Identification and partial characterization by chemical cross-linking of a binding protein for tissue-type plasminogen activator (t-PA) on rat hepatoma cells. A plasminogen activator inhibitor type 1-independent t-PA receptor.
Plasma tissue-type plasminogen activator (t-PA) is cleared rapidly in vivo by the liver. Previous studies with the human hepatoma cell line HepG2 have identified a clearance system for t-PA modulated by plasminogen activator inhibitor type 1 (PAI-1). In the present study, a rat hepatoma cell line MH1C1 is shown to contain a PAI-1-independent t-PA clearance system. At 4 degrees C, binding of 125I-t-PA to MH1C1 cells was rapid, specific, and saturable. Scatchard analysis of the binding data yielded a mean estimate of 105,000 high affinity binding sites per cell (Kd = 4.1 nM). When the bound ligand was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the majority (about 90%) of the specific binding was in the form of uncomplexed 125I-t-PA. This is in contrast to HepG2 cells in which specific binding was mainly in the form of a sodium dodecyl sulfate-stable 125I-t-PA.PAI-1 complex. When availability of matrix-associated PAI-1 was blocked by preincubation with anti-PAI-1 antibody or removed by elastase treatment, specific 125I-t-PA binding to MH1C1 cells was unaffected, whereas most of the specific 125I-t-PA binding to HepG2 cells was abolished. Furthermore, when the active site of t-PA was inactivated with diisopropyl fluorophosphate, the diisopropyl fluorophosphate-t-PA specifically competed for binding of 125I-t-PA to MH1C1 cells, but failed to block specific 125I-t-PA binding to HepG2 cells. At 37 degrees C, PAI-1-independent t-PA binding to MH1C1 cells was followed by ligand uptake and degradation with kinetics similar to that seen in HepG2 cells. Chemical cross-linking of t-PA to MH1C1 cells revealed a specific t-PA binding protein with a molecular mass of about 500,000 daltons. Ligand-receptor complexes generated by chemical cross-linking were immunoprecipitable by anti-t-PA antibody but not by anti-PAI-1 antibody, further supporting the finding that binding of t-PA to MH1C1 cells is PAI-1-independent.
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Edward Mallinckrodt Department of Pediatrics, Washington University School of Medicine St. Louis, Missouri 63110.
Morton
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Schwartz
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D000818:Animals; D001665:Binding Sites; D002462:Cell Membrane / Q000378:metabolism; D003432:Cross-Linking Reagents; D003545:Cysteine / Q000378:metabolism; D007457:Iodine Radioisotopes; D007531:Isoflurophate / Q000494:pharmacology; D007700:Kinetics; D008114:Liver Neoplasms, Experimental / Q000378:metabolism*; D015849:Plasminogen Inactivators / Q000494:pharmacology*; D051381:Rats; D011956:Receptors, Cell Surface / Q000187:drug effects / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D010959:Tissue Plasminogen Activator / Q000378:metabolism*
D003432:Cross-Linking Reagents; D007457:Iodine Radioisotopes; D015849:Plasminogen Inactivators; D011956:Receptors, Cell Surface; D011994:Recombinant Proteins; D007531:Isoflurophate; C067262:PLAT protein, human; D010959:Tissue Plasminogen Activator; D003545:Cysteine