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Receptors for plasminogen activator, urokinase, in normal and Rous sarcoma virus-transformed mouse fibroblasts
Abstract:
We have prepared a conjugate of the plasminogen activator urokinase (UK) and ferritin, which maintains fibrinolytic activity. Monolayers of BALB/c-3T3 cells and of Rous sarcoma virus-transformed highly malignant line AA12-3T3, subcultured in plasminogen-free serum, were incubated with UK-ferritin at 0 degree and processed for transmission electron microscopy. Under these conditions, both of the lines showed specific receptors on the cell surface that were distributed in singlets, in small or large clusters. In the presence of excess native UK, the binding of ferritin was reduced by 99%, indicating the interaction of UK:ferritin with a specific receptor. The ligand-receptor interaction involves the catalytic site of UK, since the binding was completely impaired by preincubation of UK:ferritin with p-aminobenzamidine, a competitive inhibitor of the catalytic site of UK. The number and density of receptors decreased about one order of magnitude on the membrane of AA12 cells when compared with normal 3T3 cells. Saturation kinetics, using 125I-labeled UK, indicate the presence of 4 X 10(4) and 2.5 X 10(3) receptors on the membrane of 3T3 and AA12 cells, respectively. At 37 degrees, UK:ferritin redistributed on the plane of the membrane, in a process which was faster in malignant than in normal cells. Ferritin particles clustered in large groups on coated areas of the surface and were internalized by adsorptive pinocytosis. After 10 min at 37 degrees, the vesicles showed a progressively deeper internalization and a fusion with lysosomes, and some were observed in the Golgi complex area. Since the experiments were planned in order to exclude the presence of protease-nexin in the incubation medium, these data suggest the existence of a plasminogen-independent novel receptor for the catalytic site of plasminogen activators, the number on the cell surface of which decreases in Rous sarcoma virus-transformed mouse fibroblasts.