Cloning and expression of the receptor for human urokinase plasminogen activator, a central molecule in cell surface, plasmin dependent proteolysis

Ann L. Roldan, Maria Vittoria Cubellis, Maria Teresa Masucci, Niels Behrendt, Leif R. Lund, Keld Danø, Ettore Appella, Francesco Blasi

Research output: Contribution to journalArticle

Abstract

The surface receptor for urokinase plasminogen activator (uPAR) has been recognized in recent years as a key molecule in regulating plasminogen mediated extracellular proteolysis. Surface plasminogen activation controls the connections between cells, basement membrane and extracellular matrix, and therefore the capacity of cells to migrate and invade neighboring tissues. We have isolated a 1.4 kb cDNA clone coding for the entire human uPAR. An oligonucleotide synthesized on the basis of the N-terminal sequence of the purified protein was used to screen a cDNA library made from SV40 transformed human fibroblasts [Okayama and Berg (1983) Mol. Cell Biol., 3, 280-289]. The cDNA encodes a protein of 313 amino acids, preceded by a 21 residue signal peptide. A hydrophobicity plot suggests the presence of a membrane spanning domain close to the C-terminus. The cDNA hybridizes to a 1.4 kb mRNA from human cells, a size very close to that of the cloned cDNA. Expression of the uPAR cDNA in mouse cells confirms that the clone is complete and expresses a functional uPA binding protein, located on the cell surface and with properties similar to the human uPAR. Caseinolytic plaque assay, immunofluorescence analysis, direct binding studies and crosslinking experiments show that the transfected mouse LB6 cells specifically bind human uPA, which in turn activates plasminogen. The Mr of the mature human receptor expressed in mouse cells is ∼ 55 000, in accordance with the naturally occurring, highly glycosylated human uPAR. The Mr calculated on the basis of the cDNA sequence, ∼35 000, agrees well with that of the deglycosylated receptor.

Original languageEnglish
Pages (from-to)467-474
Number of pages8
JournalEMBO Journal
Volume9
Issue number2
Publication statusPublished - 1990

Keywords

  • Extracellular proteolysis
  • Surface receptor
  • Urokinase plasminogen activator

ASJC Scopus subject areas

  • Genetics
  • Cell Biology

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    Roldan, A. L., Cubellis, M. V., Masucci, M. T., Behrendt, N., Lund, L. R., Danø, K., Appella, E., & Blasi, F. (1990). Cloning and expression of the receptor for human urokinase plasminogen activator, a central molecule in cell surface, plasmin dependent proteolysis. EMBO Journal, 9(2), 467-474.