Demonstration of a specific clearance receptor for tissue-type plasminogen activator on rat Novikoff hepatoma cells.
The binding, internalization, and degradation of tissue-type plasminogen activator (t-PA) were studied in a rat hepatoma (Novikoff) cell line. Binding of t-PA to specific saturable high affinity binding sites (Kd = 12 nM, 54,000 sites/cell) was followed by internalization and degradation and did not require a functional active site. The catabolism of t-PA was not inhibited by an excess of urokinase-type plasminogen activator (u-PA), and t-PA bound to Novikoff membranes was not complexed to PAI-1, suggesting a mechanism independent of PAI-1. Additionally, a mannose receptor is not involved since t-PA binding was not influenced by an excess of mannose, galactose, ovalbumin, or EDTA. Furthermore, the degradation of t-PA was not influenced by 10 mM 6-aminohexanoic acid, a lysine analogue. The t-PA receptor binds to and can be eluted from wheat germ agglutinin-Sepharose. Cross-linking of t-PA with partially purified receptor and ligand blot analysis, suggest that t-PA binds to two proteins, a principal one of 55 kDa and a minor one of 43 kDa. Novikoff cells are able also to bind (Kd = 1.4 nM, 25,000 sites/cell) and degrade u-PA. The binding was inhibited by pro-u-PA and the amino-terminal fragment of u-PA, but not by an excess of t-PA. The u-PA receptor, but not the t-PA receptor, was removed by treatment with phosphatidylinositol-specific phospholipase C. Our results show that the clearance receptor for t-PA on Novikoff cells is different from the mannose receptor and the PAI-1-dependent receptor described in other cells. The rat hepatoma cells are thus a good model to study the PAI-1 independent hepatocyte-specific clearance of t-PA.
Nguyen
G
G
Division of Hematology, Centre Hospitalia Universitaire Vaudois, Lausanne, Switzerland.
Self
S J
SJ
Camani
C
C
Kruithof
E K
EK
D015119:Aminocaproic Acid / Q000494:pharmacology; D000818:Animals; D001667:Binding, Competitive; D002462:Cell Membrane / Q000378:metabolism; D002846:Chromatography, Affinity; D004705:Endocytosis; D006801:Humans; D007700:Kinetics; D008114:Liver Neoplasms, Experimental / Q000378:metabolism*; D010446:Peptide Fragments / Q000494:pharmacology; D043265:Phosphatidylinositol Diacylglycerol-Lyase; D054801:Phosphoinositide Phospholipase C; D010727:Phosphoric Diester Hydrolases / Q000378:metabolism / Q000494:pharmacology; D051381:Rats; D011956:Receptors, Cell Surface / Q000187:drug effects / Q000302:isolation & purification / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D010959:Tissue Plasminogen Activator / Q000378:metabolism*; D014568:Urokinase-Type Plasminogen Activator / Q000378:metabolism
D010446:Peptide Fragments; D011956:Receptors, Cell Surface; D011994:Recombinant Proteins; D010727:Phosphoric Diester Hydrolases; D054801:Phosphoinositide Phospholipase C; C067262:PLAT protein, human; D010959:Tissue Plasminogen Activator; D014568:Urokinase-Type Plasminogen Activator; D043265:Phosphatidylinositol Diacylglycerol-Lyase; D015119:Aminocaproic Acid