Urokinase binding and receptor identification in cultured endothelial cells

• Published in: The Journal of biological chemistry Vol. 266; no. 32; pp. 21466 - 21473
• Main Authors: R C Haddock, M L Spell, C D Baker, 3rd, J R Grammer, J M Parks, M Speidel, F M Booyse
• Format: Journal Article
• Language: English
• Published: Bethesda, MD American Society for Biochemistry and Molecular Biology 15-11-1991
• Subjects: Animals; Biological and medical sciences; Cell receptors; Cell structures and functions; Cells, Cultured; Chromatography, Affinity; Electrophoresis, Polyacrylamide Gel; Endothelium, Vascular - metabolism; Fundamental and applied biological sciences. Psychology; Humans; Isoflurophate - pharmacology; Kinetics; Macromolecular Substances; Miscellaneous; Molecular and cellular biology; Molecular Weight; Muscle, Smooth, Vascular; plasminogen activator; Pulmonary Artery; Receptors, Cell Surface - isolation & purification; Receptors, Cell Surface - metabolism; Receptors, Urokinase Plasminogen Activator; Recombinant Proteins - metabolism; Swine; Umbilical Veins; Urokinase-Type Plasminogen Activator - metabolism; Urokinase; Membrane receptor; Vertebrata; Endothelial cell; Binding capacity; Mammalia; Enzyme; Affinity chromatography; Dissociation constant; Recombinant protein
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/S0021-9258(18)54662-7

Recombinant human single-chain urokinase (rscu-PA), two-chain urokinase (tcu-PA), and diisopropyl-fluorophosphate-treated
tcu-PA (DFP-tcu-PA) bound to cultured human and porcine endothelial cells in a rapid, saturable, dose-dependent and reversible
manner. Analysis of specific binding results in cultured human umbilical vein endothelial cells (HUVECs) gave the following
estimated values for Kd and Bmax: 0.57 +/- 0.08 nM (mean +/- S.E.) and 188,000 +/- 18,000 sites/cell for 125I-labeled rscu-PA;
0.54 +/- 0.10 nM and 132,000 +/- 23,900 sites/cells for 125I-labeled tcu-PA; 0.89 +/- 0.14 nM and 143,000 +/- 30,300 sites/cell
for 125I-labeled DFP-tcu-PA, respectively. Values for Kd were similar for primary and subcultured (six passages) HUVECs, but
Bmax values were lower in subcultured HUVECs. Similar Kd values were found in cultured porcine endothelial cells; however,
Bmax values varied depending on the endothelial cell type. All 125I-labeled urokinase forms yielded similar cross-linked approximately
110-kDa ligand-receptor complexes with cultured HUVECs, and 125I-labeled DFP-tcu-PA bound to a single major approximately
55-kDa protein in whole-cell lysates (ligand blotting/autoradiography), suggesting the presence of a single major approximately
55-kDa urokinase receptor in cultured HUVECs. The approximately 55-kDa urokinase receptor, isolated from several separate
batches of cultured HUVECs (3-5 micrograms of protein, approximately 1 x 10(9) cells), by ligand affinity chromatography,
exhibited the following properties: retained biologic activity as evidenced by its ability to bind 125I-labeled rscu-PA by
ligand blotting/autoradiography and formation of a cross-linked 125I-labeled approximately 110-kDa rscu-PA-receptor complex;
single-chain approximately 55-kDa protein, following reduction; complete conversion to and formation of a single major deglycosylated
approximately 35-kDa protein, following treatment with N-glycanase.