Identification of the urokinase receptor as an adhesion receptor for ...

THE J O U ~OFAB L I O ~ I CCHEMISTRY AL

Vol. 269,No. 51, Issue of December 23, pp. 3238s32388, 1994 Printed in U.S.A.

0 1994 by The American Society for Biochemistry and Molecular Biology, Inc

Identification of the Urokinase Receptor asan Adhesion Receptorfor Vitronectin” (Received forpublication, August 23, 1994, and in revised form, October 13, 1994)

Ying Weil, DavidA. Waltz§, Navaneetha Raon, Robert J. Drummondll, Steven Rosenbergll, and Harold A. Chapman+** From the $Departments of Medicine, Brigham and Women’s Hospital and BChildrenk Hospital, Haruard Medical School, the mivision of Biological Sciences, PhysiologyProgram, Harvard School of Public Health, Boston, Massachusetts 02115, and the IlChiron Corporation, Emeryville, California 94608

which it becomes stronglyadhesive for cells and for proUrokinase receptors, expressed on surfaces of many teinswithheparin-like motifs (Preissner and Jenne, 1991; cell types, focus to the pericellular space plasminogendependent proteolysis importantin matrix remodeling Stockmann etal., 1993; Bittorfet al., 1993). Cellsare known to and cell movement. We now report that the urokinase interact with suchvitronectinthroughsurface receptors of receptor ( u P A R ) is also a high affinity (K, .c 30 m)re- the integrin family, especially those containing the a v chain ceptor for vitronectin. Recombinant uPAR binds (Felding-Habermann and Cheresh,1993). The altered configuvitronectin in the absence of urokinase, but vitronectin ration presumably predominatesin matrix-deposited vitronecof tin; this configuration is promoted by adsorption topolystyrene binding is promoted by concurrent receptor binding eitherurokinaseorfragmentsthereofcontaining its (promoting the spreading and growth of cells cultured invitro) UPAR binding domain. Stable epithelial cell transfecas well as by denaturants such as urea and heat.A connection tantsexpressingmembrane-anchored U P A R , butnot between cellularuPA and vitronectinhas been established precells expressing soluble UP- become strongly adheviously as vitronectin canbe shown t o redistribute cell-associsive with altered morphology in the absence of urokiated uPA and to co-localize with uPA in focal contacts of cells nase. These observations identify a new class of (Ciambrone and McKeown-Longo, 1992). The molecular basis vitronectin receptor and imply a duality in function for the receptor that intrinsically links matrix adhesion tofor these observations, however, are currently unknown. Increases in cellularuPA activity have long been associated regulation of protease activity. Increases in urokinase with cellular activation, malignant transformation, and biologreceptor expressionk n o w n to be associated with celluical events characterizedby cellular movement, e.g. ovulation, laractivationandmalignanttransformationcould tumor cell metastasis, angiogenesis, and smooth muscle cell modulate cellular trafficking and function by promoting and monocytic cell migration (Reich, 1978; Blasi et al., 1987; attachment to vitronectin. Mignatti and Rifkin, 1993). More recently, cellular expression of urokinase receptorsperse have been strongly correlated with some of these same phenomena(Schlechte et al., 1989; Pepper Vitronectin is a circulating plasma protein with multiple et al., 1993). Therealization that cells exhibit cell-surface binding properties (Preissner and Jenne, 1991; Tomasini and Mosher, 1990). It is alsofound associated with cell surfaces and urokinase activity and that it occurs via binding of uPA to in loose connective tissue of many organs (Reilly and Nash, specific receptors provided an apparent explanation for these 1988). Vitronectin accumulates prominently in atherosclerotic strong associations: concentration of urokinase at the cell surface by its receptors could initiate a pericellular protease sysplaques and in extracellular matrices associated with acute tem capable of degrading extracellular matrix proteins and injury and repair and several malignant tumors (Niculescu et al., 1989; Loridon-Rosa et al., 1988; Gladson and Cheresh, thereby enhancing migration potentialand tissue remodeling 1991). In its circulating, native configuration vitronectin func- (Chapman et al., 1982; Vassalliet al., 1985). Reports that urokitions as the major binding protein of plasminogen activator nase receptors localize to sites of focal contacts in fibroblasts inhibitor type-1 (PAI-l),l the physiological inhibitor of tissue and to the leading edge of migrating monocytes support this plasminogen activator and urokinase (uPA) (Salonen et al., hypothesis (Estreicher et al., 1990; Pollanen et al., 1987). This 1989). Vitronectin also exists in an altered configuration in paradigm has been further supportedby evidence that urokinase might participate in movement and tissue remodeling indirectly through proteolytic activation of matrix metallopro* This work was supported by National Institutes of Health Grant HL44712 and grants from the Charles H. Hood Foundation (Boston, teases and cytokines (Blasi et al., 1987; Paranjpe et al., 1980; MA). The costsof publication of this article were defrayed in part by the Falcone et al., 1993). However, cellular activation and maligpayment of page charges. This article must therefore be hereby marked nant transformation are also characterizedby concurrent up“advertisement” in accordance with 18 U.S.C. Section 1734 solely to regulation of PAT-1 (Loskutoe et ai., 1989; Sumiyoshi et al., indicate this fact. 1991; Pedersen et al., 1994). Several metastatic tumor sites **To whom correspondence and reprint requests should be addressed: Respiratory Division, Brigham and Women’s Hospital, Thorn have beenobserved to containmore PAI-1than thecorrespondResearchBldg., Rm. 703, 20 Shattuck St., Boston, MA 02115. Tel.: ing primary tumors, andPAI-1 expression has been correlated 617-732-6074; Fax: 617-232-4623. et al., 1992; Jankun et al., The abbreviations used are: PAI-1, plasminogen activator inhibitor inversely with cancer survival (Sier 1993). Similarly, sites of angiogenesis and inflammation are type 1;ATF, amino-terminal fragment of urokinase; GFD, growth factor domain of urokinase; GPI, glycosylphosphatidylinositol; PI, phosphoi- characterized by marked PAI-1 accumulation (Bacharach et al., nositol-specific;uPA, urokinase plasminogen activator; uPAR,uroki1992; Bertozzi et al., 1990). The importanceof urokinase activnase receptor; s-uPAR, soluble form of uPm,VTN or V N , vitronectin; ity in embryogenesis and cell migration has been further quesPCR, polymerase chain reaction; DMEM, Dulbecco’s modified Eagle’s medium; PES, phosphate-buffered saline; BSA, bovine serum albumin; tioned by observations that deletion of the murine urokinase gene by homologous recombination does not produce a clearly wt, wild type.

32380

Urokinase Receptor Binds Vitronectin

32381

in serine for threonine and serine for asparagine transversions, respectively. For transfection, 293 cells were harvested in midlog phase, -4 million cells/ml,and washed once with DMEM before being resuspended in 0.5 mlof cold DMEM. DNAwas linearized with ClaI endonuclease,and -10 pg ofDNA was added and cells were electroporated using the Invitrogen ElectroPorator at 250 V and 250 pF.After 10min on ice,the cells were transferred to 30 ml of 37 "C prewarmed DMEM containing 10%fetal bovine serum. The transfected cells were selected as a mixture of transfectants with DMEM containing 10% fetal calf serum and 50 pg/rnl hygromycin (Sigma) 2 days after transfection. Cell Lines and Culture Conditions-The 293 cells were cultured in DMEM supplemented with penicillin(100 units/ml), streptomycin (100 and 10% FBS. pg/ml), HEPES (10 mM,pH 7.4), glutamine (5 m), Receptor-transfected 293cellswere maintained in DMEMcomplete medium containing 50 pg/ml hygromycin. Incubations were at 37 "C, 5% CO,. To collect receptor-enriched conditioned media, cellswere grown subconfluent, washed twice with serum-free DMEM, and cultured in the same medium overnight (-10 mV10 million cells). The conditioned medium was harvested, aliquoted, and frozen at -80 "C. For metabolic labeling, subconfluent cells were washedand starved in methionine-free DMEM for 30 min before 150 pCi/ml [35Slmeth~onine was added. The culture medium (4 ml) was supplemented with 5% complete DMEM after 2 h and incubated for another 12 h. The labeled conditioned medium was collected, aliquoted, and stored a t -80 "C. Baculovirus Expression of uPAR-Truncated, soluble human urokinase receptor was also expressed in Sf9 insect cells using a recombinant baculovirus as described (Goodson et al., 1994). DNA sequencing verified the correct uPAR sequence (Roldan et al., 1990). The protein was purified from the conditioned medium by conventional methods or by affinity chromatography on a column of recombinant human urokinase growth factor-like domain (residues 1-48). The purified protein was biotinylated as described previously (Kaufman et al., 1993) and separated from unmodified material on Soft-Avidin (Pierce). Domains 1and 2/3 of uPAR were prepared by limited proteolysis of purified baculovirus-derivedreceptor with trace amounts of chymotrypsin asdescribed previously(Behrendt et al., 1991). Domainswere then purified by size-exclusion chromatography (Superose 12, Pharmacia Biotech Inc.) using PBS as eluant. The eluant was monitored forabsorbance at 280 nm, and aliquots of peak fractions were analyzed by SDSEXPERIMENTALPROCEDURES PAGE. Selective poolingof fractions resulted in preparations of domain Reagents-Prourokinase, two-chain active urokinase, ATF (residues 1(approximately9000 Da) and domain 2 + 3 (approximately28,000 Da), 1-143) and GFD (residues 4-43) were the kind gifts of Dr. Jack Henkin approximately 90% pure, containing no detectable uncleaved s-uPAR (Abbott Laboratories). Phosphoinositol-specific phospholipase C (from and less than 10% cross-contamination of the respective domain. Bacillus cereus) was obtained from Sigma. Urokinase was inactivated Binding and Adhesion Assays-Untransfected 293 cells (10') or cells by treatment with diisopropyl fluorophosphate as described (Waltz et transfected with uPARwere seeded in 96-well tissue culture plates al., 1993). Vitronectin was purified from human plasma by heparin (Becton Dickinson Labware) and cultured in DMEM overnight. Cells affinity chromatography in the presence of urea (Yatohgo et al., 1988). were washed with binding buffer (DMEM,1mg/ml BSA)and incubated Native plasma vitronectin was purified as described by Mosher (Bittorf with binding buffer containing 1n~ '251-uPA(or 5 n~ '251-VTN) for1.5 h et al., 1993) and was a kind gift ofDr. Deane Mosher, Madison, WI. a t 4 "C in a totalvolume of 100 pl. After three washes with PBS, cells Urokinase and urea-purified vitronectin were iodinated by a modifica- were lysed in 0.2% SDS, 0.2% Triton X-100, 10%glycerol in PBS for 30 tion of the chloramine T method (Waltz et al., 1993).Fibrinogen and the min at 4 "C, and radioactivity was counted by a y counter. Binding of peptides GRGDSP and GRGESP were from Peninsula Laboratories Inc. VTN to phorbol 12-myristate 13-acetate-stimulated U937 cells was perMurine monoclonal anti-uPA and monoclonal mouse anti-PAI-1 anti- formed as described (Nusrat and Chapman, 1991). Nonspecificbinding body were from American Diagnostica (Greenwich, CT). Monoclonal was determined by inclusion of a 100-fold molar excess of unlabeled uPA antibodies R2 and R4, directed to domains 2/3 ofuPAR (Ronne et al., (or VTN) in the reaction mixture. 1991), were kindly provided byDr. Ebbe Ronne, Finsen Laboratory For determination of the function of soluble GAR,Nunc-Immuno (Copenhagen, Denmark). Monoclonal antibody MA13, directed to plates with 96 flat-bottomed wells (Marsh Biomedical Products, domain 1 of uPAR, wasprovided byDr. W. Knapp, University of Rochester, N Y ) were used. Pro-uPA, 10 pg/ml, in PBS, pH 6.8, were Vienna (Vienna, Austria). Monoclonal antibody CD14 was from SCT pipetted into each well. Plates were incubated at 37 "C overnight, and (Bethlehem, PA). Goat anti-rabbit lz5I-IgG,lZ6I-sodium,~-[~'S]methi- then residual binding sites were blocked with 200 pl of 1%BSA in PBS onine, and C Z - [ ~ ~ P ] ~were A T Pfrom DuPont NEN. for 1 h at room temperature. 35S-Labeled conditioned medium from uPA Receptor ExpressionPlasmids and Cell Dansfection-A cDNA of untransfected or transfected 293 cells was thawed on ice and placed into uPAR containing the full coding sequence was isolated from human coated wells.The plates were kept at 4 "C for 1h and then processed as macrophages by reverse transcription and polymerase chain reaction described above. In some experiments, bound radioactivity was eluted (PCR), as described previously (Shi et al., 1992). Primers used for am- either into acidic buffer(50 mM glycine, pH3.2) and then neutralized for plification spanned nucleotide base pairs 2 7 4 9 and 1069-1090 (Roldan subsequent experiments or directly into reduced sample buffer for SDSet al., 1990).A soluble form of uPAR was obtained by inserting a stop PAGE and autoradiography.Plates were coatedwith 5 pg/ml vitronectin codon after nucleotide 943 of the uPAR cDNA by PCR. Both PCR prod- or soluble uPAR expressed in baculovirus and analyzed as above. ucts were subcloned into pBluescript II(KS-). The expression plasmids To measure binding of biotinylated baculovirus s-uPAR, vitronectinwere constructed by inserting the XbaI-XhoI fragments from the sub- coated plates were exposed tobiotinylated baculovirus s-uPAR (diluted clones containing wild-type or soluble uPAR into NheI and XhoI sites of in PBS, 1mg/ml BSA)and washed with PBS containing 0.1%Tween 20, the vector pCEP4(Invitrogen), respectively. Bothreceptor cDNAs were and then peroxidase-labeledavidin was added (1mg/ml initial concenfullysequenced by double-stranded DNA sequencing using dideoxy tration, diluted 1:5000 in PBS, 10 mg/ml BSA). After additional washchain termination methodology (Sequenase, United States Biochemical ings, peroxidase substrate wasadded (TMB Peroxidase Substrates, Cow.). Sequencing of the soluble construct revealed two PCR-induced Kirkegaard and Perry Laboratories, Inc., Gaithersburg, MD) and alsingle base pairsubstitutions at positions 249 and 669 resulting lowed to react for 3 min. The reactions were terminated with l M H,SO,,

altered phenotype (Carmeliet et al., 1994). Taken together, these observations challenge the premise that excess cell surface urokinase activity alone underlies the strong associations between UPNUPARand the processes alluded to above. Employing modelsof human monocyte/macrophage differentiation, we have previously provided evidence that urokinase receptor binding per se, independent of enzymatic activity, modifies cellular function (Nusrat and Chapman, 1991; Waltz et al.,1993). Human ieukemic suspension cells, stimulated in vitro with vitamin D3, transforming growth factor type 01, and other cytokines, exhibit up-regulated urokinase receptor number and become strongly adhesive in the presence of uPA. Adhesion is induced not only by binding of active urokinase but also by diisopropyl fluorophosphate-inactivated enzyme and fragments of the urokinase molecule comprised simply of its receptor binding domain. Recently, we have reported that stimulated myelomonocyticcellsexposed to urokinase bind vitronectin and more specifically altered vitronectin, i.e. the matrix-like form, but not native plasma vitronectin (Waltz and Chapman, 1994).Urokinase receptor occupancy induced a high affinity, RGD- and EDTA-independentreceptoractil-ity for urea-purified vitronectin. Because of the direct parallel between the extent of urokinase binding to its receptor and increases in vitronectin binding, we questioned whether the urokinase receptor itself might contain a latent vitronectin binding site. In this reportwe provide evidencethat theurokinase receptor is also a high affinity receptor for the matrix-like form of vitronectin, and this function can be regulated by concurrent uPA receptor binding. As PAI-1 promotes turnover of uPA from its receptor and vitronectin is themajor plasma and matrix binding protein of PAI-1, these observations suggest a novel conceptualization for function of this receptor in fibrinolysis and cellular accumulation in tissues.

32382


FIG.1. Expression of human urokinsse receptor by transfected 293 cells. A, immunohlot of full-lenkc ~ l l m r n t , c’.,g. metastasis, c’vcn though this should surface proteolytic activity. Prior studicls show th:lt I ’ A I - I inhihition of receptor-hound uPA results in rapid intrrn:tliz:ltion of hound uPA.PAI-1 complexes ((’uhc.llis 1.t ( I / . , 1 9 9 0 1 . Intlwd. tvr h a v e o h s e r v e d t h a t t h r a d h r s i v c ~ n w sof myc.loid rrlls r x p r w s ing uPARis deprndcnt on continurd occupancyof ul’A :Ind t h u s down-rcgulatcd hy the prrscncr of I’A.1-1 I\Vnltz v t (11.. l 9 9 3 1 . Although the process o f intrrnalizatinn is not compl(btc.ly uncrlls this may occur fnllo\ving tr:lnsfcar of derstood. in somc uPA.PAI complexes from uPAR t o t h r Io\v tlrnsity lipoprotrin recc.ptor-related protc~inl(r-2-m:lcrnClot,ulinr c v p t o r 1 Kotlnn:Is (’1 nl., 199.7,. Vitronrctin is rrcognizrd as thc, majorhinding protein of PAI-I, and its hinding to uI’.AR could hr rspwtrtf t o hring PAI-1 in close approximation tvith uP:l. t h ( w h y p r o m o t ing inhihition and clearancr o f uI’A from t h e rrw.ptor. \f‘c post u l a t e t h a t t h i s proccss may rffrct R l o u r r avidity of rrllular attachment to vitronrctin. In this paradipn. P z l I - 1 . although decreasing uPA activity. wouldalso p r o m o t r d c ~ t a c h m r noft t h c cell from its contact site. Thus. R41-1in circumstancc~sLvhrrr sustained protrolytic activity is notvital t o movvmchnt could promote rather thanretard m i ~ ~ a t i o n . T h i s m oofd (rrpnll:ltion ~l o f the urokinasfl/vitron~~ctin rrccptor is d(*pictcd schrm:ltically surface localizationof urokinase-occupied uPAR to focal contact in Fig. 7 . Results of t h e w e x p c r i m c n t s also indicntr that undrr snmr sitesinfibroblasts(Pollanen ~t nl., 1987; Estreicher (’1 01.. intl(-pc.ntlvntly of conditionsuPARinteractstvithvitronwtin 1990). These contactsites are knowntoco-localizewith f r w o f uI1,.l, \vas nhvitronectin in adherent cell lines, and the presenceof vitronec- urokinase.RecornhinanthumanuPAR, tin has been shown to redistributecell surface uPAR receptors s e n d to hind vitronectin (Fig.4 R I. a n d rxpression of:lnchorrd uPAR in 293 cells conft.rrr.tl vitronclctin hinding potrnti:ll in thtb (Ciamhrone and McKeown-Longo, 1992). Second, these ohser-

Vitronectin BindsReceptor Urokinase INACTIVE

ACTIVE

other interactions

N

32387 lating tissue fibrinolysis and cellular accumulation in tissues, including that associated with atherogenesis and metastasis.

ACTIVE

-

Acknowledgments-We thank Drs. Deane Mosher, Ebbe Ronne, and Walter Knapp for the kind gifts of native vitronectin, R2 and R4, and MA13 monoclonal antibodies, respectively;Guo-Ping Shi for expressing a uPAR construct in FLAG vector; and Hui Xu and Lisa Natkin for technical support. We also thank Dr. Jack Henkin (AbbottLaboratories) for kindly providing uPA and fragments thereof used in this work.

uPA

REFERENCES

I

Altieri, D. C., and Edgington, T. S. (1988) J. Biol. Chem. 263, 7007-7015 Bacharach, E., Itin, A., and Keshet, E. (1992) Proc. Natl. Acad. Sei. U. S. A. 89, 10686-10690 Behrendt, N., Plough, M., Patthy, L., Houen, G., Blasi, F., and Dano, K. (1991) J. Biol. Chem. 266,7842-7847 Bertozzi, P., Astedt, B., Zenzius, L., Lynch,K., Lemaire, E , Zapol, W., and .. . .) 4..” uPA:PAI Chapman, H. A., Jr. (1990) N. Engl. J. Med. 322,890-897 LRP Bittorf, S. V., Williams, E. C., and Mosher, D. F. (1993) J. Biol. Chem. 268,24838N clearance 24846 Blasi, E , Vassalli, J. K , and Dano, K. (1987) J. Cell Biol. 104, 801-804 Bodary, S. C., and McLean, J. W. (1990) J. Biol. Chem. 266,5938-5941 uPAR:VN Bordier, C. (1981) J. Biol. Chem. 266, 1604-1607 Carmeliet, P., Schoonjans, L., Kieckens, L., Ream, B., Degen, J., Bronson, R., De clearance Vas, R., Van den Oord, J. J., Collen, D., and Mulligan, R. C. (1994)Nature 368, FIG.7. Model for regulation of the vitronectinbinding proper419424 ties of the urokinase receptor. The model illustrates that the uPA/ Chapman, H. A,, Vavrin, Z., and Hibbs, J. B. (1982) Cell 28,653-662 vitronectin receptor, uPAR, can exist in active and inactive configura- Chapman, H. A,, Bertozzi, P., Sailor, L. Z., and Nusrat,A. R. (1990)Am. J. Physiol. tions with respect to vitronectin binding, with both uPA-dependentand 269, L432438 uPA-independent pathways of activation (see “Discussion”).The fate of Ciambrone, G . J., and McKeown-Longo, P. J. (1992) J . Biol. Chem. 267, 1361713622 vitronectin.uPAR complexes, once formed, is unknown. The model in the figure depicts (with dotted lines) two likely outcomes: reversal of Cubellis, M. V., Wun, T.-C., and Blasi, F. (1990) EMBO J . 9, 1079-1085 M. L., Selvaraj, P., Mattaliano, R. J.,and Springer, T. A. (1987)Nature 329, vitronectin binding at thecell surface promoted by removal of receptor- Dustin, 846-848 bound uPA.PAI complexes via low density lipoprotein receptor-related Estreicher, A,, Muhlhauser, J., Carpentier, J. L., Orci, L., and Vassalli, J. D. (1990) protein (LRP) and, alternatively, clearance of the upAR.VN complex J . Cell Biol. 111, 783-792 from the cell surface by either endocytosis or shedding. Falcone, D. J., McCafiey, T. A., Haimovitz-Friedman, A,, and Garcia, M. (1993) J . Cell. Physiol. 155,595405 Faull, R. J., Kovach, N. L., Harlan, J. M., and Ginsberg, M. H. (1993) J. Cell Biol. 121, 155-162 absence of detectable uPA (Fig. 2). The Kd for vitronectin bind- Felding-Habermann, B., and Cheresh, D. A. (1993) Curr Opin. Cell Biol. 6, 864868 ing to thesecells was similar in the presence or absence of uPA Gladson, C. L., and Cheresh, D.A. (1991) J . Clin. Invest. 88, 1924-1932 (not shown), suggesting thatuPA stabilizes an active vitronec- Goodson, R.J., Doyle, M. V., Kaufman, S. E., and Rosenberg, S. (1994) Proc. Natl. Acad. Sci. U. S. A . 91,7129-7133 tin binding conformation rather than changing the innateafJ. J., Edelman, G. M., and Cunningham, B. A. (1986)Proc. Natl. Acad. finity of uPAR for vitronectin. Thus, as indicated in Fig. 7, we Hemperly, Sci. 5. A. 83, 9822-9826 suggest thatuPAR exists in either an active or inactive config- Hortsch, M., and Goodman, C. S. (1990)J. Biol. Chem. 265,15104-15109 uration with regard to vitronectin binding. Such active and Ishihara, A,, Hou, Y., and Jacobson, K. (1987) Proc. Natl. Acad. Sei. U. 5. A . 84, 1290-1293 inactive conformers within the integrin class of adhesion re- Jankun, J., Memck, H. W., and Goldblatt, P. J. (1993)J . Cell. Biochem. 63, 135144 ceptors are well described (Faull et al., 1993; Masumoto and Kaufman, S. E., Brown, S.,and Stauber,G . B. (1993)Anal. Biochem. 211,261-266 Hemler, 1993). In the case of uPAR, uPA appears to be a phys- Koretz, K., Moller, P., and Schwartz-Albiez, R. (1993) Eul: J. Cancer 29A, 1184iological activator of this vitronectin receptor. In addition, other 1189 soluble or membrane-associated proteins may stabilize the ac- Kounnas, M. Z., Henkin, J.,Argraves, W. S., and Strickland, D. K (1993) J . Biol. Chem. 268,21862-21867 tive state andallow for vitronectin binding, accounting for the Lisanti, M. P., Field, M. C., Caras, I. W., Menon, A. K., and Rodriguez-Boulan E. (1991) EMBO J . 10, 1969-1977 altered binding and biological properties of 293 cells transB., Vlelh,P., Cuadrado, C., and Burtin, P. (1988)Am.J. Clin. Pathol. fected with anchored uPAR. The nature of these putative in- Loridon-Rosa, 90,7-16 teractions remains to be defined, but this potential mayallow Loskutoff, D. J., Sawdey, M., and Mimuro, J. (1989) Prog. Hemostasis Thromb. 9, 87-115 uPAR to modify the function of cells, such as colon carcinoma Masumoto, A,, and Hemler, M. E. (1993) J. Biol. Chem. 268, 228-234 cells, known to express uPAR in the absence of concomitant Mignatti, P., and Rifkin, D. B. (1993) Physiol. Reu. 73,161-194 Niculescu, F., Rus, H. G., Porutiu, D., Ghiurca, V., and Vlaicu, R. (1989) uPA (Koretz et al., 1993; Pyke et al., 1991). Atherosclerosis 78, 197-203 Although the model in Fig. 7 emphasizes therole of binding Nusrat, A. R., and Chapman, H. A. (1991) J . Clin. Inuest. 87, 1091-1097 and removal of uPA from uPAR in regulatingreceptor function, Paranjpe, M., Engel, L., Young, N.,and Liotta, L. A. (1980)Life Sci. 26, 1223-1231 our observations imply a duality in function for this receptor Pedersen, H., Grondahl-Hansen, J., Francis, D., Osterlind, K., Hansen, H. H., Dan0 K, and Brunner N. (1994) Cancer Res. 54, 120-123 that directly links a protease known to disrupt cell-matrix in- Pepper, M. S., Sappino, A.-P., Stocklin, R., Montesano, R., Orci, L., and Vassalli, J.-D. (1993). J. Cell Biol. 122, 673484 teractions with a matrix protein known to promote such interM., Eriksen, J., Plesner, T., Hansen, N. E., and Dano, K. (1992) Eur J . actions. This linkage suggests to us thatfour molecules: uPA, Plough, Biochem. 208,397-404 uPAR, vitronectin, and PAI-1, constitute the core of an inte- Pollanen, J., Saksela, O., Salonen, E. M., Andreasen, P., Nielsen, L., Dano, K., and Vaheri, A. (1987) J. Cell Biol. 104, 108L1096 grated, dynamic system for modulating cell-matrix interacPreissner, K. T., and Jenne, D. (1991) Thromb. Haemostasis 66, 189-194 tions. Under some conditions, theprotease activity of the Pyke, C., Kristensen, P., Ralfkiar, E., Grondahl-Hansen, J., Eriksen, J., Blasi, F., and Dano, K. (1991)Am. J. Pathol. 138, 1059-1067 urokinase/plasmin armof this system may be more important S. A,, Mazar, A. P., Bernier, S. M., Haq, M., Bolivar, I., Henkin, J., and in cellular movement and tissue remodeling; under other con- Rabbani, Goltzman, D. (1992) J . Biol. Chem. 267, 14151-14156 ditions, the intrinsic adhesiveness of uPAR for vitronectin, per- Reich, E. (1978) in Markers of Neoplasia: Basic and Applied Aspects(Ruddon, E. W., ed) pp. 491-500, Elsevier Science Publishing Co., New York haps regulatedby repeated uPA.PAI turnover or other interacReilly, J. T., and Nash, J. R. G. (1988) J. Clin. Pathol. 41, 1269-1272 tions,may be more important.Thisdualityin function Roldan, A. L., Cubellis, M. V., Masucci, M. T., Behrendt, N., Lund, L. R., Dano, K., Appella, E., and Blasi, F. (1990) EMBO J. 9, 467474 indicates a broader role for this widely expressed receptor in E., Behrendt, N., Ellis, V., Plough, M., Dano, K., and Hoyer-Hansen, G. cell biologythan previously realized. Interrupting theadhesive Ronne, (1991) FEBS Lett. 288,233-236 properties of this receptor may offer new strategies in modu- Ruoslahti, E., and Pierschbacher, M. D. (1987) Science 238,491497

u.

32388


Salonen, E."., Vaheri, A,, Pollanen, J., Stephens, R., Andreasen, P., Mayer, M., Dam, K., Gailit, J., and Ruoslahti, E. (1989)J. Biol. Chem. 264, 6339-6343 Schlechte, W., Murano, G., and Boyd, D. (1989)Cancer Res. 49, 6064-6069 Shi, G. P., Munger, J. S., Meara, J. P., Rich, D. H., and Chapman, H. A. (1992) J. B i d . Chem. 267,7258-7262 Sier, C. F. M., Verspaget, H. W.,Vloedgraven, H. J. M., Ganesh, S., Griffioen, G., and Lamers, C. B. H. W. (1992)Cancer Res. 33,A386 Stockmann, A,, Hess, S., Declerck, P., Timpl, R., and Preissner, K. T.(1993)J . B i d . Chem. 266,2287622882 Sumiyoshi, K., Baba, S., Sakaguchi, S., Urano, T., Takada, Y,and Takada, A.

(1991)Thromb. Res. 63, 59-71 Tomasini, B. R., and Mosher, D. F. (1990)Prog. Hemostasis Thromb. 10, 269-305 Vassalli, J. D., and Belin, D. (1987)FEBS Lett. 214, 187-191 Vassalli, J. D., Baccino, D., and Belin, D. (1985)J. Cell Biol. 100,86-92 Vogel,B. E., Lee, S.-J., Hildebrand, A,, Craig, W., Pierschbacher, M. D., WongStaal, E , and Ruoslahti, E. (1993)J. Cell Biol. 121, 461-468 Waltz, D. A,, and Chapman, H. A. (1994)J. Biol. Chem. 289, 14746-14750 Waltz, D. A,, Sailor, L. Z., and Chapman, H.A. (199315.Clin. Invest. 91,1541-1552 Yatohgo, T., Izumi, M., Kashiwagi, H., and Hayashi, M. (1988)Cell Struct. Funct. 13,281-292