Skip to main content
PMC home page
The Journal of Cell Biology logoLink to The Journal of Cell Biology
. 1995 Apr 2;129(2):335–344. doi: 10.1083/jcb.129.2.335

The urokinase-type plasminogen activator receptor, a GPI-linked protein, is localized in caveolae

PMCID: PMC2199914  PMID: 7721938

Abstract

The urokinase plasminogen activator receptor (uPAR), a glycosylphosphatidylinositol-linked glycoprotein, plays a central role in the regulation of pericellular proteolysis and participates in events leading to cell activation. Here, we demonstrate that uPAR, on a human melanoma cell line, is localized in caveolae, flask-shaped microinvaginations of the plasma membrane found in a variety of cell types. Indirect immunofluorescence with anti-uPAR antibodies on the melanoma cells showed a punctated staining pattern that accumulated to stretches along sides of cell-cell contact and membrane ruffles. uPAR colocalized with caveolin, a characteristic protein in the coat of caveolae, as demonstrated by double staining with specific antibodies. Further, uPAR could be directly localized in caveolae by in vivo immunoelectron microscopy. Both uPAR and its ligand, uPA, were present in caveolae enriched low density Triton X-100 insoluble complexes, as shown by immunoblotting. From such complexes, caveolin could be coprecipitated with uPAR-specific antibodies suggesting a close spatial association between uPAR and caveolin that might have implications for the signal transduction mediated by uPAR. Further, functional studies indicated that the localization of uPAR and its ligand in caveolae enhances pericellular plasminogen activation, since treatment of the cells with drugs that interfere with the structural integrity of caveolae, such as nystatin, markedly reduced cell surface plasmin generation. Thus, caveolae promote efficient cell surface plasminogen activation by clustering uPAR, uPA, and possibly other protease receptors in one membrane compartment.

Full Text

The Full Text of this article is available as a PDF (3.3 MB).

Selected References

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

  1. Behrendt N., Ploug M., Rønne E., Høyer-Hansen G., Danø K. Cellular receptor for urokinase-type plasminogen activator: protein structure. Methods Enzymol. 1993;223:207–222. doi: 10.1016/0076-6879(93)23047-q. [DOI] [PubMed] [Google Scholar]
  2. Blasi F. Molecular mechanisms of protease-mediated tumor invasiveness. J Surg Oncol Suppl. 1993;3:21–23. [PubMed] [Google Scholar]
  3. Boyd D., Florent G., Kim P., Brattain M. Determination of the levels of urokinase and its receptor in human colon carcinoma cell lines. Cancer Res. 1988 Jun 1;48(11):3112–3116. [PubMed] [Google Scholar]
  4. Brown D. A., Rose J. K. Sorting of GPI-anchored proteins to glycolipid-enriched membrane subdomains during transport to the apical cell surface. Cell. 1992 Feb 7;68(3):533–544. doi: 10.1016/0092-8674(92)90189-j. [DOI] [PubMed] [Google Scholar]
  5. 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]
  6. Cesarman G. M., Guevara C. A., Hajjar K. A. An endothelial cell receptor for plasminogen/tissue plasminogen activator (t-PA). II. Annexin II-mediated enhancement of t-PA-dependent plasminogen activation. J Biol Chem. 1994 Aug 19;269(33):21198–21203. [PubMed] [Google Scholar]
  7. Chang W. J., Ying Y. S., Rothberg K. G., Hooper N. M., Turner A. J., Gambliel H. A., De Gunzburg J., Mumby S. M., Gilman A. G., Anderson R. G. Purification and characterization of smooth muscle cell caveolae. J Cell Biol. 1994 Jul;126(1):127–138. doi: 10.1083/jcb.126.1.127. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Del Rosso M., Fibbi G., Dini G., Grappone C., Pucci M., Caldini R., Magnelli L., Fimiani M., Lotti T., Panconesi E. Role of specific membrane receptors in urokinase-dependent migration of human keratinocytes. J Invest Dermatol. 1990 Mar;94(3):310–316. doi: 10.1111/1523-1747.ep12874442. [DOI] [PubMed] [Google Scholar]
  9. Ellis V., Pyke C., Eriksen J., Solberg H., Danø K. The urokinase receptor: involvement in cell surface proteolysis and cancer invasion. Ann N Y Acad Sci. 1992 Dec 4;667:13–31. doi: 10.1111/j.1749-6632.1992.tb51591.x. [DOI] [PubMed] [Google Scholar]
  10. 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]
  11. 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]
  12. Glenney J. R., Jr, Zokas L. Novel tyrosine kinase substrates from Rous sarcoma virus-transformed cells are present in the membrane skeleton. J Cell Biol. 1989 Jun;108(6):2401–2408. doi: 10.1083/jcb.108.6.2401. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. 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]
  14. Hajjar K. A., Jacovina A. T., Chacko J. An endothelial cell receptor for plasminogen/tissue plasminogen activator. I. Identity with annexin II. J Biol Chem. 1994 Aug 19;269(33):21191–21197. [PubMed] [Google Scholar]
  15. Hollas W., Hoosein N., Chung L. W., Mazar A., Henkin J., Kariko K., Barnathan E. S., Boyd D. Expression of urokinase and its receptor in invasive and non-invasive prostate cancer cell lines. Thromb Haemost. 1992 Dec 7;68(6):662–666. [PubMed] [Google Scholar]
  16. Hébert C. A., Baker J. B. Linkage of extracellular plasminogen activator to the fibroblast cytoskeleton: colocalization of cell surface urokinase with vinculin. J Cell Biol. 1988 Apr;106(4):1241–1247. doi: 10.1083/jcb.106.4.1241. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Keller G. A., Siegel M. W., Caras I. W. Endocytosis of glycophospholipid-anchored and transmembrane forms of CD4 by different endocytic pathways. EMBO J. 1992 Mar;11(3):863–874. doi: 10.1002/j.1460-2075.1992.tb05124.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Kirchheimer J. C., Christ G., Binder B. R. Growth stimulation of human epidermal cells by urokinase is restricted to the intact active enzyme. Eur J Biochem. 1989 Apr 15;181(1):103–107. doi: 10.1111/j.1432-1033.1989.tb14699.x. [DOI] [PubMed] [Google Scholar]
  19. Kurzchalia T. V., Dupree P., Parton R. G., Kellner R., Virta H., Lehnert M., Simons K. VIP21, a 21-kD membrane protein is an integral component of trans-Golgi-network-derived transport vesicles. J Cell Biol. 1992 Sep;118(5):1003–1014. doi: 10.1083/jcb.118.5.1003. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Lee S. W., Ellis V., Dichek D. A. Characterization of plasminogen activation by glycosylphosphatidylinositol-anchored urokinase. J Biol Chem. 1994 Jan 28;269(4):2411–2418. [PubMed] [Google Scholar]
  21. Li H., Kuo A., Kochan J., Strickland D., Kariko K., Barnathan E. S., Cines D. B. Endocytosis of urokinase-plasminogen activator inhibitor type 1 complexes bound to a chimeric transmembrane urokinase receptor. J Biol Chem. 1994 Mar 18;269(11):8153–8158. [PubMed] [Google Scholar]
  22. Lisanti M. P., Scherer P. E., Vidugiriene J., Tang Z., Hermanowski-Vosatka A., Tu Y. H., Cook R. F., Sargiacomo M. Characterization of caveolin-rich membrane domains isolated from an endothelial-rich source: implications for human disease. J Cell Biol. 1994 Jul;126(1):111–126. doi: 10.1083/jcb.126.1.111. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Lisanti M. P., Tang Z. L., Sargiacomo M. Caveolin forms a hetero-oligomeric protein complex that interacts with an apical GPI-linked protein: implications for the biogenesis of caveolae. J Cell Biol. 1993 Nov;123(3):595–604. doi: 10.1083/jcb.123.3.595. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. 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]
  25. Mayor S., Rothberg K. G., Maxfield F. R. Sequestration of GPI-anchored proteins in caveolae triggered by cross-linking. Science. 1994 Jun 24;264(5167):1948–1951. doi: 10.1126/science.7516582. [DOI] [PubMed] [Google Scholar]
  26. McNeill H., Jensen P. J. A high-affinity receptor for urokinase plasminogen activator on human keratinocytes: characterization and potential modulation during migration. Cell Regul. 1990 Oct;1(11):843–852. doi: 10.1091/mbc.1.11.843. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Miles L. A., Dahlberg C. M., Levin E. G., Plow E. F. Gangliosides interact directly with plasminogen and urokinase and may mediate binding of these fibrinolytic components to cells. Biochemistry. 1989 Nov 28;28(24):9337–9343. doi: 10.1021/bi00450a014. [DOI] [PubMed] [Google Scholar]
  28. Miles L. A., Levin E. G., Plescia J., Collen D., Plow E. F. Plasminogen receptors, urokinase receptors, and their modulation on human endothelial cells. Blood. 1988 Aug;72(2):628–635. [PubMed] [Google Scholar]
  29. 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]
  30. Mohanam S., Sawaya R., McCutcheon I., Ali-Osman F., Boyd D., Rao J. S. Modulation of in vitro invasion of human glioblastoma cells by urokinase-type plasminogen activator receptor antibody. Cancer Res. 1993 Sep 15;53(18):4143–4147. [PubMed] [Google Scholar]
  31. Montgomery A. M., De Clerck Y. A., Langley K. E., Reisfeld R. A., Mueller B. M. Melanoma-mediated dissolution of extracellular matrix: contribution of urokinase-dependent and metalloproteinase-dependent proteolytic pathways. Cancer Res. 1993 Feb 1;53(3):693–700. [PubMed] [Google Scholar]
  32. Mueller B. M., Romerdahl C. A., Trent J. M., Reisfeld R. A. Suppression of spontaneous melanoma metastasis in scid mice with an antibody to the epidermal growth factor receptor. Cancer Res. 1991 Apr 15;51(8):2193–2198. [PubMed] [Google Scholar]
  33. 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]
  34. 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]
  35. 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]
  36. Odekon L. E., Sato Y., Rifkin D. B. Urokinase-type plasminogen activator mediates basic fibroblast growth factor-induced bovine endothelial cell migration independent of its proteolytic activity. J Cell Physiol. 1992 Feb;150(2):258–263. doi: 10.1002/jcp.1041500206. [DOI] [PubMed] [Google Scholar]
  37. 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]
  38. Parton R. G. Ultrastructural localization of gangliosides; GM1 is concentrated in caveolae. J Histochem Cytochem. 1994 Feb;42(2):155–166. doi: 10.1177/42.2.8288861. [DOI] [PubMed] [Google Scholar]
  39. Pepper M. S., Sappino A. P., Stöcklin R., Montesano R., Orci L., Vassalli J. D. Upregulation of urokinase receptor expression on migrating endothelial cells. J Cell Biol. 1993 Aug;122(3):673–684. doi: 10.1083/jcb.122.3.673. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Plesner T., Ploug M., Ellis V., Rønne E., Høyer-Hansen G., Wittrup M., Pedersen T. L., Tscherning T., Danø K., Hansen N. E. The receptor for urokinase-type plasminogen activator and urokinase is translocated from two distinct intracellular compartments to the plasma membrane on stimulation of human neutrophils. Blood. 1994 Feb 1;83(3):808–815. [PubMed] [Google Scholar]
  41. 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]
  42. Pöllänen J., Hedman K., Nielsen L. S., Danø K., Vaheri A. Ultrastructural localization of plasma membrane-associated urokinase-type plasminogen activator at focal contacts. J Cell Biol. 1988 Jan;106(1):87–95. doi: 10.1083/jcb.106.1.87. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Pöllänen J., Saksela O., Salonen E. M., Andreasen P., Nielsen L., Danø K., Vaheri A. Distinct localizations of urokinase-type plasminogen activator and its type 1 inhibitor under cultured human fibroblasts and sarcoma cells. J Cell Biol. 1987 Apr;104(4):1085–1096. doi: 10.1083/jcb.104.4.1085. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Rabbani S. A., Desjardins J., Bell A. W., Banville D., Mazar A., Henkin J., Goltzman D. An amino-terminal fragment of urokinase isolated from a prostate cancer cell line (PC-3) is mitogenic for osteoblast-like cells. Biochem Biophys Res Commun. 1990 Dec 31;173(3):1058–1064. doi: 10.1016/s0006-291x(05)80893-9. [DOI] [PubMed] [Google Scholar]
  45. Rothberg K. G., Heuser J. E., Donzell W. C., Ying Y. S., Glenney J. R., Anderson R. G. Caveolin, a protein component of caveolae membrane coats. Cell. 1992 Feb 21;68(4):673–682. doi: 10.1016/0092-8674(92)90143-z. [DOI] [PubMed] [Google Scholar]
  46. Rothberg K. G., Ying Y. S., Kamen B. A., Anderson R. G. Cholesterol controls the clustering of the glycophospholipid-anchored membrane receptor for 5-methyltetrahydrofolate. J Cell Biol. 1990 Dec;111(6 Pt 2):2931–2938. doi: 10.1083/jcb.111.6.2931. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Sargiacomo M., Sudol M., Tang Z., Lisanti M. P. Signal transducing molecules and glycosyl-phosphatidylinositol-linked proteins form a caveolin-rich insoluble complex in MDCK cells. J Cell Biol. 1993 Aug;122(4):789–807. doi: 10.1083/jcb.122.4.789. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Shenoy-Scaria A. M., Kwong J., Fujita T., Olszowy M. W., Shaw A. S., Lublin D. M. Signal transduction through decay-accelerating factor. Interaction of glycosyl-phosphatidylinositol anchor and protein tyrosine kinases p56lck and p59fyn 1. J Immunol. 1992 Dec 1;149(11):3535–3541. [PubMed] [Google Scholar]
  49. Smart E. J., Foster D. C., Ying Y. S., Kamen B. A., Anderson R. G. Protein kinase C activators inhibit receptor-mediated potocytosis by preventing internalization of caveolae. J Cell Biol. 1994 Feb;124(3):307–313. doi: 10.1083/jcb.124.3.307. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Stahl A., Mueller B. M. Binding of urokinase to its receptor promotes migration and invasion of human melanoma cells in vitro. Cancer Res. 1994 Jun 1;54(11):3066–3071. [PubMed] [Google Scholar]
  51. 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]
  52. 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]
  53. 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]
  54. Thomas P. M., Samelson L. E. The glycophosphatidylinositol-anchored Thy-1 molecule interacts with the p60fyn protein tyrosine kinase in T cells. J Biol Chem. 1992 Jun 15;267(17):12317–12322. [PubMed] [Google Scholar]
  55. 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]
  56. Ying Y. S., Anderson R. G., Rothberg K. G. Each caveola contains multiple glycosyl-phosphatidylinositol-anchored membrane proteins. Cold Spring Harb Symp Quant Biol. 1992;57:593–604. doi: 10.1101/sqb.1992.057.01.065. [DOI] [PubMed] [Google Scholar]
  57. Zhang S., Laurent M., Lopez-Alemany R., Mazar A., Henkin J., Ronne E., Burtin P. Comparative localization of receptors for plasmin and for urokinase on MCF 7 cells. Exp Cell Res. 1993 Aug;207(2):290–299. doi: 10.1006/excr.1993.1196. [DOI] [PubMed] [Google Scholar]

Articles from The Journal of Cell Biology are provided here courtesy of The Rockefeller University Press

RESOURCES