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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

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  • Sune Skeldal, Denmark
  • Jakob Vejby Larsen, Denmark
  • Katrine E Pedersen, Denmark
  • Helle H Petersen, Denmark
  • Rikke Egelund
  • ,
  • Anni Christensen
  • Jan Kristian Jensen
  • Jørgen Gliemann, Denmark
  • Peter A Andreasen, Denmark
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.
Original languageEnglish
JournalF E B S Journal
Volume273
Issue22
Pages (from-to)5143-59
Number of pages17
ISSN1742-464X
DOIs
Publication statusPublished - 1 Nov 2006

    Research areas

  • Amino Acid Substitution, Animals, Binding Sites, COS Cells, Cells, Cultured, Cercopithecus aethiops, Endocytosis, Humans, Models, Molecular, Mutagenesis, Site-Directed, Mutant Proteins, Plasminogen Activator Inhibitor 1, Protein Binding, Protein Interaction Mapping, Receptors, LDL, Surface Plasmon Resonance, U937 Cells, Urokinase-Type Plasminogen Activator

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