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

• Published in: The EMBO journal Vol. 9; no. 2; pp. 467 - 474
• Main Authors: Roldan, A.L., Cubellis, M.V., Masucci, M.T., Behrendt, N., Lund, L.R., Danø, K., Appella, E., Blasi, F.
• Format: Journal Article
• Language: English
• Published: London Nature Publishing Group 01.02.1990
• Subjects: Amino Acid Sequence; Animals; Base Sequence; Biological and medical sciences; Blotting, Northern; Cloning, Molecular; Enzyme Precursors - metabolism; Fluorescent Antibody Technique; Fundamental and applied biological sciences. Psychology; Gene Expression; Gene Library; genes; Genes. Genome; Humans; Kinetics; L Cells - metabolism; man; Mice; Molecular and cellular biology; Molecular genetics; Molecular Sequence Data; Oligonucleotide Probes; plasminogen activator receptors; Plasminogen Activators - metabolism; Receptors, Cell Surface - genetics; Receptors, Cell Surface - metabolism; Receptors, Urokinase Plasminogen Activator; RNA - genetics; RNA - isolation & purification; Transfection; Human; Radiolabelling; Nucleotide sequence; Gel electrophoresis; Enzyme; Molecular interaction; Hydrophobicity; Gene expression; Cell surface; Northern blotting; Urokinase; Transfection; Complementary DNA; Molecular cloning; Immunofluorescence; Biological receptor
• ISSN: 0261-4189; 1460-2075
• DOI: 10.1002/j.1460-2075.1990.tb08132.x

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 cross‐linking 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 approximately 55,000, in accordance with the naturally occurring, highly glycosylated human uPAR. The Mr calculated on the basis of the cDNA sequence, approximately 35,000, agrees well with that of the deglycosylated receptor.