Plasmnogen activator lnhmrror-1 (pai-1) a molecular switch that contains a cryptic high affinity binding site for the low density upoprotein receptor related protein (lrf)

Previously, we demonstrated that thrombin in complex with PAI-1 was endocytosed and degraded by cells via LRP much more efficiently then either thrombin alone or thrombin in complex with other proteinase inhibitors (J.B.C. (1996) 271:8215). Therefore, to test the hypothesis that PAI-1 binds specifically to LRP with high affinity, and to characterize this binding interaction, a unique PAI-1 mutant containing a consensus peptide sequence for heart muscle kinase (HMK) has been created by PCR The mutant, HMK-PAI-1, can be specifically radiolabeled with 32P to high specific activity and is indistinguishable from wild-type PAI-1 (wtPAI-1) with respect to inhibitory activity, and binding to vitronectin or LRP. Analysis of the endocytosis and degradation of different conformational forms of HMK-PAI-1 by mouse embryonic fibroblasts demonstrates that only HMK-PAI-1 in complex with a proteinase is efficiently internalized. Direct binding and competitive inhibition assays of HMK-PAI-1 with purified LRP indicates that a high affinity binding site for LRP is generated on PAI-1 upon complex formation with either uPA or trypsin, and that this site is not expressed on either the enzyme alone or on the active, latent or elastase cleaved forms of PAI-1. Together, these data indicate that PAI-1 contains a high affinity binding site for LRP. However, in uncomplexed PAI-1 this binding site is cryptic, and thus only following inhibition of a proteinase will the PAI-1 :proteinase complex be cleared. This conformational control of PAI-1's interaction with a non-proteinase ligand is remarkably similar to PAI-1's association with the matrix protein vitronectin which is also confomationally controlled (J.B.C. (1997) 272:7676). However, in the case of vitronectin only the active form of PAI-1 binds with high affinity. This suggests that under normal physiological conditions deposition of PAI-1 at site of tissue injury will result in the association of active PAI-1 with vitronectin in subcellular matrix However, as proteinase concentration increases locally, as would be expected during wound healing, PAI-1 present in the matrix will inhibit these active enzymes and in doing so loose its affinity for the matrix while simultaneously gaining affinity for LRP. This suggests that PAI-1 may act as a molecular switch that that either localizes to the extra cellular matrix or to the endocytic compartment depending on its conformation which in turn is regulated by its activity state.