Plasminogen activator inhibitor-1 contains a cryptic high affinity binding site for the low density lipoprotein receptor-related protein.
Much of the controversy surrounding the binding of plasminogen activator inhibitor-1 (PAI-1) to the low density lipoprotein receptor-related protein (LRP) may be due to the labile structure of PAI-1 and the distinct conformations that it can adopt. To examine this possibility and to test the hypothesis that PAI-1 contains a specific high affinity binding site for LRP, a sensitive and quantitative assay for PAI-1 binding to LRP was developed. This assay utilizes a unique PAI-1 mutant that was constructed with a hexapeptide tag at the NH2 terminus, which is recognized by the protein kinase, heart muscle kinase and can be specifically labeled with 32P. Our results show that only 32P-PAI-1 in complex with a proteinase binds LRP with high affinity and is efficiently endocytosed by cells, indicating that a high affinity site for LRP is generated on PAI-1 only when in complex with a proteinase. In addition, PAI-1 in complex with different proteinases is shown to cross-compete for LRP binding, demonstrating that the binding site is independent of the proteinase and therefore must reside on the PAI-1 portion of the complex. Finally, mutagenesis of PAI-1 results in loss of LRP binding, confirming that the high affinity binding site is located on PAI-1 and suggesting that the LRP binding site lays within a region of PAI-1 previously shown to contain the heparin binding domain.
Stefansson
S
S
Departments of Biochemistry, J. H. Holland Laboratory, American Red Cross, Rockville, Maryland 20855, USA.
Muhammad
S
S
Cheng
X F
XF
Battey
F D
FD
Strickland
D K
DK
Lawrence
D A
DA
D000818:Animals; D001665:Binding Sites; D001667:Binding, Competitive; D001692:Biological Transport; D004705:Endocytosis; D026503:Low Density Lipoprotein Receptor-Related Protein-1; D051379:Mice; D009154:Mutation; D017395:Plasminogen Activator Inhibitor 1 / Q000235:genetics / Q000378:metabolism*; D010960:Plasminogen Activators / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D011971:Receptors, Immunologic / Q000378:metabolism*; D014357:Trypsin / Q000378:metabolism; D014568:Urokinase-Type Plasminogen Activator / Q000378:metabolism
D026503:Low Density Lipoprotein Receptor-Related Protein-1; D017395:Plasminogen Activator Inhibitor 1; D011971:Receptors, Immunologic; D010960:Plasminogen Activators; D014357:Trypsin; D014568:Urokinase-Type Plasminogen Activator