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BM 06.022) with human umbilical vein endothelial cells
12
32
ORAL COMMUNICATIONS l-2: Plasminogen
COMPARISON PLASMINOGEN PLASMINOGEN
Activators
OF
THE INTERACTION OF TISSUE TYPE ACTIVATOR AND RECOMBINANT ACTIVATOR (r-PA/BM 06.022) WITH HUMAN
UMBILICAL VEIN ENDOTHELIAL CELLS ‘Mulder M, ‘Kohnert U, ‘Fischer S, and ‘Verheijen JH ‘Gnublus Institute, TNO-PG. PO Box 430, NL-2300 AP Leiden, The Netherlands, ‘Boehrlnger Mannheim GmbH, Biochemical Research Center Penzberg, Nonnenwald 2, D-81 22 Penrberg, Germany. The interaction of rt-PA and E.coli-expressed recombinant plasminogen activator (r-PA) comprising kringle 2 and proteases domains of human t-PA, with human umbilical vein endothelial cells was compared. At 4°C and at 37°C both radiolabeled rt-PA and r-PA bound to the cells, although the latter with a two-fold lower affinrty. At 37°C the binding of rtPA and r-PA was followed by internalisation and subsequently by degradation, which has to our knowledge not yet been reported. The binding of both rt-PA and r-PA was inhibited in the presence respectively
of a 300.fold excess of unlabeled rt-PA and r-PA, to 53 and 40% of the control value, indicating
that part of the binding is specific. Also the degradation of rtPA was partly specific, while the degradation of r-PA was almost completely non-specific. The degradation of both
33 A SINGLE AMINO ACID SUBSTITUTION Asn 22 Tyr IN THE GROWTH FACTOR DOMAIN OF U-PA INHIBITS THE BINDING TO ITS RECEPTOR u-PAR M. Lansink. P.H.A. Ouax. A.H.F. Bakker and J.H. Verheiien, Gaubius Laboratory,TNO-PG, Leiden, the Netherlands Urokinase-type plasminogen activator (u-PA), esp. when it is bound to its receptor (U-PAR) is playing a major role in local proteolytic processes occurring during cell migration and tumor cell invasion. The receptor binding domain of u-PA has been localized to a region in the growth factor domain between residues 20 and 32. Since the binding of u-PA to u-PAR is rather species specific, we have used the differences in amino acid sequences between various species to determine how to construct a non-receptor binding human u-PA variant. We have substituted Asn 22 by its mouse analog Tyr by the use of site directed mutagenesis. This mutant u-PA had similar
34 SINGLE CHAIN UROKINASE TYPE PLASMINOGEN ACTIVATOR
(SCU-PA) COMPLEXES WITH AND IS INHIBITED BY PLASMINOGEN ACTIVATOR INHIBITOR TYPE 1 (PAI-1). Manchanda N and Schwartz BS Department of Medicine, University of Wisconsin, Madison, WI, USA.
Because the enzymatic activity of scu-PA is required ior cell surface plasminogen activation, we utilized recombinant proteins to study the regulation of this enzymatic activity by PAI-1. PAI- inhibited the PA activities of both native scu-PA and the mutant G~u'~ scu-PA (not cleaved by plasmin to tcu-PA) in a concentration dependent This inhibition was evident in the manner. functional '*?-Fibrin plate assay, and the plasmin independent "'I-plasminogencleavage assay. AntiPAI- antibodies blocked this inhibition:control antibodies did not. Inhibition of scu-PA by PAI1 occurred in the context of SDS-stable complexes being formed between the two molecules, as incubation of '251-scu-PAwith PAI- yielded 1251-
proteins lysosomal
was partially degradation.
inhibited Cross
by chloroquine, competition
indicating experiments
indicated that rt-PA and r-PA share, at least partly, the same bInding sites. However the binding and association of radiolabeled rt-PA and r-PA could only be inhibited to 50% of the control value. The binding of rt-PA and r-PA was also inhibited in the presence of 75 nM of urokinase (to 49 and 57% of the control had been functionally
value, respectively). R-PA and r-PA that inactivated with D-phenylalanyl-L-prolyl-
L-arginlne chloromethyl ketone exhibited a reduced cell assoctation and degradation when compared with non-treated rt-PA and r-PA. These experiments suggest a role for PAI-1, In conclusion, these results indicate that both I-t-PA and r-PA bind to endothelial cells, and are subsequently internalized and degraded. The binding, internalisation and degradation of r-PA was relatively lower. Part of the interaction of rt-PA and r-PA with the cells is mediated through PAI-1. However, besides PAM1 other binding sites exist which may be different for rt-PA and r-PA. It remains to be established whether the vast area of endothelial cells in the vascular tree may contribute to the blnding and degradation of the plasminogen activators in viva.
plasminogen activator characteristics as wildtype u-PA concerning specific activity and interaction with PAI-1. However, in cross-linking experiments using DFP-treated 1251-labeled mutant U-PA and lysates of various cell lines including U937, PMA treated endothelial cells and mouse LB6 cells transfected with human U-PAR cDNA no uPA/u-PAR complexes could be observed. In direct binding experiments DFP-treated ‘251-labeled mutant U-PA could not bind to PMA-treated endothelial cells whereas wildtype u-PA did bind. Furthermore, binding of wildtype u-PA to the receptor on LB6 cells was not prevented in the presence of 25 fold excess of mutant u-PA. By the use of ligand blotting, however, very weak binding of mutant u-PA to human u-PAR could be observed. We conclude that Asn 22 in human u-PA comprises a key determinant’ in the receptor binding of u-PA.
labelled complexes of 100 kD on SDS-PAGE. In contrast, although PAI- inhibited the enzymatic activity of scu-PA, it did not form SDS-stable complexes with 1251-~~~-PA. Formation of 'z51-scuPAaPAI-1 complexes was time and concentration dependent. In addition, tcu-PA and scu-PA competed with one another for complex formation with PAI-1. Interestingly, although all enzymatic activity within a population of scu-PA molecules was inhibited by PAI-1, only a minority of lz51scu-PA molecules formed SDS-stable complexes with PAI-1, even at a 100 fold molar excess of inhibitor. This property was uni~,~,,to.othe scuPA.PAI-1 interaction, as ’ ’ molar concentrations of tcu-PA showed complete complex formation with PAI-1. Vitronectin, the physiological PAIbinding protein did not influence the formation of SDS-stable complexes between '251-scu-PA and PAI-1. We hypothesize that this interaction may play a role in regulating the enzymatic activity of scu-PA.
32
ORAL COMMUNICATIONS l-2: Plasminogen
COMPARISON PLASMINOGEN PLASMINOGEN
Activators
OF
THE INTERACTION OF TISSUE TYPE ACTIVATOR AND RECOMBINANT ACTIVATOR (r-PA/BM 06.022) WITH HUMAN
UMBILICAL VEIN ENDOTHELIAL CELLS ‘Mulder M, ‘Kohnert U, ‘Fischer S, and ‘Verheijen JH ‘Gnublus Institute, TNO-PG. PO Box 430, NL-2300 AP Leiden, The Netherlands, ‘Boehrlnger Mannheim GmbH, Biochemical Research Center Penzberg, Nonnenwald 2, D-81 22 Penrberg, Germany. The interaction of rt-PA and E.coli-expressed recombinant plasminogen activator (r-PA) comprising kringle 2 and proteases domains of human t-PA, with human umbilical vein endothelial cells was compared. At 4°C and at 37°C both radiolabeled rt-PA and r-PA bound to the cells, although the latter with a two-fold lower affinrty. At 37°C the binding of rtPA and r-PA was followed by internalisation and subsequently by degradation, which has to our knowledge not yet been reported. The binding of both rt-PA and r-PA was inhibited in the presence respectively
of a 300.fold excess of unlabeled rt-PA and r-PA, to 53 and 40% of the control value, indicating
that part of the binding is specific. Also the degradation of rtPA was partly specific, while the degradation of r-PA was almost completely non-specific. The degradation of both
33 A SINGLE AMINO ACID SUBSTITUTION Asn 22 Tyr IN THE GROWTH FACTOR DOMAIN OF U-PA INHIBITS THE BINDING TO ITS RECEPTOR u-PAR M. Lansink. P.H.A. Ouax. A.H.F. Bakker and J.H. Verheiien, Gaubius Laboratory,TNO-PG, Leiden, the Netherlands Urokinase-type plasminogen activator (u-PA), esp. when it is bound to its receptor (U-PAR) is playing a major role in local proteolytic processes occurring during cell migration and tumor cell invasion. The receptor binding domain of u-PA has been localized to a region in the growth factor domain between residues 20 and 32. Since the binding of u-PA to u-PAR is rather species specific, we have used the differences in amino acid sequences between various species to determine how to construct a non-receptor binding human u-PA variant. We have substituted Asn 22 by its mouse analog Tyr by the use of site directed mutagenesis. This mutant u-PA had similar
34 SINGLE CHAIN UROKINASE TYPE PLASMINOGEN ACTIVATOR
(SCU-PA) COMPLEXES WITH AND IS INHIBITED BY PLASMINOGEN ACTIVATOR INHIBITOR TYPE 1 (PAI-1). Manchanda N and Schwartz BS Department of Medicine, University of Wisconsin, Madison, WI, USA.
Because the enzymatic activity of scu-PA is required ior cell surface plasminogen activation, we utilized recombinant proteins to study the regulation of this enzymatic activity by PAI-1. PAI- inhibited the PA activities of both native scu-PA and the mutant G~u'~ scu-PA (not cleaved by plasmin to tcu-PA) in a concentration dependent This inhibition was evident in the manner. functional '*?-Fibrin plate assay, and the plasmin independent "'I-plasminogencleavage assay. AntiPAI- antibodies blocked this inhibition:control antibodies did not. Inhibition of scu-PA by PAI1 occurred in the context of SDS-stable complexes being formed between the two molecules, as incubation of '251-scu-PAwith PAI- yielded 1251-
proteins lysosomal
was partially degradation.
inhibited Cross
by chloroquine, competition
indicating experiments
indicated that rt-PA and r-PA share, at least partly, the same bInding sites. However the binding and association of radiolabeled rt-PA and r-PA could only be inhibited to 50% of the control value. The binding of rt-PA and r-PA was also inhibited in the presence of 75 nM of urokinase (to 49 and 57% of the control had been functionally
value, respectively). R-PA and r-PA that inactivated with D-phenylalanyl-L-prolyl-
L-arginlne chloromethyl ketone exhibited a reduced cell assoctation and degradation when compared with non-treated rt-PA and r-PA. These experiments suggest a role for PAI-1, In conclusion, these results indicate that both I-t-PA and r-PA bind to endothelial cells, and are subsequently internalized and degraded. The binding, internalisation and degradation of r-PA was relatively lower. Part of the interaction of rt-PA and r-PA with the cells is mediated through PAI-1. However, besides PAM1 other binding sites exist which may be different for rt-PA and r-PA. It remains to be established whether the vast area of endothelial cells in the vascular tree may contribute to the blnding and degradation of the plasminogen activators in viva.
plasminogen activator characteristics as wildtype u-PA concerning specific activity and interaction with PAI-1. However, in cross-linking experiments using DFP-treated 1251-labeled mutant U-PA and lysates of various cell lines including U937, PMA treated endothelial cells and mouse LB6 cells transfected with human U-PAR cDNA no uPA/u-PAR complexes could be observed. In direct binding experiments DFP-treated ‘251-labeled mutant U-PA could not bind to PMA-treated endothelial cells whereas wildtype u-PA did bind. Furthermore, binding of wildtype u-PA to the receptor on LB6 cells was not prevented in the presence of 25 fold excess of mutant u-PA. By the use of ligand blotting, however, very weak binding of mutant u-PA to human u-PAR could be observed. We conclude that Asn 22 in human u-PA comprises a key determinant’ in the receptor binding of u-PA.
labelled complexes of 100 kD on SDS-PAGE. In contrast, although PAI- inhibited the enzymatic activity of scu-PA, it did not form SDS-stable complexes with 1251-~~~-PA. Formation of 'z51-scuPAaPAI-1 complexes was time and concentration dependent. In addition, tcu-PA and scu-PA competed with one another for complex formation with PAI-1. Interestingly, although all enzymatic activity within a population of scu-PA molecules was inhibited by PAI-1, only a minority of lz51scu-PA molecules formed SDS-stable complexes with PAI-1, even at a 100 fold molar excess of inhibitor. This property was uni~,~,,to.othe scuPA.PAI-1 interaction, as ’ ’ molar concentrations of tcu-PA showed complete complex formation with PAI-1. Vitronectin, the physiological PAIbinding protein did not influence the formation of SDS-stable complexes between '251-scu-PA and PAI-1. We hypothesize that this interaction may play a role in regulating the enzymatic activity of scu-PA.