Binding of urokinase-type plasminogen activator-plasminogen activator inhibitor-1 complex to the endocytosis receptors alpha2-macroglobulin receptor/low-density lipoprotein receptor-related protein and very-low-density lipoprotein receptor involves basic residues in the inhibitor.
The complex of the type-1 plasminogen activator inhibitor (PAI-1) and its target proteinases, the urokinase and tissue-type plasminogen activators (uPA and tPA), but not the free components, bind with high affinity to the endocytosis receptors alpha2-macroglobulin receptor/low-density lipoprotein receptor-related protein (alpha2MR/LRP) and very-low-density lipoprotein receptor (VLDLR). To characterize the molecular interaction between the complexes and the receptors, alanine codons were introduced into the human PAI-1 cDNA to replace the four basic residues, Arg-78, Lys-82, Arg-120 and Lys-124, as double mutations. The purified recombinant mutant proteins, rPAI-1/R78A-K124A and rPAI-1/K82A-R120A, produced by the yeast Pichia pastoris, were indistinghuisable from wild-type recombinant and natural human PAI-1 with respect to inhibitory activity against uPA, stability of SDS-resistant complexes with uPA, and vitronectin binding. Radiolabelled mutant uPA.PAI-1 complexes bound with a 10- to 20-fold, and 3- to 7-fold reduced affinity to purified alpha2MR/LRP and VLDLR respectively. alpha2MR/LRP-mediated endocytosis of the mutant complexes by COS-1 cells was reduced to 48 and 38% of the level of endocytosis of wild-type PAI-1. Binding of the mutant complexes to the uPA receptor was not affected. These findings suggest that the binding mode of the uPA.PAI-1 complex to both alpha2MR/LRP and VLDLR is similar. The four residues are surface exposed in the region defined by alpha-helix D and beta-strand 1A in the serine protease inhibitor (serpin) structure. Our study represents the first identification of residues in a surface region implicated in molecular recognition of protease.serpin complexes by endocytosis receptors of the low-density lipoprotein receptor family.
Rodenburg
K W
KW
Department of Molecular and Structural Biology, Aarhus University, 10 Gustav Wieds Vej, DK 8000 Arhus C, Denmark.
Kjoller
L
L
Petersen
H H
HH
Andreasen
P A
PA
D000818:Animals; D019556:COS Cells; D004591:Electrophoresis, Polyacrylamide Gel; D004705:Endocytosis / Q000502:physiology*; D006801:Humans; D015151:Immunoblotting; D007457:Iodine Radioisotopes; D007700:Kinetics; D026503:Low Density Lipoprotein Receptor-Related Protein-1; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009994:Osmolar Concentration; D010843:Pichia / Q000235:genetics; D017395:Plasminogen Activator Inhibitor 1 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D017433:Protein Structure, Secondary; D011971:Receptors, Immunologic / Q000378:metabolism*; D011973:Receptors, LDL / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D017421:Sequence Analysis; D015843:Serpins / Q000737:chemistry; D014568:Urokinase-Type Plasminogen Activator / Q000378:metabolism*; D019096:Vitronectin / Q000378:metabolism
D007457:Iodine Radioisotopes; D026503:Low Density Lipoprotein Receptor-Related Protein-1; D017395:Plasminogen Activator Inhibitor 1; D011971:Receptors, Immunologic; D011973:Receptors, LDL; D011994:Recombinant Proteins; D015843:Serpins; C036053:VLDL receptor; D019096:Vitronectin; D014568:Urokinase-Type Plasminogen Activator