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.