title: Binding areas of urokinase-type plasminogen activator-plasminogen activator inhibitor-1 complex for endocytosis receptors of the low-density lipoprotein receptor family, determined by site-directed mutagenesis.
abstract: Some endocytosis receptors related to the low-density lipoprotein receptor, including low-density lipoprotein receptor-related protein-1A, very-low-density lipoprotein receptor, and sorting protein-related receptor, bind protease-inhibitor complexes, including urokinase-type plasminogen activator (uPA), plasminogen activator inhibitor-1 (PAI-1), and the uPA-PAI-1 complex. The unique capacity of these receptors for high-affinity binding of many structurally unrelated ligands renders mapping of receptor-binding surfaces of serpin and serine protease ligands a special challenge. We have mapped the receptor-binding area of the uPA-PAI-1 complex by site-directed mutagenesis. Substitution of a cluster of basic residues near the 37-loop and 60-loop of uPA reduced the receptor-binding affinity of the uPA-PAI-1 complex approximately twofold. Deletion of the N-terminal growth factor domain of uPA reduced the affinity 2-4-fold, depending on the receptor, and deletion of both the growth factor domain and the kringle reduced the affinity sevenfold. The binding affinity of the uPA-PAI-1 complex to the receptors was greatly reduced by substitution of basic and hydrophobic residues in alpha-helix D and alpha-helix E of PAI-1. The localization of the implicated residues in the 3D structures of uPA and PAI-1 shows that they form a continuous receptor-binding area spanning the serpin as well as the A-chain and the serine protease domain of uPA. Our results suggest that the 10-100-fold higher affinity of the uPA-PAI-1 complex compared with the free components depends on the bonus effect of bringing the binding areas on uPA and PAI-1 together on the same binding entity.
authors.affiliation: Department of Molecular Biology, University of Aarhus, Denmark.
authors.forename: Sune
authors.initials: S
authors.lastname: Skeldal
authors.forename: Jakob V
authors.initials: JV
authors.lastname: Larsen
authors.forename: Katrine E
authors.initials: KE
authors.lastname: Pedersen
authors.forename: Helle H
authors.initials: HH
authors.lastname: Petersen
authors.forename: Rikke
authors.initials: R
authors.lastname: Egelund
authors.forename: Anni
authors.initials: A
authors.lastname: Christensen
authors.forename: Jan K
authors.initials: JK
authors.lastname: Jensen
authors.forename: Jørgen
authors.initials: J
authors.lastname: Gliemann
authors.forename: Peter A
authors.initials: PA
authors.lastname: Andreasen
mesh_terms: D019943:Amino Acid Substitution / Q000502:physiology; D000818:Animals; D001665:Binding Sites; D019556:COS Cells; D002478:Cells, Cultured; D002522:Chlorocebus aethiops; D004705:Endocytosis / Q000502:physiology; D006801:Humans; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed / Q000379:methods*; D050505:Mutant Proteins / Q000378:metabolism; D017395:Plasminogen Activator Inhibitor 1 / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D025941:Protein Interaction Mapping / Q000379:methods; D011973:Receptors, LDL / Q000378:metabolism*; D020349:Surface Plasmon Resonance / Q000379:methods; D020298:U937 Cells; D014568:Urokinase-Type Plasminogen Activator / Q000235:genetics / Q000378:metabolism*
chemical_list: D050505:Mutant Proteins; D017395:Plasminogen Activator Inhibitor 1; D011973:Receptors, LDL; D014568:Urokinase-Type Plasminogen Activator