Regions involved in binding of urokinase-type-1 inhibitor complex and pro-urokinase to the endocytic alpha 2-macroglobulin receptor/low density lipoprotein receptor-related protein. Evidence that the urokinase receptor protects pro-urokinase against binding to the endocytic receptor.
The alpha 2-macroglobulin receptor/low density lipoprotein receptor-related protein (alpha 2MR/LRP) binds several ligands, including complex between the two chain urokinase-type plasminogen activator (uPA) and type-1 plasminogen activator inhibitor (PAI-1), and the single chain zymogen pro-urokinase (pro-uPA). We have used truncated variants of uPA and PAI-1 as well as Fab fragments of monoclonal antibodies with known epitopes to identify regions in the uPA.PAI-1 complex and in pro-uPA involved in binding to alpha 2MR/LRP.uPA.PAI-1 complex bound with high affinity (EC50 about 0.4 nM) via contacts in the PAI-1 moiety as well as the uPA serine proteinase domain and the uPAA chain. Pro-uPA bound with lower affinity (EC50 about 10 nM), and efficient binding to alpha 2MR/LRP was dependent on contact with both the A chain and the serine proteinase domain. We analyzed the effect of complex formation with the urokinase receptor since this is the primary target for binding of uPA.PAI-1 and pro-uPA at the cell surface, and since it has been demonstrated that urokinase receptor-bound uPA.PAI-1 complex is internalized following interaction with alpha 2 MR/LRP (Nykjaer, A., Petersen, C. M., Møller, B., Jensen, P.H., Moestrup, S.K., Holtet, T.L., Etzerodt M., Thøgersen, H.C., Munch, M., Andreasen, P.A., and Gliemann, J. (1992) J. Biol. Chem. 267, 14543-14546). Soluble recombinant urokinase receptor blocked the binding of pro-uPA to alpha 2MR/LRP but caused only a slight reduction in the affinity for binding of uPA.PAI-1. Moreover, pro-uPA bound to the urokinase receptor at the cell surface was not internalized and degraded unless activated to uPA and complexed with PAI-1. We conclude that pro-uPA is protected against degradation via alpha 2MR/LRP when bound to uPAR due to shielding of a binding contact in the A chain, whereas the affinity of uPAR-bound uPA.PAI-1 complex for binding to alpha 2MR/LRP remains sufficient to allow rapid internalization and degradation.
Nykjaer
A
A
Department of Medical Biochemistry, University of Aarhus, Denmark.
Kjøller
L
L
Cohen
R L
RL
Lawrence
D A
DA
Garni-Wagner
B A
BA
Todd
R F
RF
van Zonneveld
A J
AJ
Gliemann
J
J
Andreasen
P A
PA
D016475:3T3 Cells; D000818:Animals; D000911:Antibodies, Monoclonal; D004705:Endocytosis / Q000502:physiology*; D004792:Enzyme Precursors / Q000378:metabolism*; D006801:Humans; D007140:Immunoglobulin Fab Fragments; D026503:Low Density Lipoprotein Receptor-Related Protein-1; D051379:Mice; D008954:Models, Biological; D017395:Plasminogen Activator Inhibitor 1 / Q000276:immunology / Q000378:metabolism*; D010960:Plasminogen Activators / Q000378:metabolism; D011485:Protein Binding; D011956:Receptors, Cell Surface / Q000378:metabolism; D011971:Receptors, Immunologic / Q000378:metabolism*; D011973:Receptors, LDL / Q000378:metabolism*; D055293:Receptors, Urokinase Plasminogen Activator; D011994:Recombinant Proteins / Q000276:immunology / Q000378:metabolism; D013329:Structure-Activity Relationship; D014568:Urokinase-Type Plasminogen Activator / Q000276:immunology / Q000378:metabolism*
D000911:Antibodies, Monoclonal; D004792:Enzyme Precursors; D007140:Immunoglobulin Fab Fragments; D026503:Low Density Lipoprotein Receptor-Related Protein-1; C524898:PLAUR protein, human; D017395:Plasminogen Activator Inhibitor 1; C524899:Plaur protein, mouse; D011956:Receptors, Cell Surface; D011971:Receptors, Immunologic; D011973:Receptors, LDL; D055293:Receptors, Urokinase Plasminogen Activator; D011994:Recombinant Proteins; D010960:Plasminogen Activators; D014568:Urokinase-Type Plasminogen Activator; C067551:saruplase