uPAR interacts with integrins on apposing cells (trans-interaction). These unexpected findings may help to clarify the complex role of uPAR in signal transduction, inflammation, and cancer.


    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Materials

TS2/16, an activating anti-beta 1 mAb (23), was obtained from American Type Culture Collection (Manassas, VA). AIIB2, a function-blocking anti-beta 1 mAb (24), was obtained from Developmental Studies Hybridoma Bank (Iowa City). IA. P1B5 (anti-alpha 3) (25) was a gift from E. Wayner and W. G. Carter (University of Washington, Seattle, WA). SG73 (anti-alpha 4) (26) and KH72 (anti-alpha 5) were provided by K. Miyake (Saga Medical School, Saga, Japan). 135-13C (anti-alpha 6) (27) was obtained from S. J. Kennel (Oak Ridge National Laboratory, Oak Ridge, TN), and Y9A2 (anti-alpha 9) (28) was obtained from D. Sheppard (University of California, San Francisco, CA). 7E3 (anti-beta 3) (29) was provided by B. S. Coller (Mount Sinai Hospital, New York, NY). 8C8 (anti-alpha 2) and 15 (anti-beta 3) were provided by M. H. Ginsberg (The Scripps Research Institute). Anti-uPAR monoclonal antibody (3B10) (30) was provided by R. F. Todd III (University of Michigan Medical Center, Ann Arbor, MI).

Recombinant VCAM-1-mouse Ck chain fusion protein was provided by Novartis (Basel, Switzerland). Recombinant domain 2 and domain 3 forms of soluble uPAR generated in Chinese hamster ovary (CHO) cells were prepared as described previously (31). GRGDS and GRGES peptides were purchased from Advanced ChemTech (Louisville, KY). alpha -Bungarotoxin and rabbit IgG were obtained from Sigma. Human fibrinogen was obtained from Enzyme Research Laboratories (South Bend, IN).

CHO cells and human erythroleukemia K562 cells were obtained from the American Type Culture Collection. Integrin alpha 5beta 1-deficinet B2 variant CHO cells (32) were provided by R.L. Juliano (University of North Carolina, Chapel Hill, NC), and Jurkat human T-cell leukemic cells were provided by M. H. Ginsberg (The Scripps Research Institute). CHO cells expressing human alpha 9 (designated alpha 9-CHO) (33) were provided by D. Sheppard (University of California, San Francisco, CA). CHO cells expressing other human integrin alpha  and/or beta  subunits (wild type and mutant) have been described (34). B2 cells expressing human alpha 2, alpha 3, and alpha 4 (designated alpha 2-B2, alpha 3-B2, and alpha 4-B2 cells, respectively) were prepared as described for CHO cells expressing these integrins (34). These cells express human alpha /hamster beta 1 or hamster alpha v/human beta 3 hybrid. K562 cells expressing recombinant human alpha 4 have been described (35). K562 cells expressing recombinant human alpha 9 (alpha 9-K562) have been described (36). K562 cells expressing recombinant human alpha vbeta 3 (alpha vbeta 3-K562) (37) are a gift from E. J. Brown (Washington University, St Louis, MO).

CHO cells expressing the three domain forms of human uPAR (designated HuPAR-CHO) were prepared by transfecting full-length human uPAR cDNA (provided by L. A. Miles, The Scripps Research Institute) into a pCDNA3 vector (Invitrogen) together with a plasmid containing a neomycin-resistant gene. Cells were selected with G-418 (0.7 mg/ml medium). Approximately 50% of cells stably expressed uPAR after selection with mAb 3B10. CHO cells stably expressing uPAR were sorted by FACStar (Becton-Dickinson) to obtain cells homogeneously expressing uPAR at a high level.

Methods

Production of suPAR in Drosophila S2 Cells-- cDNAs encoding soluble wild-type uPAR (amino acids 1-277) and soluble domains 2 and 3 (amino acids 88-277) were generated by polymerase chain reaction using pTracer-full-length uPAR as a template. The fragments were digested with BglII and Xho and sub-cloned into the expression vector (pMT/BiP/V5, Invitrogen). Soluble suPAR domains were expressed in Drosophila Schneider S2 cells (DES system, Invitrogen) as described by the manufacturer.

Purification of suPAR Expressed in S2 Cells-- Small scale preparations of suPAR and variants were purified from the medium using a polyclonal anti-uPAR antibody affinity column. For large scale preparations, S2 cell culture supernatants were filtered, and the medium were loaded onto a 40-g hydroxyapatite column equilibrated with 10 mM K2HPO4, pH 7.0. The column was then eluted with a gradient of 10-200 mM K2HPO4, pH 7.0, and the suPAR-containing fractions were identified by Western blotting. Fractions containing monomeric suPAR eluted between 50 and 120 mM K2HPO4. Fractions containing monomeric suPAR were pooled, concentrated, and further purified using C8 reverse-phase HPLC. Crude suPAR (20 mg of total protein) was loaded in a volume of 2 ml onto a semi-preparative (10 × 250 cm) C8 column and eluted at a flow rate of 4 ml/min with a linear gradient of 0-70% solvent B, where solvent A was 100% H2O, 0.1% trifluoroacetic acid, and solvent B was 100% acetonitrile, 0.1% trifluoroacetic acid. suPAR eluted as a single broad peak under these conditions with a retention time of ~27 min. SDS-polyacrylamide gel electrophoresis analysis of suPAR purified in this manner demonstrated a single major peak at 35 kDa under nonreducing conditions. Also observed was a slight laddering effect with three to four minor higher molecular weight species. These were determined to be SDS-stable aggregates of suPAR, which disappeared when SDS-polyacrylamide gel electrophoresis was performed under reducing conditions. Matrix-assisted laser desorption ionization-time of flight mass spectrometry revealed a single broad peak with an average mass of 34,797 Da. The predicted mass based on the amino acid sequence is 30,672 Da, indicating the presence of about 4 kDa in glycosylation. Expression levels of suPAR were typically 30 mg/liter determined by enzyme-linked immunosorbent assay. Purification yielded about 10-12 mg of pure suPAR protein per liter of culture supernatant.

Enzymatic Digestion of Wild-type suPAR-- suPAR was digested with chymotrypsin to generate the soluble D1 and D2D3 fragments. Chymotrypsin was added to suPAR (1 mg/ml in phosphate-buffered saline) at a final molar ratio of suPAR:chymotrypsin of 1000:1, and the digest was allowed to proceed for 2 h at room temperature. The digest was quenched by addition of Pefablock (100 µM final concentration). The D1 and D2D3 fragments were separated using C8 reverse phase-HPLC. The D1 and D2D3 fragments eluted with a retention time of ~27 and 25 min, respectively. The purified D1 fragment was sequenced, and a single N terminus was observed beginning with RS (representing the two extra amino acids present in this construct) followed by LR, amino acids 1 and 2 in the mature suPAR sequence. Sequencing of the D2D3 fragment revealed a single N terminus as well, beginning with amino acids SRS, corresponding to amino acids 88-90 in the mature suPAR sequence. Matrix-assisted laser desorption ionization-time of flight mass spectral analysis revealed a single peak for the D1 fragment (mass = 11,041 Da) and several peaks for the D2D3 fragment (mass = 23,846; 23,691; 22,936; 22,800). These were presumed to be glycosylation isoforms, although additional digestion of the D2D3 fragment from the C terminus could not be excluded. However, no consensus chymotryptic cleavage site that could result in the observed mass differences is present at the C terminus of the D2D3 fragment.

Affinity-purified Anti-suPAR-- A polyclonal antibody against suPAR was prepared as described (38). Briefly, recombinant suPAR was expressed in SP2/0 cells and purified using a single-chain uPA-Sepharose column. Serum was collected from rabbits immunized with purified suPAR, and the IgG was obtained by a 50% ammonium sulfate precipitation. This material was dialyzed (10,000×) against phosphate-buffered saline and further purified using a suPAR-Sepharose column to generate affinity-purified anti-suPAR IgG.

Adhesion Assays-- Adhesion assays were performed as described previously (34). Briefly, wells in 96-well Immulon-2 microtiter plates (Dynatech Laboratories, Chantilly, VA) were coated with 100 µl of phosphate-buffered saline (10 mM phosphate buffer, 0.15 M NaCl, pH 7.4) containing substrates at a concentration of 0.2-5 µg/ml and were incubated overnight at 4 °C. Remaining protein-binding sites were blocked by incubating with 0.2% bovine serum albumin (Calbiochem) for 1 h at room temperature. Cells (105 cells/well) in 100 µl of Hepes-Tyrode buffer (10 mM HEPES, 150 mM NaCl, 12 mM NaHCO3, 0.4 mM NaH2PO4, 2.5 mM KCl, 0.1% glucose, 0.02% bovine serum albumin) supplemented with 2 mM MgCl2 were added to the wells and incubated at 37 °C for 1 h, unless stated otherwise. After nonbound cells were removed by rinsing the wells with the same buffer, bound cells were quantified by measuring endogenous phosphatase activity (39). Antibodies were used at 250× dilution of ascites (TS2/16, 8C8, P1B5, SG73, KH72, and 135-13C), at 4 µg/ml (AIIB2, 7E3, and Y9A2), and at 3.4 µg/ml (anti-uPAR and rabbit IgG). Data are shown as means ± S.D. of three independent experiments.

Cell-Cell Binding Assay-- Integrin-transfected, mock-transfected (vector only), or parental K562 cells were labeled with 2',7'-bis-(2-carboxyethyl)-5(6)-carboxyfluorescein acetoxymethyl ester (Molecular Probes, Eugene, OR) according to the manufacturer's instructions. The labeled cells (105 cells/well) were added to the monolayer of parent, mock-transfected, or HuPAR-CHO cells and incubated for 30 min at 37 °C. After the wells were rinsed with medium to remove unbound cells, bound cells were quantified by assaying fluorescence (excitation 485 nm, emission 530 nm) using an FL500 microplate fluorescence reader (Bio Tek Instruments, Winooski, VT). Antibodies were used at the same concentrations as described above. Data are shown as means ± S.D. of three independent experiments.

Other Methods-- Site-directed mutagenesis (40), flow cytometric analysis (41), and transfection (41) were performed as described in the cited references.


    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Binding of Recombinant suPAR to beta 1 and beta 3 Integrins-- To study whether and how integrins are involved in uPAR-mediated signal transduction, we first asked whether recombinant soluble uPAR fragments interact with beta 1 and beta 3 integrins. It has been proposed that the activity of suPAR requires chymotrypsin cleavage between the N-terminal domains 1 and 2 (14). We used several different suPAR fragments that were expressed in Drosophila S2 cells (domain 1, domains 2 and 3, or full-length suPAR containing domains 1, 2, and 3, designated D1, D2D3, D1D2D3, respectively) and domains 2 and 3 expressed in CHO cells. Unexpectedly, these suPAR fragments supported adhesion of both alpha 4- and beta 3-CHO cells expressing recombinant human alpha 4/hamster beta 1 (alpha 4beta 1) and hamster alpha v/human beta 3 hybrid (alpha vbeta 3), respectively, but not CHO cells (Fig. 1A). Parental CHO cells express endogenous alpha 5beta 1, alpha vbeta 1, and alpha vbeta 5 but not alpha 4beta 1 or alpha vbeta 3 integrins (42). It appears that suPAR expressed in Drosophila and CHO cells supports adhesion of alpha 4- and beta 3-CHO cells at comparable levels. We used suPAR D2D3 expressed in CHO cells (43) for further characterization of suPAR-integrin interaction throughout this study. The rationale of using D2D3 is that removal of D1 leaves uPAR D2D3 that is capable of signal transduction without uPA (14), and thus suPAR D2D3-integrin interaction is likely to be biologically relevant.



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Fig. 1.   Integrin-mediated cell adhesion to recombinant soluble human uPAR. A, alpha 4-CHO cells (closed columns), beta 3-CHO cells (open columns), and CHO cells (shaded columns) were tested for their ability to adhere to immobilized recombinant suPAR (at the coating concentration of 5 µg/ml). suPAR domain 1 (D1), domains 2 and 3 (D2D3), and domains 1, 2, and 3 (D1D2D3) were expressed in Drosophila S2 cells or in CHO cells. B and C, CHO cells expressing different human integrins or CHO B2 variant cells lacking endogenous alpha 5beta 1 were tested for their ability to adhere to immobilized recombinant suPAR D2D3 synthesized in CHO cells (up to 5 µg/ml). D, the effect of function-blocking anti-integrin antibodies on adhesion to suPAR D2D3 was studied. The concentration of suPAR D2D3 used for coating was 5 µg/ml. Bovine serum albumin, closed columns; suPAR D2D3, open columns; and suPAR D2D3 plus function-blocking antibodies, shaded columns. The antibodies used were SG73 (anti-alpha 4) for alpha 4-CHO cells, KH72 (anti-alpha 5) for CHO cells, 135-13C (anti-alpha 6) for alpha 6-CHO cells, Y9A2 (anti-alpha 9) for alpha 9-CHO cells, and 7E3 (anti-beta 3) for beta 3-CHO cells. E, the effect of affinity-purified anti-uPAR antibody on alpha vbeta 3-suPAR D2D3 interaction was studied. Polyclonal anti-uPAR or control rabbit IgG was included in the assay medium at 3.4 µg/ml. SuPAR D2D3 was used at a coating concentration of 5 µg/ml. F, the capability of alpha -bungarotoxin, a protein structurally similar to uPAR, to support alpha vbeta 3-mediated cell adhesion was studied as a function of substrate concentration. SuPAR D2D3 and alpha -bungarotoxin were coated up to 5 µg/ml.

We next studied the specificity of suPAR D2D3 binding to beta 1 and beta 3 integrins. We used CHO cells expressing different human alpha  subunits (alpha 2, alpha 3, alpha 6, and alpha 9, designated alpha 2-, alpha 3-, alpha 6-, and alpha 9-CHO cells, respectively) in addition to alpha 4- and beta 3-CHO cells. These transfectants express human alpha /hamster beta 1 hybrids (alpha 2beta 1, alpha 3beta 1, alpha 6beta 1, or alpha 9beta 1, respectively). We found that beta 3-, alpha 4-, alpha 9-, and alpha 6-CHO cells adhered to uPAR in a dose-dependent manner (Fig. 1, B and C). However, other transfectants (alpha 2- and alpha 3-CHO cells), parental CHO cells, CHO B2 variant cells lacking endogenous alpha 5beta 1 (32) (Fig. 1, B and C), and mock-transfected CHO cells (data not shown) adhered only weakly under the conditions used. The adhesion of alpha 4-, alpha 6-, alpha 9-, and beta 3-CHO cells to uPAR was blocked by specific function-blocking antibodies (SG73 to alpha 4, 135-13C to alpha 6, Y9A2 to alpha 9, and 7E3 to beta 3) (Fig. 1D). These results suggest that suPAR D2D3 specifically binds to several integrins (alpha 4beta 1, alpha 6beta 1, alpha 9beta 1, and alpha vbeta 3).

We further tested the specificity of interaction using antibodies against uPAR. We found that antibodies against uPAR effectively blocked suPAR binding to alpha vbeta 3 (Fig. 1E). We then tested whether alpha -bungarotoxin, which has a "three-finger protein" structure similar to the uPAR structure (44), "nonspecifically" binds to alpha vbeta 3. The toxin showed only weak, if any, affinity to alpha vbeta 3 (Fig. 1F). These results suggest that suPAR binding to integrins is specific to the uPAR sequence and structure.

Effect of Cations and beta 1 Integrin Activation and Inactivation on uPAR Binding-- Ligand binding to integrins is tightly regulated by activation/inactivation of integrins through inside-out signal transduction (45). beta 3-CHO cells significantly adhere to suPAR D2D3 in the presence of Mg2+ but not in the presence of Ca2+ (Fig. 2A). CHO cells do not adhere to suPAR in either condition. These results suggest that alpha vbeta 3 requires Mg2+ for binding to suPAR D2D3 under the conditions employed. Although alpha 2-CHO, alpha 3-CHO, and CHO cells do not strongly adhere to suPAR D2D3 under the assay conditions used, it is still possible that binding of alpha 2beta 1, alpha 3beta 1, and alpha 5beta 1 may require more activation. Therefore, we studied whether Mn2+ (0.1 mM), which universally activates integrins, facilitates uPAR binding to these integrins (Fig. 2A). We found that alpha 5beta 1-deficient B2 variant CHO cells did not significantly adhere to suPAR D2D3 in the presence of Mn2+, but parental CHO cells did. We found that B2 cells expressing human alpha 3 or alpha 4 (designated alpha 3- or alpha 4-B2 cells, respectively) also adhered to suPAR D2D3 in the presence of Mn2+. alpha 2-B2 cells did not adhere to suPAR D2D3 under any conditions tested. Ca2+ did not significantly support adhesion of these cells. These results suggest that alpha 3beta 1, alpha 4beta 1, and alpha 5beta 1 but not alpha 2beta 1 bind to suPAR D2D3 in an activation-dependent manner.



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Fig. 2.   Effects of cations and anti-integrin activating or function-blocking antibodies on suPAR binding. A, the effect of cations on adhesion to suPAR D2D3 was studied. Ca2+ (2 mM), Mg2+ (2 mM), or Mn2+ (0.1 mM) was added to Hepes-Tyrode buffer. The concentration of suPAR D2D3 used for coating was 5 µg/ml. Antibodies used were KH72 (anti-alpha 5) for CHO, 8C8 (anti-alpha 2) for alpha 2-B2, P1B5 (anti-alpha 3) for alpha 3-B2, and SG73 (anti-alpha 4) for alpha 4-B2. B, Jurkat T-cell leukemic cells were tested for adhesion to suPAR D2D3. The concentration of suPAR D2D3 used for coating was 2 µg/ml. Antibodies used were TS2/16 (anti-beta 1, activating), AIIB2 (anti-beta 1, function-blocking), SG73 (anti-alpha 4), and KH72 (anti-alpha 5).

We then asked whether the activation status of beta 1 integrins that is required for uPAR binding to natural integrins is similar to the requirements for the recombinant integrins that we studied. To answer this question, we studied uPAR binding to nonrecombinant integrins on Jurkat human T-cell leukemia cells (alpha 4beta 1+, alpha 5beta 1+). We can stimulate or suppress beta 1 integrin-ligand interaction from outside cells using anti-beta 1 antibodies (e.g. TS2/16, activating; AIIB2, inhibiting) (reviewed in Ref. 46). We found that adhesion of Jurkat cells to suPAR D2D3 is stimulated by TS2/16 and blocked completely by AIIB2 and SG73 (anti-alpha 4 mAb) and partially by KH72 (anti-alpha 5 mAb) (Fig. 2B). These results suggest that nonrecombinant alpha 4beta 1 and alpha 5beta 1 in Jurkat cells may interact with suPAR D2D3 in an activation-dependent manner.

Does uPAR Share Common Binding Sites in Integrins with Known Integrin Ligands?-- We then asked whether uPAR competes with VCAM-1, a known alpha 4beta 1 ligand, for binding to alpha 4beta 1. To address this question, we examined the adhesion of alpha 4-B2 cells to suPAR D2D3 in the presence of soluble VCAM-1. We used B2 cells to completely eliminate the contribution of alpha 5beta 1 in uPAR binding. We found that adhesion of alpha 4-B2 cells to suPAR D2D3 was blocked by VCAM-1 in a dose-dependent manner (Fig. 3A) but not by irrelevant ligand fibrinogen. These results suggest that the inhibitory effect of VCAM-1 is specific to alpha 4beta 1-suPAR D2D3 interaction, and thus suPAR and VCAM-1 compete for binding to alpha 4beta 1. We also studied the effect of the GRGDS peptide, a widely distributed integrin-binding motif, on uPAR-alpha vbeta 3 interaction (Fig. 3B). We found that GRGDS peptide completely blocked adhesion of beta 3-CHO cells to suPAR D2D3, but control GRGES peptide did not, suggesting that uPAR and the ligand-derived RGD motif compete for binding to alpha vbeta 3. Thus it is highly likely that the uPAR-binding sites in these integrins may overlap with those of known integrin ligands.



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Fig. 3.   Competition between uPAR and VCAM-1 for binding to alpha 4beta 1 (A) or between uPAR and RGD peptide for binding to alpha vbeta 3 (B). A, the effect of soluble VCAM-1 on alpha 4beta 1-suPAR D2D3 interaction was studied. The concentration of suPAR D2D3 used for coating was 5 µg/ml. The alpha 5beta 1-deficient CHO B2 variant cells expressing human alpha 4/hamster beta 1 (a receptor for VCAM-1) (alpha 4-B2) were used instead of alpha 4-CHO cells. Fibrinogen, an irrelevant ligand, was used as a negative control. VCAM-1 and fibrinogen were added at the final concentrations of 1, 5, or 10 µg/ml in the incubation medium. The numbers in parentheses are concentrations of proteins (µg/ml). Fg, fibrinogen. B, the effect of GRGDS or GRGES peptide on alpha vbeta 3-suPAR interaction was studied with beta 3-CHO cells. The concentration of suPAR D2D3 used for coating was 5 µg/ml. GRGDS or GRGES peptide was added at 10 or 100 µM final concentration in the incubation medium. The numbers in parentheses are concentrations of proteins (µM).

We next studied whether uPAR binding is inhibited by mutations in these integrins that block binding of known ligands. We have previously reported that the mutation to Ala of several amino acid residues, Tyr-187, Trp-188, and Gly-190 in alpha 4 (designated Y187A, W188A, and G190A mutations, respectively), blocks VCAM-1 and fibronectin connecting segment-1 (CS-1) peptide binding to alpha 4beta 1 (47). These residues are located within the putative ligand-binding site in alpha 4, and the corresponding residues in alpha IIb or alpha 5 have also been reported to be critical for ligand binding to alpha IIbbeta 3 or alpha 5beta 1 (47, 48). We studied whether these alpha 4 mutations block adhesion of alpha 4beta 1 to suPAR D2D3. We found that these mutations blocked uPAR-alpha 4beta 1 interaction, but several other mutations did not (Fig. 4). The overall effect of these mutations on uPAR binding to alpha 4beta 1 is similar to their effect on VCAM-1 or CS-1 binding to alpha 4beta 1. These results suggest that the uPAR-binding site in alpha 4 overlaps with those for VCAM-1 and CS-1.



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Fig. 4.   Critical residues in alpha 4 for uPAR binding. CHO cells expressing different alpha 4 mutants were tested for their ability to adhere to suPAR D2D3 (closed columns) and VCAM-1 (open columns). Mutation to Ala at positions 187 (Tyr), 188 (Trp), and 190 (Gly) within the putative ligand-binding sites of alpha 4 blocks alpha 4beta 1-VCAM-1 binding; the data on VCAM-1 binding are from a previous publication (47) and are shown for comparison. The concentration of substrates used for coating was 3 µg/ml for suPAR D2D3 and 0.25 µg/ml for VCAM-1-Ck fusion protein. Goat anti-mouse Ck antibody was used to facilitate VCAM-1-Ck coating (47). wt, wild type.

The Asp-130 residue of beta 1 (42, 49) and the corresponding Asp residues in beta 2 (50), beta 3 (Asp-119) (51), and beta 6 (52) have been reported to be critical for ligand binding. We tested whether mutation of Asp-119 to Ala in beta 3 affected suPAR D2D3 binding to alpha vbeta 3. The wild type or Asp-119 to Ala (D119A) dominant-negative mutant of human beta 3 was transiently expressed in CHO cells, and the ability of cells to adhere to suPAR D2D3 was determined. Wild-type or mutant beta 3 is expressed as hamster alpha v/human beta 3 hybrid. We found that cells transiently expressing the alpha vbeta 3(D119A) mutant did not adhere to suPAR D2D3, although cells expressing wild-type alpha vbeta 3 did (Fig. 5A). The levels of adhesion (~22%) of wild-type beta 3 are lower than in Fig. 1 but are substantial, considering that only 35% of added cells express human beta 3. Note that the nonfunctional beta 3(D119A) mutant was expressed on the surface of cells at a comparable level.



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Fig. 5.   Conserved Asp residues in the beta  subunits are critical for uPAR binding. A, the wild type or Asp-119 to Ala (D119A) dominant-negative mutant of human beta 3 was transiently expressed in CHO cells. Expression vector (pBJ-1) was transiently transfected in CHO cells as control. Transfected wild-type or mutant beta 3 is expressed as hamster alpha v/human beta 3 hybrid. Forty-eight h after transfection, the ability of cells to adhere to suPAR D2D3 was determined. The level of beta 3 expression is 35.8% in cells expressing wild-type beta 3, 43.8% in cells expressing mutant beta 3, and 8.8% (background) in mock-transfected cells with anti-human beta 3 antibody (mAb 15). The concentration of suPAR D2D3 used for coating was 5 µg/ml. B, the wild type or Asp-130 to Ala (D130A) dominant-negative mutant of human beta 1 was expressed in clonal CHO cells together with human alpha 4. The resulting alpha 4beta 1- and alpha 4beta 1(D130A)-CHO cells were tested for their capacity to adhere to suPAR D2D3. The expression levels of human alpha 4 and beta 1 are comparable. alpha 4beta 1(D130A)-CHO cells show lower adhesion to suPAR D2D3 than do alpha 4beta 1- or alpha 4-CHO cells (dominant-negative effect). It should be noted that the remaining binding in alpha 4beta 1(D130A)-CHO cells to suPAR D2D3 is due to endogenous intact hamster beta 1.

We also studied the effect of the corresponding Asp-130 to Ala (D130A) mutation in beta 1 on suPAR D2D3 binding to alpha 4beta 1. For this purpose we transfected wild-type human beta 1 or beta 1(D130A) mutant into cells stably expressing human alpha 4 (49). Cells stably expressing both human alpha 4 and beta 1 or beta 1(D130A) were cloned to obtain cells expressing beta 1 or beta 1(D130A) at high levels (49) (designated alpha 4beta 1- and alpha 4beta 1(D130A)-CHO cells, respectively). The expression levels of wild-type and mutant beta 1 in alpha 4beta 1 and alpha 4beta 1(D130A) are comparable, and the beta 1(D130A) mutant shows a dominant-negative effect on VCAM-1 and CS-1 binding to alpha 4beta 1 (49). We found that the alpha 4beta 1(D130A) mutant bound far less well to uPAR than did alpha 4beta 1-CHO (dominant-negative effect) (Fig. 5B). The residual adhesion of alpha 4beta 1(D130A)-CHO cells to suPAR D2D3 may be due to endogenous intact hamster beta 1/human alpha 4 complex. These results suggest that Asp-130 of beta 1 is critical for uPAR binding to alpha 4beta 1 and that the conserved Asp residues in beta 1 and beta 3, which are required for known integrin ligand binding, are critical for uPAR binding as well. Taken together, these results strongly suggest that uPAR is an integrin ligand.

uPAR-Integrin Trans-interaction Supports Cell-Cell Adhesion-- uPAR has been proposed to interact with integrins on the same cells (cis-interaction) (reviewed in Ref. 1). Because the present results suggest that uPAR may interact with integrins as a ligand, it is possible that uPAR may interact with integrins on apposing cells (trans-interaction), analogous to the interaction between VCAM-1 and alpha 4beta 1. To determine whether uPAR interacts in-trans with integrins, we tested whether K562 erythroleukemic cells expressing recombinant alpha 4beta 1 (designated alpha 4-K562 cells) interact with unsorted CHO cells expressing human uPAR (the three-domain form, designated HuPAR-CHO cells) (Fig. 6A). We found that alpha 4-K562 cells showed significantly greater adhesion to HuPAR-CHO cells than to parental CHO cells and that this adhesion was blocked by SG73 (anti-alpha 4). Mock-transfected and parental K562 or CHO cells generated essentially the same results, and the data with parental K562 or CHO cells are shown. Endogenous alpha 5beta 1 in K562 cells and endogenous hamster uPAR in CHO cells (53) may explain the background binding of K562 cells to CHO cells. We obtained essentially the same results with K562 cells expressing recombinant alpha 9beta 1 and alpha vbeta 3 (designated alpha 9- and alpha vbeta 3-K562 cells, respectively) and cloned HuPAR-CHO cells (Fig. 6, B and C). Y9A2 and 7E3 blocked binding of alpha 9- and alpha vbeta 3-K562 cells to cloned HuPAR-CHO cells, respectively, suggesting that these interactions are specific to the respective integrins. These results suggest that GPI-anchored uPAR specifically interacts with several integrins on the apposing cells and supports cell-cell interaction.



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Fig. 6.   uPAR-mediated cell-cell interaction. A, fluorescence-labeled parent K562 cells or K562 cells expressing recombinant human alpha 4 (alpha 4-K562) were incubated with a monolayer of CHO cells expressing human uPAR (HuPAR-CHO) or CHO cells in RPMI 1640 medium. Labeled alpha 4-K562 cells were incubated with HuPAR-CHO cells in the presence of mAbs against integrin alpha 4 (SG73). The HuPAR cells used were not clonal (~50% of the population was positive in human uPAR expression). B and C, fluorescence-labeled parent K562 cells or K562 cells expressing recombinant human alpha 9 (alpha 9-K562) (B) or alpha vbeta 3 (alpha vbeta 3-K562) (C) were incubated with a monolayer of HuPAR-CHO or CHO cells in Hepes-Tyrode buffer supplemented with 2 mM Mg2+. Labeled alpha 9- or alpha vbeta 3-K562 cells were incubated with HuPAR-CHO cells in the presence of mAbs (Y9A2 to alpha 9, or 7E3 to beta 3), respectively. HuPAR-CHO cells used in B and C are clonal. Very similar results were obtained with parent or mock-transfected CHO or K562 cells in A, B, and C.



    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

uPAR as a Ligand for Several Non-I Domain Integrins-- The present study for the first time establishes that uPAR is a ligand for several beta 1 and beta 3 integrins. We demonstrated that 1) these integrins adhere to suPAR D2D3 in a dose-dependent and cation-dependent manner and 2) specific antibodies against these integrins or uPAR block these interactions. Another structurally related protein, alpha -bungarotoxin, showed only weak integrin binding relative to suPAR D2D3, suggesting that binding to integrins is a specific property of uPAR. We also demonstrated that the uPAR-binding sites in alpha 4beta 1 and alpha vbeta 3 overlap with the previously reported putative ligand-binding site in these integrins. This is based on the observations that 3) soluble ligands for alpha 4beta 1 and alpha vbeta 3 compete with suPAR D2D3 for binding to these integrins and 4) the known integrin mutations that block ligand binding to alpha 4beta 1 and alpha vbeta 3 block binding of suPAR D2D3 to these integrins as well. These results suggest that uPAR may directly compete with other ligands for binding to these integrins, rather than only operating indirectly by regulating the binding affinity of integrins to other ligands as an integrin-associated protein. These unexpected results are consistent with the observation that soluble uPAR blocks fibronectin binding to alpha 5beta 1 (17) but do not necessarily agree with the current view that uPAR interacts with integrins exclusively as an associated protein rather than as a ligand (reviewed in Ref. 1). Integrin specificity experiments were performed using suPAR D2D3. It is possible that if we had used suPAR D1 or full-length suPAR (D1D2D3), differences in integrin specificity or avidity would have become apparent. Although all of the recombinant suPAR fragments used supported the alpha 4beta 1- and alpha vbeta 3-mediated cell adhesion to the same extent, it is not yet conclusive whether each of the domains contains similar or distinct integrin-binding sites. Detailed binding kinetic studies using individual soluble domains would be required to address this question. Also, there are known differences in uPAR glycosylation among mammalian cells and between some mammalian tumor cells and S2 cells. It would be interesting to study the influence of glycosylation, if any, on avidity or specificity in future experiments.

We also demonstrated that uPAR may mediate cell-cell adhesion by trans-interaction with integrins (Figs. 6 and 7B). This broadens the currently held concept of uPAR-integrin interactions, in which uPAR is proposed to interact exclusively with integrins residing on the same cell (cis-interaction) as an "associated protein" that mediates signal transduction directly or through the mediation of a distinct transmembrane adapter protein. Thus, the current model holds that all of the molecules that are involved in the uPAR signaling pathway are located on the same cell surface as uPAR (Fig. 7A) (reviewed in Refs. 1 and 54). The present results do not rule out such uPAR-integrin cis-interactions. However, the present results predict that if uPAR interacts with integrins on the same cell surface (cis-interaction), uPAR may bind as a ligand rather than as an associated protein (Fig. 7C) and that signal transduction events are mediated through the engaged integrin.



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Fig. 7.   Models of interaction between uPAR and integrins. The current view of uPAR signaling is based on association of uPAR with integrins in-cis as an associated protein (A). We have shown that uPAR may interact with integrins in-trans (B). If uPAR interacts with integrins in-cis, uPAR may bind to the ligand-binding site of integrins (C). It has been shown that soluble uPAR may bind to integrins (D). The present study suggests that uPAR is a ligand but not an associate protein of integrins, suggesting that uPAR (GPI-anchored or soluble) binding to integrins may induce signal transduction through integrin pathways.

It has been reported that association of uPAR with integrins induces tyrosine phosphorylation of focal adhesion kinase, paxillin, p130(cas), and mitogen-activated protein kinase (13). These signaling molecules have also been identified as being involved in signaling through integrins. If uPAR binds to integrins as a ligand, it is not difficult to imagine that GPI-anchored uPAR may transduce signals through the integrin pathway without the help of an additional putative transmembrane adapter molecule. Phosphorylation of these proteins may be induced through integrins on uPAR-integrin trans-interaction. The trans-interaction model (Fig. 7B) predicts that tyrosine phosphorylation of proteins and the resulting induction of gene expression occur in the cells that have integrin receptors but not in the cells in which uPAR alone is expressed. In uPAR-integrin-mediated cell-cell interaction, both of the apposing cells may express uPAR and integrins, and uPAR-integrin interaction may occur reciprocally; thus tyrosine phosphorylation might occur on both sides.

Implication of uPAR as an Integrin Ligand in Pathological Situations-- It has been reported that down-regulation of uPAR makes a human epidermoid carcinoma Hep3 dormant in vivo (6, 7) as a result of a reduced proliferation rate rather than an increased apoptotic rate (7). It has been proposed that reduction of uPAR expression makes cells either incapable of responding to a signal, or unable to generate a sufficient signal, to propel them through G0/G1 in vivo (10). However, cells expressing less than 50% of the normal level of uPAR grow indistinguishably from parental cells in culture (10). It is unclear why the altered uPAR expression level affects the proliferation of cancer cells in vivo but not in culture. We suspect that uPAR-integrin trans-interaction, which occurs during three-dimensional growth in tumor mass in vivo but may not occur in culture, may contribute to this discrepancy. Trans-interactions between uPAR and integrins as described in this study have the potential to transduce proliferative signals within tumor masses through integrin-dependent pathways. It is possible that uPAR may also function as a ligand for integrin through cis-interactions (Fig. 7C), thereby providing autocrine-type proliferative signals. If this is the case, cancer cells that express high levels of uPAR may have the potential to grow faster than cells that express uPAR at lower levels.

Soluble uPAR is present in ascites of ovarian cancer patients (55). Soluble uPAR levels in plasma increase in patients with rheumatoid arthritis (56), ovarian cancer (57), and leukemia (58). Soluble uPAR levels correlate with resistance to chemotherapy in leukemia (58) and with tumor volume in animal models (59). It has also been shown that soluble uPAR induces extracellular regulated kinase activation when added to uPAR-low Hep3 cells (10). The present study suggests that soluble uPAR might also interact as a ligand with integrins on leukemic cells, solid tumor cells, or inflammatory leukocytes and transduce proliferative signals (Fig. 7D). Thus binding of GPI-anchored uPAR (trans-interaction) or soluble uPAR to integrins as a ligand is a potential therapeutic target in cancer, inflammation, or other pathological situations.

It has been reported that leukocyte rolling and adhesion dramatically increase in mesenteric postcapillary vessels in adjuvant-induced chronic vasculitis in rats (60). An anti-integrin alpha 4 antibody significantly blocks this leukocyte rolling and adhesion. Interestingly, this alpha 4-dependent interaction is not dependent on VCAM-1 or the CS-1 region of fibronectin. It has also been reported that platelet and endothelial uPAR is involved in the survival of platelets in the circulation in mice (61). Injection of tumor necrosis factor increases the number of platelets in the lung alveolar capillaries in wild-type mice, but platelet trapping is insignificant in mice deficient in uPAR. uPAR may be a candidate integrin ligand in these cases. It is possible that uPAR-integrin trans-interaction may be involved in these leukocyte-endothelium and platelet-endothelium interactions during inflammation.

In summary, we have shown that uPAR is a ligand for several integrins and mediates cell-cell adhesion through trans-interactions with these integrins. These findings also help to clarify how uPAR binding to integrins as a ligand transduces signals through integrin pathways. In addition, the present findings predict that interaction of uPAR with integrin as a ligand (cis or trans) may be involved in transduction of proliferative or activating signals in cancer and inflammatory cells. Additional studies will be required to clarify the role of uPAR-integrin interactions in these processes.


    ACKNOWLEDGEMENTS

We thank E. Brown, W. G. Carter, B. S. Coller, M. H. Ginsberg, R. L. Juliano, S. J. Kennel, L. A. Miles, K. Miyake, D. Sheppard, R. F. Todd III, and E. Wayner for valuable reagents. We also thank A. Kuo for assistance in preparing suPAR and W. Puzon-McLaughlin for assistance in preparing HuPAR-CHO cells.


    FOOTNOTES

* This work was supported by National Institutes of Health Grants GM47175 and GM49899 (to Y. T.) and HL60169 (to D. C.). This is publication 13186-VB from The Scripps Research Institute.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: Dept. of Vascular Biology, CAL-10, The Scripps Research Institute, 10550 N. Torrey Pines Rd., La Jolla, CA 92037. Tel.: 858-784-7636; Fax: 858-784-7645; E-mail: takada@scripps.edu.

Published, JBC Papers in Press, October 26, 2000, DOI 10.1074/jbc.M008220200


    ABBREVIATIONS

The abbreviations used are: uPA, urokinase-type plasminogen activator; uPAR, uPA receptor; GPI, glycosyl phosphatidylinositol; suPAR, soluble uPAR; mAb, monoclonal antibody; VCAM-1, vascular cellular adhesion molecule-1; CHO, Chinese hamster ovary; HPLC, high pressure liquid chromatography; HuPAR, human uPAR; CS-1, connecting segment-1.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES


1. Chapman, H., Wei, Y., Simon, D., and Waltz, D. (1999) Thromb. Haemostasis 82, 291-297[Medline] [Order article via Infotrieve]
2. Blasi, F. (1999) Thromb. Haemostasis 82, 298-304[Medline] [Order article via Infotrieve]
3. Mazar, A., Henkin, J., and Goldfarb, H. (1999) Angiogenesis 3, 15-32[CrossRef]
4. Crowley, C. W., Cohen, R. L., Lucas, B. K., Liu, G., Shuman, M. A., and Levinson, A. D. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 5021-5025[Abstract]
5. Min, H. Y., Doyle, L. V., Vitt, C. R., Zandonella, C. L., Stratton-Thomas, J. R., Shuman, M. A., and Rosenberg, S. (1996) Cancer Res. 56, 2428-2433[Abstract]
6. Kook, Y. H., Adamski, J., Zelent, A., and Ossowski, L. (1994) EMBO J. 13, 3983-3991[Abstract]
7. Yu, W., Kim, J., and Ossowski, L. (1997) J. Cell Biol. 137, 767-777[Abstract/Free Full Text]
8. Wei, Y., Yang, X., Liu, Q., Wilkins, J. A., and Chapman, H. A. (1999) J. Cell Biol. 144, 1285-1294[Abstract/Free Full Text]
9. Yebra, M., Goretzki, L., Pfeifer, M., and Mueller, B. M. (1999) Exp. Cell Res. 250, 231-240[CrossRef][Medline] [Order article via Infotrieve]
10. Aguirre Ghiso, J. A., Kovalski, K., and Ossowski, L. (1999) J. Cell Biol. 147, 89-104[Abstract/Free Full Text]
11. Konakova, M., Hucho, F., and Schleuning, W. D. (1998) Eur. J. Biochem. 253, 421-429[Abstract]
12. Nguyen, D. H., Hussaini, I. M., and Gonias, S. L. (1998) J. Biol. Chem. 273, 8502-8507[Abstract/Free Full Text]
13. Tang, H., Kerins, D. M., Hao, Q., Inagami, T., and Vaughan, D. E. (1998) J. Biol. Chem. 273, 18268-18272[Abstract/Free Full Text]
14. Resnati, M., Guttinger, M., Valcamonica, S., Sidenius, N., Blasi, F., and Fazioli, F. (1996) EMBO J. 15, 1572-1582[Abstract]
15. Xue, W., Kindzelskii, A. L., Todd, R. F., III, and Petty, H. R. (1994) J. Immunol. 152, 4630-4640[Abstract/Free Full Text]
16. Xue, W., Mizukami, I., Todd, R. F., III, and Petty, H. R. (1997) Cancer Res. 57, 1682-1689[Abstract]
17. Wei, Y., Lukashev, M., Simon, D. I., Bodary, S. C., Rosenberg, S., Doyle, M. V., and Chapman, H. A. (1996) Science 273, 1551-1555[Abstract]
18. Wary, K. K., Mainiero, F., Isakoff, S. J., Marcantonio, E. E., and Giancotti, F. G. (1996) Cell 87, 733-743[Medline] [Order article via Infotrieve]
19. Wary, K. K., Mariotti, A., Zurzolo, C., and Giancotti, F. G. (1998) Cell 94, 625-634[Medline] [Order article via Infotrieve]
20. Mainiero, F., Murgia, C., Wary, K. K., Curatola, A. M., Pepe, A., Blumemberg, M., Westwick, J. K., Der, C. J., and Giancotti, F. G. (1997) EMBO J. 16, 2365-2375[Abstract/Free Full Text]
21. Miyamoto, S., Teramoto, H., Gutkind, J. S., and Yamada, K. M. (1996) J. Cell Biol. 135, 1633-1642[Abstract]
22. Yamada, K. M., and Geiger, B. (1997) Curr. Opin. Cell Biol. 9, 76-85[CrossRef][Medline] [Order article via Infotrieve]
23. Hemler, M. E., Sanchez-Madrid, F., Flotte, T. J., Krensky, A. M., Burakoff, S. J., Bhan, A. K., Springer, T. A., and Strominger, J. L. (1984) J. Immunol. 132, 3011-3018[Abstract/Free Full Text]
24. Hall, D. E., Reichardt, L. F., Crowley, B., Holley, B., Moezzi, H., Sonnenberg, A., and Damsky, C. H. (1990) J. Cell Biol. 110, 2175-2184[Abstract]
25. Wayner, E. A., and Carter, W. G. (1987) J. Cell Biol. 105, 1873-1884[Abstract]
26. Miyake, K., Hasunuma, Y., Yagita, H., and Kimoto, M. (1992) J. Cell Biol. 119, 653-662[Abstract]
27. Falcioni, R., Sacchi, A., Resau, J., and Kennel, S. (1988) Cancer Res. 48, 816-821[Abstract]
28. Wang, A., Yokosaki, Y., Ferrando, R., Balmes, J., and Sheppard, D. (1996) Am. J. Respir. Cell Mol. Biol. 15, 664-672[Abstract]
29. Coller, B. S. (1985) J. Clin. Invest. 76, 101-108[Medline] [Order article via Infotrieve]
30. Min, H. Y., Semnani, R., Mizukami, I. F., Watt, K., Todd, R. F. d., and Liu, D. Y. (1992) J. Immunol. 148, 3636-3642[Abstract/Free Full Text]
31. Ploug, M., Rahbek-Nielsen, H., Nielsen, P. F., Roepstorff, P., and Dano, K. (1998) J. Biol. Chem. 273, 13933-13943[Abstract/Free Full Text]
32. Schreiner, C. L., Bauer, J. S., Danilov, Y. N., Hussein, S., Sczekan, M., and Juliano, R. L. (1989) J. Cell Biol. 109, 3157-3167[Abstract]
33. Yokosaki, Y., Palmer, E., Prieto, A., Crossin, K., Bourdon, M., Pytela, R., and Sheppard, D. (1994) J. Biol. Chem. 269, 26691-26696[Abstract/Free Full Text]
34. Zhang, X. P., Kamata, T., Yokoyama, K., Puzon-McLaughlin, W., and Takada, Y. (1998) J. Biol. Chem. 273, 7345-7350[Abstract/Free Full Text]
35. Matsuura, N., Puzon-McLaughlin, W., Irie, A., Morikawa, Y., Kakudo, K., and Takada, Y. (1996) Am. J. Pathol. 148, 55-61[Abstract]
36. Eto, K., Puzon-McLaughlin, W., Sheppard, D., Sehara-Fujisawa, A., Zhang, X. P., and Takada, Y. (2000) J. Biol. Chem. 275, 34922-34930[Abstract/Free Full Text]
37. Blystone, S., Graham, I., Lindberg, F., and Brown, E. (1994) J. Cell Biol. 127, 1129-1137[Abstract]
38. Gum, R., Juarez, J., Allgayer, H., Mazar, A., Wang, Y., and Boyd, D. (1998) Oncogene 17, 213-225[CrossRef][Medline] [Order article via Infotrieve]
39. Prater, C. A., Plotkin, J., Jaye, D., and Frazier, W. A. (1991) J. Cell Biol. 112, 1031-1040[Abstract]
40. Deng, W. P., and Nickoloff, J. A. (1992) Anal. Biochem. 200, 81-88[Medline] [Order article via Infotrieve]
41. Takada, Y., and Puzon, W. (1993) J. Biol. Chem. 268, 17597-17601[Abstract/Free Full Text]
42. Takada, Y., Ylanne, J., Mandelman, D., Puzon, W., and Ginsberg, M. (1992) J. Cell Biol. 119, 913-921[Abstract]
43. Higazi, A. A. R., Mazar, A., Wang, J., Quan, N., Griffin, R., Reilly, R., Henkin, J., and Cines, D. B. (1997) J. Biol. Chem. 272, 5348-5353[Abstract/Free Full Text]
44. Tsetlin, V. (1999) Eur. J. Biochem. 264, 281-286[Abstract/Free Full Text]
45. Giancotti, F. G., and Ruoslahti, E. (1999) Science 285, 1028-1032[Abstract/Free Full Text]
46. Takada, Y., Kamata, T., Irie, A., Puzon-McLaughlin, W., and Zhang, X.-P. (1997) Matrix Biol. 16, 143-151[Medline] [Order article via Infotrieve]
47. Irie, A., Kamata, T., Puzon-McLaughlin, W., and Takada, Y. (1995) EMBO J. 14, 5542-5549[Abstract]
48. Kamata, T., Irie, A., and Takada, Y. (1996) J. Biol. Chem. 271, 18610-18615[Abstract/Free Full Text]
49. Kamata, T., Puzon, W., and Takada, Y. (1995) Biochem. J. 305, 945-951[Medline] [Order article via Infotrieve]
50. Bajt, M., and Loftus, J. (1994) J. Biol. Chem. 269, 20913-20919[Abstract/Free Full Text]
51. Loftus, J. C., O'Toole, T. E., Plow, E. F., Glass, A., Frelinger, A. L., and Ginsberg, M. H. (1990) Science 249, 915-918[Medline] [Order article via Infotrieve]
52. Huang, X., Chen, A., Agrez, M., and Sheppard, D. (1995) Am. J. Respir. Cell Mol. Biol. 13, 245-251[Abstract]
53. Fowler, B., Mackman, N., Parmer, R. J., and Miles, L. A. (1998) Thromb. Haemostasis 80, 148-154[Medline] [Order article via Infotrieve]
54. Ghosh, S., Brown, R., Jones, J. C., Ellerbroek, S. M., and Stack, M. S. (2000) J. Biol. Chem. 275, 23869-23876[Abstract/Free Full Text]
55. Pedersen, N., Schmitt, M., Ronne, E., Nicoletti, M. I., Hoyer-Hansen, G., Conese, M., Giavazzi, R., Dano, K., Kuhn, W., Janicke, F., and Blasi, F. (1993) J. Clin. Invest. 92, 2160-2167[Medline] [Order article via Infotrieve]
56. Slot, O., Brunner, N., Locht, H., Oxholm, P., and Stephens, R. W. (1999) Ann. Rheum. Dis. 58, 488-492[Abstract/Free Full Text]
57. Sier, C. F., Stephens, R., Bizik, J., Mariani, A., Bassan, M., Pedersen, N., Frigerio, L., Ferrari, A., Dano, K., Brunner, N., and Blasi, F. (1998) Cancer Res. 58, 1843-1849[Abstract]
58. Mustjoki, S., Alitalo, R., Stephens, R. W., and Vaheri, A. (1999) Thromb. Haemostasis 81, 705-710[Medline] [Order article via Infotrieve]
59. Holst-Hansen, C., Hamers, M. J., Johannessen, B. E., Brunner, N., and Stephens, R. W. (1999) Br. J. Cancer 81, 203-211[CrossRef][Medline] [Order article via Infotrieve]
60. Johnston, B., Chee, A., Issekutz, T. B., Ugarova, T., Fox-Robichaud, A., Hickey, M. J., and Kubes, P. (2000) J. Immunol. 164, 3337-3344[Abstract/Free Full Text]
61. Piguet, P. F., Vesin, C., Donati, Y., Tacchini-Cottier, F., Belin, D., and Barazzone, C. (1999) Circulation 99, 3315-3321[Abstract/Free Full Text]


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