Download PDF
Thromb Haemost 2002; 88(02): 298-306
DOI: 10.1055/s-0037-1613202
In Focus
Schattauer GmbH

Metalloproteases Cleave the Urokinase-Type Plasminogen Activator Receptor in the D1-D2 Linker Region and Expose Epitopes not Present in the intact Soluble Receptor

Annapaola Andolfo
2   Department of Molecular Pathology and Medicine, Molecular Genetics Unit, DIBIT, San Raffaele Scientific Institute, Milan, Italy
,
William R. English
1   School of Biological Sciences, University of East Anglia, Norwich, UK
,
Massimo Resnati
2   Department of Molecular Pathology and Medicine, Molecular Genetics Unit, DIBIT, San Raffaele Scientific Institute, Milan, Italy
,
Gillian Murphy
1   School of Biological Sciences, University of East Anglia, Norwich, UK
,
Francesco Blasi
2   Department of Molecular Pathology and Medicine, Molecular Genetics Unit, DIBIT, San Raffaele Scientific Institute, Milan, Italy
,
Nicolai Sidenius
2   Department of Molecular Pathology and Medicine, Molecular Genetics Unit, DIBIT, San Raffaele Scientific Institute, Milan, Italy
› Author Affiliations
Further Information

Publication History

Received 11 February 2002

Accepted after revision 07 May 2002

Publication Date:
07 December 2017 (online)

Also available at
    Permissions and Reprints

Summary

Proteolytic cleavage of the urokinase plasminogen activator receptor (uPA(R)) prevents the binding of uPA and vitronectin while generating biologically active uPAR fragments. We have recently shown that matrix metalloproteinase-12 (MMP-12) releases cellular uPAR-antigen from stimulated human micro-vascular endothelial cells providing a novel feedback mechanism between the plasminogen activation and MMP systems. We now show that MMP-12 and other MMPs directly and efficiently cleave uPAR at the Thr86||Tyr87 peptide bond located in the linker region connecting uPAR domains 1 and 2, releasing the major ligand binding domain 1 from the rest of the receptor. The possible biological importance of uPAR cleavage by MMPs is supported by the observation that also murine uPAR is cleaved by MMP-12 (at the Pro89||Gln90 peptide bond), despite the limited sequence homology between the linker regions. Using an antibody raised against the human uPAR linker region we show that this region of uPAR, which contains the chemotactic SRSRY epitope, is exposed upon MMP cleavage.

Keywords

uPAR - MMP - cleavage - fragments
 

  • References

  • 1 Andreasen PA, Kjøller L, Christensen L, Duffy MJ. The urokinase-type plasminogen activator system in cancer metastasis: a review. Int J Cancer 1997; 72: 1-22.
    CrossrefPubMedGoogle Scholar
  • 2 Blasi F. uPA, uPAR, PAI-1: key intersection of proteolytic, adhesive and chemotactic highways?. Immunol Today 1997; 18: 415-7.
    CrossrefPubMedGoogle Scholar
  • 3 Ploug M, Rønne E, Behrendt N, Jensen AL, Blasi F, Danø K. Cellular receptor for urokinase plasminogen activator. Carboxyl-terminal processing and membrane anchoring by glycosyl-phosphatidylinositol. J Biol Chem 1991; 266: 1926-33.
    PubMedGoogle Scholar
  • 4 Høyer-Hansen G, Rønne E, Solberg H. et al. Urokinase plasminogen activator cleaves its cell surface receptor releasing the ligand-binding domain. J Biol Chem 1992; 267: 18224-9.
    PubMedGoogle Scholar
  • 5 Solberg H, Rømer J, Brünner N. et al. A cleaved form of the receptor for urokinase-type plasminogen activator in invasive transplanted human and murine tumors. Int J Cancer 1994; 58: 877-81.
    CrossrefPubMedGoogle Scholar
  • 6 Ragno P, Montuori N, Covelli B, Høyer-Hansen G, Rossi G. Differential expression of a truncated form of the urokinase-type plasminogen-activator receptor in normal and tumor thyroid cells. Cancer Res 1998; 58: 1315-9.
    PubMedGoogle Scholar
  • 7 Sidenius N, Sier CF, Blasi F. Shedding and cleavage of the urokinase receptor (uPAR): identification and characterisation of uPAR fragments in vitro and in vivo. FEBS Lett 2000; 475: 52-6.
    CrossrefPubMedGoogle Scholar
  • 8 Wahlberg K, Høyer-Hansen G, Casslèn B. Soluble receptor for urokinase plasminogen activator in both full-length and a cleaved form is present in high concentration in cystic fluid from ovarian cancer. Cancer Res 1998; 58: 3294-8.
    PubMedGoogle Scholar
  • 9 Sier CF, Sidenius N, Mariani A. et al. Presence of urokinase-type plasminogen activator receptor in urine of cancer patients and its possible clinical relevance. Lab Invest 1999; 79: 717-22.
    PubMedGoogle Scholar
  • 10 Mustjoki S, Sidenius N, Sier CFM. et al. Levels of soluble urokinase receptor correlates with number of circulating tumor cells in acute myeloid leukemia and decrease rapidly during chemotherapy. Cancer Res. 2000 in press..
    PubMedGoogle Scholar
  • 11 Mustjoki S, Sidenius N, Vaheri A. Enhanced release of soluble urokinase receptor by endothelial cells in contact with peripheral blood cells. FEBS Lett. 2000 in press..
    PubMedGoogle Scholar
  • 12 Behrendt N, Ploug M, Patthy L, Houen G, Blasi F, Danø K. The ligandbinding domain of the cell surface receptor for urokinase-type plasminogen activator. J Biol Chem 1991; 266: 7842-7.
    PubMedGoogle Scholar
  • 13 Høyer-Hansen G, Behrendt N, Ploug M, Danø K, Preissner KT. The intact urokinase receptor is required for efficient vitronectin binding: receptor cleavage prevents ligand interaction. FEBS Lett 1997; 420: 79-85.
    CrossrefPubMedGoogle Scholar
  • 14 Sidenius N, Blasi F. Domain 1 of the urokinase receptor (uPAR) is required for uPAR-mediated cell binding to vitronectin. FEBS Lett 2000; 470: 40-6.
    CrossrefPubMedGoogle Scholar
  • 15 Fazioli F, Resnati M, Sidenius N, Higashimoto Y, Appella E, Blasi F. A urokinase-sensitive region of the human urokinase receptor is responsible for its chemotactic activity. EMBO J 1997; 16: 7279-86.
    CrossrefPubMedGoogle Scholar
  • 16 Resnati M, Pallavicini I, Wang JM. et al. The fibrinolytic receptor for urokinase activates the G protein-coupled chemotactic receptor FPRL1/LXA4R. Proc Natl Acad Sci USA 2002; 99: 1359-64.
    CrossrefPubMedGoogle Scholar
  • 17 Plesner T, Behrendt N, Ploug M. Structure, function and expression on blood and bone marrow cells of the urokinase-type plasminogen activator receptor, uPAR. Stem Cells 1997; 15: 398-408.
    CrossrefPubMedGoogle Scholar
  • 18 Resnati M, Guttinger M, Valcamonica S, Sidenius N, Blasi F, Fazioli F. Proteolytic cleavage of the urokinase receptor substitutes for the agonist-induced chemotactic effect. EMBO J 1996; 15: 1572-82.
    PubMedGoogle Scholar
  • 19 Degryse B, Resnati M, Rabbani SA, Villa A, Fazioli F, Blasi F. Src-Dependence and Pertussis-Toxin Sensitivity of Urokinase Receptor-Dependent Chemotaxis and Cytoskeleton Reorganization in Rat Smooth Muscle Cells. Blood 1999; 94: 1-15.
    PubMedGoogle Scholar
  • 20 Koolwijk P, Sidenius N, Peters E. et al. Proteolysis of the urokinase-type plasminogen activator receptor by metalloproteinase-12: implication for angiogenesis in fibrin matrices. Blood 2001; 97: 3123-31.
    CrossrefPubMedGoogle Scholar
  • 21 Murphy G, Willenbrock F. Tissue inhibitors of matrix metalloendopeptidases. Methods Enzymol 1995; 248: 496-510.
    PubMedGoogle Scholar
  • 22 Knäuper V, Murphy G, Tschesche H. Activation of human neutrophil procollagenase by stromelysin 2. Eur J Biochem 1996; 235: 187-91.
    CrossrefPubMedGoogle Scholar
  • 23 Knäuper V, Lûpez COtin, Smith B, Knight G, Murphy G. Biochemical characterization of human collagenase-3. J Biol Chem 1996; 271: 1544-50.
    CrossrefPubMedGoogle Scholar
  • 24 English WR, Holtz B, Vogt G, Knäuper V, Murphy G. Characterization of the role of the “MT-loop”: an eight-amino acid insertion specific to progelatinase A (MMP2) activating membrane-type matrix metalloproteinases. J Biol Chem 2001; 276: 42018-26.
    CrossrefPubMedGoogle Scholar
  • 25 Stracke JO, Fosang AJ, Last K. et al. Matrix metalloproteinases 19 and 20 cleave aggrecan and cartilage oligomeric matrix protein (COMP). FEBS Lett 2000; 478: 52-6.
    CrossrefPubMedGoogle Scholar
  • 26 Parkar AA, Stow MD, Smith K. et al. Large-scale expression, refolding, and purification of the catalytic domain of human macrophage metalloelastase (MMP-12) in Escherichia coli. Protein Expr Purif 2000; 20: 152-61.
    CrossrefPubMedGoogle Scholar
  • 27 English WR, Puente XS, Freije JM. et al. Membrane type 4 matrix metalloproteinase (MMP17) has tumor necrosis factor-alpha convertase activity but does not activate pro-MMP2. J Biol Chem 2000; 275: 14046-55.
    CrossrefPubMedGoogle Scholar
  • 28 English WR, Velasco G, Stracke JO, Knäuper V, Murphy G. Catalytic activities of membrane-type 6 matrix metalloproteinase (MMP25). FEBS Lett 2001; 491: 137-42.
    CrossrefPubMedGoogle Scholar
  • 29 Masucci MT, Pedersen N, Blasi F. A soluble, ligand binding mutant of the human urokinase plasminogen activator receptor. J Biol Chem 1991; 266: 8655-8.
    PubMedGoogle Scholar
  • 30 Kyhse JAndersen. Electroblotting of multiple gels: a simple apparatus without buffer tank for rapid transfer of proteins from polyacrylamide to nitrocellulose. J Biochem Biophys Methods 1984; 10: 203-9.
    CrossrefPubMedGoogle Scholar
  • 31 Vorm O, Mann M. Improved mass accuracy in matrix assisted laser desorption/ionization time-of-flight mass spectrometry of peptides. J Am Soc Mass Spectrom 1994; 05: 955-8.
    CrossrefPubMedGoogle Scholar
  • 32 Roldan AL, Cubellis MV, Masucci MT. et al. Cloning and expression of the receptor for human urokinase plasminogen activator, a central molecule in cell surface, plasmin dependent proteolysis. [published erratum appears in EMBO J 1990 May; 9 (5): 1674] EMBO J 1990; 09: 467-74.
    PubMedGoogle Scholar
  • 33 Kristensen P, Eriksen J, Blasi F, Dan K. Two alternatively spliced mouse urokinase receptor mRNAs with different histological localization in the gastrointestinal tract. J Cell Biol 1991; 115: 1763-71.
    CrossrefPubMedGoogle Scholar
  • 34 Høyer-Hansen G, Pessara U, Holm A. et al. Urokinase-catalysed cleavage of the urokinase receptor requires an intact glycolipid anchor. Biochem J 2001; 358: 673-9.
    CrossrefPubMedGoogle Scholar
  • 35 Okada Y, Gonoji Y, Naka K. et al. Matrix metalloproteinase 9 (92-kDa gelatinase/type IV collagenase) from HT 1080 human fibrosarcoma cells. Purification and activation of the precursor and enzymic properties. J Biol Chem 1992; 267: 21712-9.
    PubMedGoogle Scholar
  • 36 Suzuki K, Enghild JJ, Morodomi T, Salvesen G, Nagase H. Mechanisms of activation of tissue procollagenase by matrix metalloproteinase 3 (stromelysin). Biochemistry 1990; 29: 10261-70.
    CrossrefPubMedGoogle Scholar
  • 37 Eeckhout Y, Vaes G. Further studies on the activation of procollagenase, the latent precursor of bone collagenase. Effects of lysosomal cathepsin B, plasmin and kallikrein, and spontaneous activation. Biochem J 1977; 166: 21-31.
    CrossrefPubMedGoogle Scholar
  • 38 He CS, Wilhelm SM, Pentland AP. et al. Tissue cooperation in a proteolytic cascade activating human interstitial collagenase. Proc Natl Acad Sci USA 1989; 86: 2632-6.
    CrossrefPubMedGoogle Scholar
  • 39 Keski-Oja J, Lohi J, Tuuttila A, Tryggvason K, Vartio T. Proteolytic processing of the 72,000-Da type IV collagenase by urokinase plasminogen activator. Exp Cell Res 1992; 202: 471-6.
    CrossrefPubMedGoogle Scholar
  • 40 Lijnen HR, Arza B, Van Hoef B, Collen D, Declerck PJ. Inactivation of plasminogen activator inhibitor-1 by specific proteolysis with stromelysin-1 (MMP-3). J Biol Chem 2000; 275: 37645-50.
    CrossrefPubMedGoogle Scholar
  • 41 Lijnen HR, Van Hoef B, Collen D. Inactivation of the serpin alpha(2)-antiplasmin by stromelysin-1. Biochim Biophys Acta 2001; 1547: 206-13.
    CrossrefPubMedGoogle Scholar
  • 42 Ugwu F, Van Hoef B, Bini A, Collen D, Lijnen HR. Proteolytic cleavage of urokinase-type plasminogen activator by stromelysin-1 (MMP-3). Biochemistry 1998; 37: 7231-6.
    CrossrefPubMedGoogle Scholar
  • 43 Ugwu F, Lemmens G, Collen D, Lijnen HR. Modulation of cell-associated plasminogen activation by stromelysin-1 (MMP-3). Thromb Haemost 1999; 82: 1127-31.
    Article in Thieme ConnectPubMedGoogle Scholar
  • 44 Lijnen HR, Ugwu F, Bini A, Collen D. Generation of an angiostatin-like fragment from plasminogen by stromelysin-1 (MMP-3). Biochemistry 1998; 37: 4699-702.
    CrossrefPubMedGoogle Scholar
  • 45 Sier CFM, Stephens RW, Bizik J. et al. The level of urokinase-type plasminogen activator receptor is increased in serum of ovarian cancer patients. Cancer Res 1998; 58: 1843-9.
    PubMedGoogle Scholar
  • 46 Stephens RW, Nielsen HJ, Christensen IJ. et al. Plasma urokinase receptor levels in patients with colorectal cancer: relationship to prognosis. J Natl Cancer Inst 1999; 91: 869-74.
    CrossrefPubMedGoogle Scholar
  • 47 Sidenius N, Sier CF, Ullum H. et al. Serum level of soluble urokinase-type plasminogen activator receptor is a strong and independent predictor of survival in human immunodeficiency virus infection. Blood 2000; 96: 4091-5.
    PubMedGoogle Scholar
  • 48 Slot S, Brünner N, Locht H, Oxholm P, Stephens RW. Soluble urokinase plasminogen activator receptor in plasma of patients with inflammatory rheumatic disorders: increased concentrations in rheumatoid arthritis. Ann Rheum Dis 1999; 58: 488-92.
    CrossrefPubMedGoogle Scholar
  • 49 Ploug M, Ellis V. Structure-function relationships in the receptor for urokinase-type plasminogen activator Comparison to other members of the Ly-6 family and snake venom alpha-neurotoxins. FEBS Lett 1994; 349: 163-8.
    CrossrefPubMedGoogle Scholar