Binding of human urokinase-type plasminogen activator to its receptor: Residues involved in species specificity and binding

Binding of human urokinase-type plasminogen activator to its receptor: Residues involved in species specificity and binding

Quax, P.H.A.
Grimbergen, J.M.
Lansink, M.
Bakker, A.H.F.
Blatter, M.C.
Belin, D.
van Hinsbergh, V.W.M.
Verheijen, J.H.
Gaubius Instituut TNO

1998

Urokinase-type plasminogen activator (UPA), particularly when bound to its receptor (UPAR), is thought to play a major role in local proteolytic processes, thus facilitating cell migration as may occur during angiogenesis, neointima and atherosclerotic plaque formation, and tumor cell invasion. To facilitate understanding of the need and function of the UPA/UPAR interaction in cell migration and vascular remodeling, we changed several amino acid residues in UPA so as to interfere with its interaction with its receptor. The receptor-binding domain of UPA has been localized to a region in the growth factor domain between residues 20 and 32. Since the binding of UPA to appears to be species specific, we used the differences in amino acid sequences in the growth factor domain of UPA between various species to construct a human UPA variant that does not bind to the human UPAR. We substituted Asn22 for its mouse equivalent Tyr by site-directed mutagenesis. This mutant UPA had similar plasminogen activator characteristics as wild- type UPA, including its specific activity and interaction with plasminogen activator inhibitor-1. However, no UPA/UPAR complexes could be observed in cross-linking experiments using DFP-treated 125I-labeled wild-type mutant UPA and lysates of various cells, including U937 histiocytic lymphoma cells, phorbol myristate acetate - treated human ECs, and mouse LB6 cells transfected with human UPAR cDNA. In direct binding experiments, DFP-treated 125I-labeled mutant UPA could not bind to phorbol myristate acetate- treated ECs, whereas wild-type UPA did bind. Furthermore, a 25-fold excess of wild-type UPA completely prevented the binding of DFP-treated 125I- labeled wild type UPA to the human receptor on transfected LB6 cells, whereas an equal amount of mutant UPA had only a veyr small effect. In ligand blotting assays, very weak binding of mutant UPA to human UPAR counld be observed. Changing Asn22 into the other amino acid residues alanine or glutamine had no effect on binding to UPAR on human ECs. The functional integrity of the growth factor domain in the non-receptor binding Asn22Tyr mutant is suggested by the fact that binding of this mutant to a murine UPAR can be restored after additional mutations in the growth factor domain, Asn27,His29,Trp30 to Arg27,Arg29,Arg30. We conclude that Ash22 and Asn27,His29,Trp30 in human UPA are key determinants in the species-specific binding of the enzyme to its receptor and that changing Asn22 into Tyr results in a UPA mutant with strongly reduced binding to UPAR.

Health
Site-directed mutagenesis
Urokinase-type plasminogen activator
Urokinase-type plasminogen activator receptor
Amino Acid Sequence
Animals
Asparagine
Binding, Competitive
Cells, Cultured
Cricetinae
Endothelium, Vascular
Humans
Ligands
Mice
Molecular Sequence Data
Mutagenesis, Site-Directed
Receptors, Cell Surface
Sequence Homology, Amino Acid
Species Specificity
Swine
Tyrosine
Urinary Plasminogen Activator

http://resolver.tudelft.nl/uuid:32bf0d55-3d94-45fe-b82e-67e942680cf4

https://doi.org/10.1161/01.atv.18.5.693

234507

1079-5642

Arteriosclerosis, Thrombosis, and Vascular Biology, 18 (5), 693-701

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