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 activat or 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 dis tinct conformations that it can adopt, To examine this possibility and to test the hypothesis that PAI-1 contains a specific high affinity b inding site for LRP, a sensitive and quantitative assay for PAI-1 bind ing to LRP was developed, This assay utilizes a unique PAI-1 mutant th at was constructed with a hexapeptide tag at the NH2 terminus, which i s recognized by the protein kinase, heart muscle kinase and can be spe cifically labeled with P-32. Our results show that only P-32-PAI-1 in complex with a proteinase binds LRP with high affinity and is efficien tly endocytosed by cells, indicating that a high affinity site for LRP is generated on PAI-1 only when in complex with a proteinase. In addi tion, PAI-1 in complex with different proteinases is shown to cross-co mpete for LRP binding, demonstrating that the binding site is independ ent of the proteinase and therefore must reside on the PAI-1 portion o f the complex. Finally, mutagenesis of PAI-1 results in loss of LRP bi nding, confirming that the high affinity binding site is located on PA I-1 and suggesting that the LRP binding site lays within a region of P AI-1 previously shown to contain the heparin binding domain.