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236 Candidate cell surface plasminogen receptors
101
234UROKINASE BINDING TO ITS CELLULAR RECEPTORS IS MODULATED BY GANGLIOSIDES. L. A. Miles, E. 6. Levin and E. F. Plow l??se;r;hA Institute of Scripps Clinic, La Jolla,
,
. . .
Cellular receptors for urokinase are widely distributed and contribute to the regulation of fibrinolysis. Gangliosides, sialic acid containing glycosphingolipids, are also widely distributed and serve as receptors for a variety of ligands. We have tested whether gangliosides are involved in or contribute to urokinase receptor function. A panel of representative gangliosides inhibited urokinase binding to U937 monocytoid cells. The inhibition was structurally specific as the inhibitory gangliosides GDla, GM1 and GTlb, with four neutral sugar residues, were more effective than GM2, with three neutral sugar residues. GM3, with only two neutral sugars and lacking the N-acetyl-D-galactosamine moiety, was
235BINDING
AND DEGRADATION OF TISSUE-TYPE PLASMINOGEN ACTIVATOR BY THE HEPATOMA CELL LINE HEP G2 (l), T.J.C. van Berkel (2) and D.C. M. Otter Rijken (1) Gaubius Institute TNO. Leiden (1) and Div. of Univ. of Leiden the Biopharm. BFW, (21, Netherlands. Tissue-type plasminogen activator (t-PA) is rapidly cleared from the blood circulation by the high affinity uptake liver. Recently a novel system on rat hepatocytes has been postulated. Here we studied the catabolism of t-PA by the human hepatoma ce&,$line Hep G2. Hep G2 cells were I-labelled t-PA (Bowes melaincubated with noma). Binding was measured at 4°C and degradation at 37°C and both are expressed 1% a percentage of the concentration of added I-t-PA (15 PM). Binding was time dependent and reached a maximum (2.2%) after 2 hrs. Excess of unlabelled t-PA (800 nM) inhibited binding and degradation by about 60%. To study the remaining 40% of the binding
236CANDIDATE
CELL SURFACE PLASMINOGEN RECEPTORS. E. F. Plow and L. A. Miles Research Institute of Scripps Clinic, La Jolla, CA, U.S.A. Plasminogen receptors, recognizing the lysine binding sites in the ligand, are expressed at very high density on circulating and adherent cells. As several components can interact with these plasminogen structures, we have considered the potential of both proteinaceous and nonproteinaceous cell surface molecules to serve as plasminogen receptors. Based on studies with synthetic peptides, proteins with carboxy-terminal lysine residues could provide plasminogen binding sites. Peptides with carboxy-terminal lysine residues inhibited plasminogen binding by 55-85% at mM concentrations while peptides with amino terminal or internal lysine residues inhibited binding by I 2% at the same concentrations. The peptides with carboxy-terminal lysines were more potent than free lysine or poly-L-lysine. As
minimally inhibitory. The individual hexose or lipid moieties of gangliosidesgalactose, Nacetyl-D-galactosamine, N-acetylneuraminic acid, ceramides, and galactocerebrosides, were noninhibitory. The gangliosides did not inhibit binding of another ligand, a monoclonal antibody to the MHC complex, but did interfere with urokinase binding to granulocytes and endothelial cells, indicating a specific effect on urokinase:receptor interactions. 1251-urokinase bound directly to insolubilized gangliosides. This binding was specific and saturable as it was 50% inhibited by 11 nM unlabeled urokinase but was not inhibited by 10 pM transferrin or albumin. In addition, the soluble gangliosides that inhibited urokin se binding to cells also with !2gIcompeted urokinase for binding to insolubilized gangliosides. When U937 cells were fed gangliosides, the extent of urokinase binding increased 2-4fold. These results suggest that cellular gangliosides may mediate or modulate the interaction of urokinase with a variety of cells.
I-t-PA was pre-incubated with 0.2 mM D-Phe-Pro-Arg-CH CL (PPACK) preventing complex formation with p 1 asminogen activator inhibitors. To avoid the development of an extracellular matrix containing t-PA-binding proteins such as fibrin, fibronectin and PAI-1, we also cultured the hepatoma cells with heparin (5 ug/ml). As a consequence, nonspecific binding decreased from 40 to 15%. so, under the present incubation circumstances 25% of the t-PA binding to Hep G2 cells should be ascribed to binding to the extracellular matrix. After yz:;y att l&yty~;~ degradation of k2! ff,_od, sine, was 1.2%. Degradation could be inhibited by NH Cl (10 mM), chloroquine (100 uM) and NaN mt49. N-acetylgalactosamine (10 mM), ovalbumia i:i uM) and asialofetuin (1P25uM) did not affect degradation or binding of I-t-PA. Our results suggest a specific mechanism for the binding and degradation of t-PA by Hep G2 cells, which is not mediated by known carbohydrate receptors for galactose or mannose.
nonproteinaceous candidate plasminogen receptors, we found that a panel of representative gangliosides (sialic acid-containing glycosphingolipids) inhibited plasminogen binding to cells in a specific structurally manner. For example, ganglioside, GDla inhibited binding by? 90% at a concentration where GM3 was noninhibitory. Individual hexose and lipid components of gangliosides were also noninhibitory. In a purified plasminogen bound specifically and system, saturably to insolubilized gangliosides. Taken together, these results suggest that more than one cell surface component, either proteinaceous or nonproteinaceous in character, can interact with plasminogen and could serve as cell sur ce &Ireceptors. Consistent with this possibility, plasminogen bound to more than one cellular constituent after electrophoresis of cell extracts in ligand blotting experiments. Thus, a family of cell surface constituents may serve as plasminogen receptors by interacting with the ligand with a similar recognition specificity and affinity.
234UROKINASE BINDING TO ITS CELLULAR RECEPTORS IS MODULATED BY GANGLIOSIDES. L. A. Miles, E. 6. Levin and E. F. Plow l??se;r;hA Institute of Scripps Clinic, La Jolla,
,
. . .
Cellular receptors for urokinase are widely distributed and contribute to the regulation of fibrinolysis. Gangliosides, sialic acid containing glycosphingolipids, are also widely distributed and serve as receptors for a variety of ligands. We have tested whether gangliosides are involved in or contribute to urokinase receptor function. A panel of representative gangliosides inhibited urokinase binding to U937 monocytoid cells. The inhibition was structurally specific as the inhibitory gangliosides GDla, GM1 and GTlb, with four neutral sugar residues, were more effective than GM2, with three neutral sugar residues. GM3, with only two neutral sugars and lacking the N-acetyl-D-galactosamine moiety, was
235BINDING
AND DEGRADATION OF TISSUE-TYPE PLASMINOGEN ACTIVATOR BY THE HEPATOMA CELL LINE HEP G2 (l), T.J.C. van Berkel (2) and D.C. M. Otter Rijken (1) Gaubius Institute TNO. Leiden (1) and Div. of Univ. of Leiden the Biopharm. BFW, (21, Netherlands. Tissue-type plasminogen activator (t-PA) is rapidly cleared from the blood circulation by the high affinity uptake liver. Recently a novel system on rat hepatocytes has been postulated. Here we studied the catabolism of t-PA by the human hepatoma ce&,$line Hep G2. Hep G2 cells were I-labelled t-PA (Bowes melaincubated with noma). Binding was measured at 4°C and degradation at 37°C and both are expressed 1% a percentage of the concentration of added I-t-PA (15 PM). Binding was time dependent and reached a maximum (2.2%) after 2 hrs. Excess of unlabelled t-PA (800 nM) inhibited binding and degradation by about 60%. To study the remaining 40% of the binding
236CANDIDATE
CELL SURFACE PLASMINOGEN RECEPTORS. E. F. Plow and L. A. Miles Research Institute of Scripps Clinic, La Jolla, CA, U.S.A. Plasminogen receptors, recognizing the lysine binding sites in the ligand, are expressed at very high density on circulating and adherent cells. As several components can interact with these plasminogen structures, we have considered the potential of both proteinaceous and nonproteinaceous cell surface molecules to serve as plasminogen receptors. Based on studies with synthetic peptides, proteins with carboxy-terminal lysine residues could provide plasminogen binding sites. Peptides with carboxy-terminal lysine residues inhibited plasminogen binding by 55-85% at mM concentrations while peptides with amino terminal or internal lysine residues inhibited binding by I 2% at the same concentrations. The peptides with carboxy-terminal lysines were more potent than free lysine or poly-L-lysine. As
minimally inhibitory. The individual hexose or lipid moieties of gangliosidesgalactose, Nacetyl-D-galactosamine, N-acetylneuraminic acid, ceramides, and galactocerebrosides, were noninhibitory. The gangliosides did not inhibit binding of another ligand, a monoclonal antibody to the MHC complex, but did interfere with urokinase binding to granulocytes and endothelial cells, indicating a specific effect on urokinase:receptor interactions. 1251-urokinase bound directly to insolubilized gangliosides. This binding was specific and saturable as it was 50% inhibited by 11 nM unlabeled urokinase but was not inhibited by 10 pM transferrin or albumin. In addition, the soluble gangliosides that inhibited urokin se binding to cells also with !2gIcompeted urokinase for binding to insolubilized gangliosides. When U937 cells were fed gangliosides, the extent of urokinase binding increased 2-4fold. These results suggest that cellular gangliosides may mediate or modulate the interaction of urokinase with a variety of cells.
I-t-PA was pre-incubated with 0.2 mM D-Phe-Pro-Arg-CH CL (PPACK) preventing complex formation with p 1 asminogen activator inhibitors. To avoid the development of an extracellular matrix containing t-PA-binding proteins such as fibrin, fibronectin and PAI-1, we also cultured the hepatoma cells with heparin (5 ug/ml). As a consequence, nonspecific binding decreased from 40 to 15%. so, under the present incubation circumstances 25% of the t-PA binding to Hep G2 cells should be ascribed to binding to the extracellular matrix. After yz:;y att l&yty~;~ degradation of k2! ff,_od, sine, was 1.2%. Degradation could be inhibited by NH Cl (10 mM), chloroquine (100 uM) and NaN mt49. N-acetylgalactosamine (10 mM), ovalbumia i:i uM) and asialofetuin (1P25uM) did not affect degradation or binding of I-t-PA. Our results suggest a specific mechanism for the binding and degradation of t-PA by Hep G2 cells, which is not mediated by known carbohydrate receptors for galactose or mannose.
nonproteinaceous candidate plasminogen receptors, we found that a panel of representative gangliosides (sialic acid-containing glycosphingolipids) inhibited plasminogen binding to cells in a specific structurally manner. For example, ganglioside, GDla inhibited binding by? 90% at a concentration where GM3 was noninhibitory. Individual hexose and lipid components of gangliosides were also noninhibitory. In a purified plasminogen bound specifically and system, saturably to insolubilized gangliosides. Taken together, these results suggest that more than one cell surface component, either proteinaceous or nonproteinaceous in character, can interact with plasminogen and could serve as cell sur ce &Ireceptors. Consistent with this possibility, plasminogen bound to more than one cellular constituent after electrophoresis of cell extracts in ligand blotting experiments. Thus, a family of cell surface constituents may serve as plasminogen receptors by interacting with the ligand with a similar recognition specificity and affinity.