Skip to main content
NCBI home page
Biochemical Journal logoLink to Biochemical Journal
. 2001 Sep 15;358(Pt 3):673–679. doi: 10.1042/0264-6021:3580673

Urokinase-catalysed cleavage of the urokinase receptor requires an intact glycolipid anchor.

G Høyer-Hansen 1, U Pessara 1, A Holm 1, J Pass 1, U Weidle 1, K Danø 1, N Behrendt 1
PMCID: PMC1222101  PMID: 11535128

Abstract

Urokinase (uPA) has the striking ability to cleave its receptor, uPAR, thereby inactivating the binding potential of this molecule. Here we demonstrate that the glycosylphosphatidylinositol (GPI) anchor of uPAR, which is attached to the third domain, is an important determinant in governing this reaction, even though the actual cleavage occurs between the first and second domains. Purified full-length GPI-anchored uPAR (GPI-uPAR) proved much more susceptible to uPA-mediated cleavage than recombinant truncated soluble uPAR (suPAR), which lacks the glycolipid anchor. This was not a general difference in proteolytic susceptibility since GPI-uPAR and suPAR were cleaved with equal efficiency by plasmin. Since the amino acid sequences of GPI-uPAR and suPAR are identical except for the C-terminal truncation, the different cleavage patterns suggest that the two uPAR variants differ in the conformation or the flexibility of the linker region between domains 1 and 2. This was supported by the fact that an antibody to the peptide AVTYSRSRYLE, amino acids 84-94 in the linker region, recognizes GPI-uPAR but not suPAR. This difference in the linker region is thus caused by a difference in a remote hydrophobic region. In accordance with this model, when the hydrophobic lipid moiety was removed from the glycolipid anchor by phospholipase C, low concentrations of uPA could no longer cleave the modified GPI-uPAR and the reactivity to the peptide antibody was greatly decreased. Naturally occurring suPAR, purified from plasma, was found to have a similar resistance to uPA cleavage as phospholipase C-treated GPI-uPAR and recombinant suPAR.

Full Text

The Full Text of this article is available as a PDF (181.7 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Andreasen P. A., Kjøller L., Christensen L., Duffy M. J. The urokinase-type plasminogen activator system in cancer metastasis: a review. Int J Cancer. 1997 Jul 3;72(1):1–22. doi: 10.1002/(sici)1097-0215(19970703)72:1<1::aid-ijc1>3.0.co;2-z. [DOI] [PubMed] [Google Scholar]
  2. Behrendt N., Ploug M., Patthy L., Houen G., Blasi F., Danø K. The ligand-binding domain of the cell surface receptor for urokinase-type plasminogen activator. J Biol Chem. 1991 Apr 25;266(12):7842–7847. [PubMed] [Google Scholar]
  3. Behrendt N., Ronne E., Dano K. Domain interplay in the urokinase receptor. Requirement for the third domain in high affinity ligand binding and demonstration of ligand contact sites in distinct receptor domains. J Biol Chem. 1996 Sep 13;271(37):22885–22894. doi: 10.1074/jbc.271.37.22885. [DOI] [PubMed] [Google Scholar]
  4. Behrendt N., Rønne E., Ploug M., Petri T., Løber D., Nielsen L. S., Schleuning W. D., Blasi F., Appella E., Danø K. The human receptor for urokinase plasminogen activator. NH2-terminal amino acid sequence and glycosylation variants. J Biol Chem. 1990 Apr 15;265(11):6453–6460. [PubMed] [Google Scholar]
  5. Degryse B., Resnati M., Rabbani S. A., 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 Jul 15;94(2):649–662. [PubMed] [Google Scholar]
  6. Durbin H., Young S., Stewart L. M., Wrba F., Rowan A. J., Snary D., Bodmer W. F. An epitope on carcinoembryonic antigen defined by the clinically relevant antibody PR1A3. Proc Natl Acad Sci U S A. 1994 May 10;91(10):4313–4317. doi: 10.1073/pnas.91.10.4313. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Ellis V., Scully M. F., Kakkar V. V. Plasminogen activation initiated by single-chain urokinase-type plasminogen activator. Potentiation by U937 monocytes. J Biol Chem. 1989 Feb 5;264(4):2185–2188. [PubMed] [Google Scholar]
  8. English A., Kosoy R., Pawlinski R., Bamezai A. A monoclonal antibody against the 66-kDa protein expressed in mouse spleen and thymus inhibits Ly-6A.2-dependent cell-cell adhesion. J Immunol. 2000 Oct 1;165(7):3763–3771. doi: 10.4049/jimmunol.165.7.3763. [DOI] [PubMed] [Google Scholar]
  9. 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 Dec 15;16(24):7279–7286. doi: 10.1093/emboj/16.24.7279. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Høyer-Hansen G., Behrendt N., Ploug M., Danø K., Preissner K. T. The intact urokinase receptor is required for efficient vitronectin binding: receptor cleavage prevents ligand interaction. FEBS Lett. 1997 Dec 22;420(1):79–85. doi: 10.1016/s0014-5793(97)01491-9. [DOI] [PubMed] [Google Scholar]
  11. Høyer-Hansen G., Ploug M., Behrendt N., Rønne E., Danø K. Cell-surface acceleration of urokinase-catalyzed receptor cleavage. Eur J Biochem. 1997 Jan 15;243(1-2):21–26. doi: 10.1111/j.1432-1033.1997.0021a.x. [DOI] [PubMed] [Google Scholar]
  12. Høyer-Hansen G., Rønne E., Solberg H., Behrendt N., Ploug M., Lund L. R., Ellis V., Danø K. Urokinase plasminogen activator cleaves its cell surface receptor releasing the ligand-binding domain. J Biol Chem. 1992 Sep 5;267(25):18224–18229. [PubMed] [Google Scholar]
  13. Kaltoft K., Nielsen L. S., Zeuthen J., Danø K. Monoclonal antibody that specifically inhibits a human Mr 52,000 plasminogen-activating enzyme. Proc Natl Acad Sci U S A. 1982 Jun;79(12):3720–3723. doi: 10.1073/pnas.79.12.3720. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Kanse S. M., Kost C., Wilhelm O. G., Andreasen P. A., Preissner K. T. The urokinase receptor is a major vitronectin-binding protein on endothelial cells. Exp Cell Res. 1996 May 1;224(2):344–353. doi: 10.1006/excr.1996.0144. [DOI] [PubMed] [Google Scholar]
  15. Kaufman R. J., Sharp P. A. Amplification and expression of sequences cotransfected with a modular dihydrofolate reductase complementary dna gene. J Mol Biol. 1982 Aug 25;159(4):601–621. doi: 10.1016/0022-2836(82)90103-6. [DOI] [PubMed] [Google Scholar]
  16. Kukulansky T., Abramovitch S., Hollander N. Cleavage of the glycosylphosphatidylinositol anchor affects the reactivity of thy-1 with antibodies. J Immunol. 1999 May 15;162(10):5993–5997. [PubMed] [Google Scholar]
  17. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  18. List K., Høyer-Hansen G., Rønne E., Danø K., Behrendt N. Different mechanisms are involved in the antibody mediated inhibition of ligand binding to the urokinase receptor: a study based on biosensor technology. J Immunol Methods. 1999 Jan 1;222(1-2):125–133. doi: 10.1016/s0022-1759(98)00189-6. [DOI] [PubMed] [Google Scholar]
  19. Low M. G. The glycosyl-phosphatidylinositol anchor of membrane proteins. Biochim Biophys Acta. 1989 Dec 6;988(3):427–454. doi: 10.1016/0304-4157(89)90014-2. [DOI] [PubMed] [Google Scholar]
  20. Nguyen D. H., Webb D. J., Catling A. D., Song Q., Dhakephalkar A., Weber M. J., Ravichandran K. S., Gonias S. L. Urokinase-type plasminogen activator stimulates the Ras/Extracellular signal-regulated kinase (ERK) signaling pathway and MCF-7 cell migration by a mechanism that requires focal adhesion kinase, Src, and Shc. Rapid dissociation of GRB2/Sps-Shc complex is associated with the transient phosphorylation of ERK in urokinase-treated cells. J Biol Chem. 2000 Jun 23;275(25):19382–19388. doi: 10.1074/jbc.M909575199. [DOI] [PubMed] [Google Scholar]
  21. Nielsen L. S., Grøndahl-Hansen J., Andreasen P. A., Skriver L., Zeuthen J., Danø K. Enzyme-linked immunosorbent assay for human urokinase-type plasminogen activator and its proenzyme using a combination of monoclonal and polyclonal antibodies. J Immunoassay. 1986;7(3):209–228. doi: 10.1080/01971528608060467. [DOI] [PubMed] [Google Scholar]
  22. O'Hare K., Benoist C., Breathnach R. Transformation of mouse fibroblasts to methotrexate resistance by a recombinant plasmid expressing a prokaryotic dihydrofolate reductase. Proc Natl Acad Sci U S A. 1981 Mar;78(3):1527–1531. doi: 10.1073/pnas.78.3.1527. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Pedersen N., Schmitt M., Rønne E., Nicoletti M. I., Høyer-Hansen G., Conese M., Giavazzi R., Dano K., Kuhn W., Jänicke F. A ligand-free, soluble urokinase receptor is present in the ascitic fluid from patients with ovarian cancer. J Clin Invest. 1993 Nov;92(5):2160–2167. doi: 10.1172/JCI116817. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Ploug M., Ellis V., Danø K. Ligand interaction between urokinase-type plasminogen activator and its receptor probed with 8-anilino-1-naphthalenesulfonate. Evidence for a hydrophobic binding site exposed only on the intact receptor. Biochemistry. 1994 Aug 2;33(30):8991–8997. doi: 10.1021/bi00196a017. [DOI] [PubMed] [Google Scholar]
  25. 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 Aug 1;349(2):163–168. doi: 10.1016/0014-5793(94)00674-1. [DOI] [PubMed] [Google Scholar]
  26. Ploug M., Kjalke M., Rønne E., Weidle U., Høyer-Hansen G., Danø K. Localization of the disulfide bonds in the NH2-terminal domain of the cellular receptor for human urokinase-type plasminogen activator. A domain structure belonging to a novel superfamily of glycolipid-anchored membrane proteins. J Biol Chem. 1993 Aug 15;268(23):17539–17546. [PubMed] [Google Scholar]
  27. Ploug M., Rahbek-Nielsen H., Ellis V., Roepstorff P., Danø K. Chemical modification of the urokinase-type plasminogen activator and its receptor using tetranitromethane. Evidence for the involvement of specific tyrosine residues in both molecules during receptor-ligand interaction. Biochemistry. 1995 Oct 3;34(39):12524–12534. doi: 10.1021/bi00039a006. [DOI] [PubMed] [Google Scholar]
  28. Ploug M., Rønne E., Behrendt N., Jensen A. L., Blasi F., Danø K. Cellular receptor for urokinase plasminogen activator. Carboxyl-terminal processing and membrane anchoring by glycosyl-phosphatidylinositol. J Biol Chem. 1991 Jan 25;266(3):1926–1933. [PubMed] [Google Scholar]
  29. Posnett D. N., McGrath H., Tam J. P. A novel method for producing anti-peptide antibodies. Production of site-specific antibodies to the T cell antigen receptor beta-chain. J Biol Chem. 1988 Feb 5;263(4):1719–1725. [PubMed] [Google Scholar]
  30. Ragno P., Montuori N., Covelli B., Hoyer-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 Mar 15;58(6):1315–1319. [PubMed] [Google Scholar]
  31. 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 Apr 1;15(7):1572–1582. [PMC free article] [PubMed] [Google Scholar]
  32. Roldan A. L., Cubellis M. V., Masucci M. T., Behrendt N., Lund L. R., Danø K., Appella E., Blasi F. Cloning and expression of the receptor for human urokinase plasminogen activator, a central molecule in cell surface, plasmin dependent proteolysis. EMBO J. 1990 Feb;9(2):467–474. doi: 10.1002/j.1460-2075.1990.tb08132.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Rønne E., Behrendt N., Ellis V., Ploug M., Danø K., Høyer-Hansen G. Cell-induced potentiation of the plasminogen activation system is abolished by a monoclonal antibody that recognizes the NH2-terminal domain of the urokinase receptor. FEBS Lett. 1991 Aug 19;288(1-2):233–236. doi: 10.1016/0014-5793(91)81042-7. [DOI] [PubMed] [Google Scholar]
  34. Rønne E., Behrendt N., Ploug M., Nielsen H. J., Wöllisch E., Weidle U., Danø K., Høyer-Hansen G. Quantitation of the receptor for urokinase plasminogen activator by enzyme-linked immunosorbent assay. J Immunol Methods. 1994 Jan 3;167(1-2):91–101. doi: 10.1016/0022-1759(94)90078-7. [DOI] [PubMed] [Google Scholar]
  35. Rønne E., Pappot H., Grøndahl-Hansen J., Høyer-Hansen G., Plesner T., Hansen N. E., Danø K. The receptor for urokinase plasminogen activator is present in plasma from healthy donors and elevated in patients with paroxysmal nocturnal haemoglobinuria. Br J Haematol. 1995 Mar;89(3):576–581. doi: 10.1111/j.1365-2141.1995.tb08366.x. [DOI] [PubMed] [Google Scholar]
  36. Solberg H., Løber D., Eriksen J., Ploug M., Rønne E., Behrendt N., Danø K., Høyer-Hansen G. Identification and characterization of the murine cell surface receptor for the urokinase-type plasminogen activator. Eur J Biochem. 1992 Apr 15;205(2):451–458. doi: 10.1111/j.1432-1033.1992.tb16799.x. [DOI] [PubMed] [Google Scholar]
  37. Solberg H., Rømer J., Brünner N., Holm A., Sidenius N., Danø K., Høyer-Hansen G. A cleaved form of the receptor for urokinase-type plasminogen activator in invasive transplanted human and murine tumors. Int J Cancer. 1994 Sep 15;58(6):877–881. doi: 10.1002/ijc.2910580622. [DOI] [PubMed] [Google Scholar]
  38. Stephens R. W., Nielsen H. J., Christensen I. J., Thorlacius-Ussing O., Sørensen S., Danø K., Brünner N. Plasma urokinase receptor levels in patients with colorectal cancer: relationship to prognosis. J Natl Cancer Inst. 1999 May 19;91(10):869–874. doi: 10.1093/jnci/91.10.869. [DOI] [PubMed] [Google Scholar]
  39. Stoppelli M. P., Corti A., Soffientini A., Cassani G., Blasi F., Assoian R. K. Differentiation-enhanced binding of the amino-terminal fragment of human urokinase plasminogen activator to a specific receptor on U937 monocytes. Proc Natl Acad Sci U S A. 1985 Aug;82(15):4939–4943. doi: 10.1073/pnas.82.15.4939. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Wahlberg K., Høyer-Hansen G., Casslén 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 Aug 1;58(15):3294–3298. [PubMed] [Google Scholar]
  41. Wei Y., Waltz D. A., Rao N., Drummond R. J., Rosenberg S., Chapman H. A. Identification of the urokinase receptor as an adhesion receptor for vitronectin. J Biol Chem. 1994 Dec 23;269(51):32380–32388. [PubMed] [Google Scholar]
  42. Weidle U. H., Buckel P., Mattes R. A protease-hypersensitive deletion derivative of human tissue-type plasminogen activator. Gene. 1988 Dec 20;73(2):439–447. doi: 10.1016/0378-1119(88)90508-2. [DOI] [PubMed] [Google Scholar]
  43. Wilhelm O. G., Wilhelm S., Escott G. M., Lutz V., Magdolen V., Schmitt M., Rifkin D. B., Wilson E. L., Graeff H., Brunner G. Cellular glycosylphosphatidylinositol-specific phospholipase D regulates urokinase receptor shedding and cell surface expression. J Cell Physiol. 1999 Aug;180(2):225–235. doi: 10.1002/(SICI)1097-4652(199908)180:2<225::AID-JCP10>3.0.CO;2-2. [DOI] [PubMed] [Google Scholar]
  44. Zhou H. M., Nichols A., Meda P., Vassalli J. D. Urokinase-type plasminogen activator and its receptor synergize to promote pathogenic proteolysis. EMBO J. 2000 Sep 1;19(17):4817–4826. doi: 10.1093/emboj/19.17.4817. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Biochemical Journal are provided here courtesy of The Biochemical Society

RESOURCES