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. 1996 Apr 1;15(7):1572–1582.

Proteolytic cleavage of the urokinase receptor substitutes for the agonist-induced chemotactic effect.

M Resnati 1, M Guttinger 1, S Valcamonica 1, N Sidenius 1, F Blasi 1, F Fazioli 1
PMCID: PMC450067  PMID: 8612581

Abstract

Physiological concentrations of urokinase plasminogen activator (uPA) stimulated a chemotactic response in human monocytic THP-1 through binding to the urokinase receptor (uPAR). The effect did not require the protease moiety of uPA, as stimulation was achieved also with the N-terminal fragment (ATF), while the 33 kDa low molecular weight uPA was ineffective. Co-immunoprecipitation experiments showed association of uPAR with intracellular kinase(s), as demonstrated by in vitro kinase assays. Use of specific antibodies identified p56/p59hck as a kinase associated with uPAR in THP-1 cell extracts. Upon addition of ATF, p56/p59hck activity was stimulated within 2 min and returned to normal after 30 min. Since uPAR lacks an intracellular domain capable of interacting with intracellular kinase, activation of p56/p59hck must require a transmembrane adaptor. Evidence for this was strongly supported by the finding that a soluble form of uPAR (suPAR) was capable of inducing chemotaxis not only in THP-1 cells but also in cells lacking endogenous uPAR (IC50, 5 pM). However, activity of suPAR require chymotrypsin cleavage between the N-terminal domain D1 and D2 + D3. Chymotrypsin-cleaved suPAR also induced activation of p56/p59hck in THP-1 cells, with a time course comparable with ATF. Our data show that uPA-induced signal transduction takes place via uPAR, involves activation of intracellular tyrosine kinase(s) and requires an as yet undefined adaptor capable of connecting the extracellular ligand binding uPAR to intracellular transducer(s).

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Selected References

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

  1. Akiyama T., Ogawara H. Use and specificity of genistein as inhibitor of protein-tyrosine kinases. Methods Enzymol. 1991;201:362–370. doi: 10.1016/0076-6879(91)01032-w. [DOI] [PubMed] [Google Scholar]
  2. Appella E., Robinson E. A., Ullrich S. J., Stoppelli M. P., Corti A., Cassani G., Blasi F. The receptor-binding sequence of urokinase. A biological function for the growth-factor module of proteases. J Biol Chem. 1987 Apr 5;262(10):4437–4440. [PubMed] [Google Scholar]
  3. Auwerx J. The human leukemia cell line, THP-1: a multifacetted model for the study of monocyte-macrophage differentiation. Experientia. 1991 Jan 15;47(1):22–31. doi: 10.1007/BF02041244. [DOI] [PubMed] [Google Scholar]
  4. 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]
  5. Bohuslav J., Horejsí V., Hansmann C., Stöckl J., Weidle U. H., Majdic O., Bartke I., Knapp W., Stockinger H. Urokinase plasminogen activator receptor, beta 2-integrins, and Src-kinases within a single receptor complex of human monocytes. J Exp Med. 1995 Apr 1;181(4):1381–1390. doi: 10.1084/jem.181.4.1381. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Bugge T. H., Suh T. T., Flick M. J., Daugherty C. C., Rømer J., Solberg H., Ellis V., Danø K., Degen J. L. The receptor for urokinase-type plasminogen activator is not essential for mouse development or fertility. J Biol Chem. 1995 Jul 14;270(28):16886–16894. doi: 10.1074/jbc.270.28.16886. [DOI] [PubMed] [Google Scholar]
  7. Busso N., Masur S. K., Lazega D., Waxman S., Ossowski L. Induction of cell migration by pro-urokinase binding to its receptor: possible mechanism for signal transduction in human epithelial cells. J Cell Biol. 1994 Jul;126(1):259–270. doi: 10.1083/jcb.126.1.259. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Conese M., Olson D., Blasi F. Protease nexin-1-urokinase complexes are internalized and degraded through a mechanism that requires both urokinase receptor and alpha 2-macroglobulin receptor. J Biol Chem. 1994 Jul 8;269(27):17886–17892. [PubMed] [Google Scholar]
  9. Cooper J. A., Esch F. S., Taylor S. S., Hunter T. Phosphorylation sites in enolase and lactate dehydrogenase utilized by tyrosine protein kinases in vivo and in vitro. J Biol Chem. 1984 Jun 25;259(12):7835–7841. [PubMed] [Google Scholar]
  10. Cubellis M. V., Andreasen P., Ragno P., Mayer M., Danø K., Blasi F. Accessibility of receptor-bound urokinase to type-1 plasminogen activator inhibitor. Proc Natl Acad Sci U S A. 1989 Jul;86(13):4828–4832. doi: 10.1073/pnas.86.13.4828. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Del Rosso M., Anichini E., Pedersen N., Blasi F., Fibbi G., Pucci M., Ruggiero M. Urokinase-urokinase receptor interaction: non-mitogenic signal transduction in human epidermal cells. Biochem Biophys Res Commun. 1993 Jan 29;190(2):347–352. doi: 10.1006/bbrc.1993.1054. [DOI] [PubMed] [Google Scholar]
  12. Dumler I., Petri T., Schleuning W. D. Interaction of urokinase-type plasminogenactivator (u-PA) with its cellular receptor (u-PAR) induces phosphorylation on tyrosine of a 38 kDa protein. FEBS Lett. 1993 May 3;322(1):37–40. doi: 10.1016/0014-5793(93)81106-a. [DOI] [PubMed] [Google Scholar]
  13. 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]
  14. Ellis V., Wun T. C., Behrendt N., Rønne E., Danø K. Inhibition of receptor-bound urokinase by plasminogen-activator inhibitors. J Biol Chem. 1990 Jun 15;265(17):9904–9908. [PubMed] [Google Scholar]
  15. Estreicher A., Mühlhauser J., Carpentier J. L., Orci L., Vassalli J. D. The receptor for urokinase type plasminogen activator polarizes expression of the protease to the leading edge of migrating monocytes and promotes degradation of enzyme inhibitor complexes. J Cell Biol. 1990 Aug;111(2):783–792. doi: 10.1083/jcb.111.2.783. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Estreicher A., Wohlwend A., Belin D., Schleuning W. D., Vassalli J. D. Characterization of the cellular binding site for the urokinase-type plasminogen activator. J Biol Chem. 1989 Jan 15;264(2):1180–1189. [PubMed] [Google Scholar]
  17. Fazioli F., Blasi F. Urokinase-type plasminogen activator and its receptor: new targets for anti-metastatic therapy? Trends Pharmacol Sci. 1994 Jan;15(1):25–29. doi: 10.1016/0165-6147(94)90130-9. [DOI] [PubMed] [Google Scholar]
  18. Fibbi G., Ziche M., Morbidelli L., Magnelli L., Del Rosso M. Interaction of urokinase with specific receptors stimulates mobilization of bovine adrenal capillary endothelial cells. Exp Cell Res. 1988 Dec;179(2):385–395. doi: 10.1016/0014-4827(88)90277-7. [DOI] [PubMed] [Google Scholar]
  19. Gross T. J., Sitrin R. G. The THP-1 cell line is a urokinase-secreting mononuclear phagocyte with a novel defect in the production of plasminogen activator inhibitor-2. J Immunol. 1990 Mar 1;144(5):1873–1879. [PubMed] [Google Scholar]
  20. Gudewicz P. W., Gilboa N. Human urokinase-type plasminogen activator stimulates chemotaxis of human neutrophils. Biochem Biophys Res Commun. 1987 Sep 30;147(3):1176–1181. doi: 10.1016/s0006-291x(87)80193-6. [DOI] [PubMed] [Google Scholar]
  21. Gyetko M. R., Todd R. F., 3rd, Wilkinson C. C., Sitrin R. G. The urokinase receptor is required for human monocyte chemotaxis in vitro. J Clin Invest. 1994 Apr;93(4):1380–1387. doi: 10.1172/JCI117114. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Herz J., Clouthier D. E., Hammer R. E. LDL receptor-related protein internalizes and degrades uPA-PAI-1 complexes and is essential for embryo implantation. Cell. 1992 Oct 30;71(3):411–421. doi: 10.1016/0092-8674(92)90511-a. [DOI] [PubMed] [Google Scholar]
  23. 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]
  24. Limongi P., Resnati M., Hernandez-Marrero L., Cremona O., Blasi F., Fazioli F. Biosynthesis and apical localization of the urokinase receptor in polarized MDCK epithelial cells. FEBS Lett. 1995 Aug 7;369(2-3):207–211. doi: 10.1016/0014-5793(95)00742-r. [DOI] [PubMed] [Google Scholar]
  25. Manchanda N., Schwartz B. S. Single chain urokinase. Augmentation of enzymatic activity upon binding to monocytes. J Biol Chem. 1991 Aug 5;266(22):14580–14584. [PubMed] [Google Scholar]
  26. Masucci M. T., Pedersen N., Blasi F. A soluble, ligand binding mutant of the human urokinase plasminogen activator receptor. J Biol Chem. 1991 May 15;266(14):8655–8658. [PubMed] [Google Scholar]
  27. Mignatti P., Rifkin D. B. Biology and biochemistry of proteinases in tumor invasion. Physiol Rev. 1993 Jan;73(1):161–195. doi: 10.1152/physrev.1993.73.1.161. [DOI] [PubMed] [Google Scholar]
  28. Min H. Y., Semnani R., Mizukami I. F., Watt K., Todd R. F., 3rd, Liu D. Y. cDNA for Mo3, a monocyte activation antigen, encodes the human receptor for urokinase plasminogen activator. J Immunol. 1992 Jun 1;148(11):3636–3642. [PubMed] [Google Scholar]
  29. Myöhänen H. T., Stephens R. W., Hedman K., Tapiovaara H., Rønne E., Høyer-Hansen G., Danø K., Vaheri A. Distribution and lateral mobility of the urokinase-receptor complex at the cell surface. J Histochem Cytochem. 1993 Sep;41(9):1291–1301. doi: 10.1177/41.9.8394852. [DOI] [PubMed] [Google Scholar]
  30. Naldini L., Tamagnone L., Vigna E., Sachs M., Hartmann G., Birchmeier W., Daikuhara Y., Tsubouchi H., Blasi F., Comoglio P. M. Extracellular proteolytic cleavage by urokinase is required for activation of hepatocyte growth factor/scatter factor. EMBO J. 1992 Dec;11(13):4825–4833. doi: 10.1002/j.1460-2075.1992.tb05588.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Naldini L., Vigna E., Bardelli A., Follenzi A., Galimi F., Comoglio P. M. Biological activation of pro-HGF (hepatocyte growth factor) by urokinase is controlled by a stoichiometric reaction. J Biol Chem. 1995 Jan 13;270(2):603–611. doi: 10.1074/jbc.270.2.603. [DOI] [PubMed] [Google Scholar]
  32. Nielsen L. S., Kellerman G. M., Behrendt N., Picone R., Danø K., Blasi F. A 55,000-60,000 Mr receptor protein for urokinase-type plasminogen activator. Identification in human tumor cell lines and partial purification. J Biol Chem. 1988 Feb 15;263(5):2358–2363. [PubMed] [Google Scholar]
  33. Nykjaer A., Petersen C. M., Møller B., Jensen P. H., Moestrup S. K., Holtet T. L., Etzerodt M., Thøgersen H. C., Munch M., Andreasen P. A. Purified alpha 2-macroglobulin receptor/LDL receptor-related protein binds urokinase.plasminogen activator inhibitor type-1 complex. Evidence that the alpha 2-macroglobulin receptor mediates cellular degradation of urokinase receptor-bound complexes. J Biol Chem. 1992 Jul 25;267(21):14543–14546. [PubMed] [Google Scholar]
  34. Olson D., Pöllänen J., Høyer-Hansen G., Rønne E., Sakaguchi K., Wun T. C., Appella E., Danø K., Blasi F. Internalization of the urokinase-plasminogen activator inhibitor type-1 complex is mediated by the urokinase receptor. J Biol Chem. 1992 May 5;267(13):9129–9133. [PubMed] [Google Scholar]
  35. Pang D. T., Sharma B. R., Shafer J. A. Purification of the catalytically active phosphorylated form of insulin receptor kinase by affinity chromatography with O-phosphotyrosyl-binding antibodies. Arch Biochem Biophys. 1985 Oct;242(1):176–186. doi: 10.1016/0003-9861(85)90491-6. [DOI] [PubMed] [Google Scholar]
  36. 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]
  37. 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]
  38. Quax P. H., Pedersen N., Masucci M. T., Weening-Verhoeff E. J., Danø K., Verheijen J. H., Blasi F. Complementation between urokinase-producing and receptor-producing cells in extracellular matrix degradation. Cell Regul. 1991 Oct;2(10):793–803. doi: 10.1091/mbc.2.10.793. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Quigley J. P., Gold L. I., Schwimmer R., Sullivan L. M. Limited cleavage of cellular fibronectin by plasminogen activator purified from transformed cells. Proc Natl Acad Sci U S A. 1987 May;84(9):2776–2780. doi: 10.1073/pnas.84.9.2776. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Robinson P. J. Phosphatidylinositol membrane anchors and T-cell activation. Immunol Today. 1991 Jan;12(1):35–41. doi: 10.1016/0167-5699(91)90110-F. [DOI] [PubMed] [Google Scholar]
  41. 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]
  42. 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]
  43. 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]
  44. Stefanová I., Horejsí V., Ansotegui I. J., Knapp W., Stockinger H. GPI-anchored cell-surface molecules complexed to protein tyrosine kinases. Science. 1991 Nov 15;254(5034):1016–1019. doi: 10.1126/science.1719635. [DOI] [PubMed] [Google Scholar]
  45. 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]
  46. Stoppelli M. P., Tacchetti C., Cubellis M. V., Corti A., Hearing V. J., Cassani G., Appella E., Blasi F. Autocrine saturation of pro-urokinase receptors on human A431 cells. Cell. 1986 Jun 6;45(5):675–684. doi: 10.1016/0092-8674(86)90782-8. [DOI] [PubMed] [Google Scholar]
  47. Uehara Y., Fukazawa H. Use and selectivity of herbimycin A as inhibitor of protein-tyrosine kinases. Methods Enzymol. 1991;201:370–379. doi: 10.1016/0076-6879(91)01033-x. [DOI] [PubMed] [Google Scholar]
  48. Vassalli J. D., Baccino D., Belin D. A cellular binding site for the Mr 55,000 form of the human plasminogen activator, urokinase. J Cell Biol. 1985 Jan;100(1):86–92. doi: 10.1083/jcb.100.1.86. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Vu T. K., Hung D. T., Wheaton V. I., Coughlin S. R. Molecular cloning of a functional thrombin receptor reveals a novel proteolytic mechanism of receptor activation. Cell. 1991 Mar 22;64(6):1057–1068. doi: 10.1016/0092-8674(91)90261-v. [DOI] [PubMed] [Google Scholar]

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