Plasminogen Activator Inhibitor-1 Contains a Cryptic High Affinity Binding Site for the Low Density Lipoprotein Receptor-related Protein

Abstract

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 NH 2 terminus, which is recognized by the protein kinase, heart muscle kinase and can be specifically labeled with 32 P. Our results show that only 32 P-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. The serine proteinase inhibitor (serpin)1 PAI-1 is the most efficient in vivo inhibitor known of both tissue-type plasminogen activator (tPA) and urokinase-type plasminogen activator (uPA) (1). Under normal physiological conditions PAI-1 expression is primarily limited to smooth muscle cells and megacaryocytes. However, many cells induce PAI-1 expression in response to cytokines associated with defensive processes such as inflammation (2). The active form of PAI-1 is thought to have its reactive center loop (RCL) fully exposed and thus available for interaction with proteinases (3, 4). However, the active conformation is unstable and decays to an inactive latent conformation which in turn can be partially reactivated by treatment with denaturants (5). Cleaved PAI-1 can be generated either by deacylation of the covalent enzyme-inhibitor complex (3, 6) or by reaction with a non-target protease such as elastase, which cleaves the RCL at a site other than the P1-P1Ј reactive center bond (7). In the latent and cleaved forms of PAI-1, the RCL is fully inserted into ␤-sheet A of the inhibitor, making these forms of PAI-1 inactive against target proteinases (8, 9). Upon complex formation with a target proteinase the RCL is cleaved at the P1-P1Ј reactive center bond and the aminoterminal portion of the RCL is inserted into ␤-sheet A (3, 10). Although the precise extent of RCL insertion in the stable complex is not known, recent data suggest that the insertion may be less than that of the latent and cleaved forms of PAI-1 (4, 11, 12). The integration of the RCL into ␤-sheet A is an essential step in the serpin inhibitory mechanism and results in a conversio...

Differential Regulation of Extracellular Tissue Inhibitor of Metalloproteinases-3 Levels by Cell Membrane-bound and Shed Low Density Lipoprotein Receptor-related Protein 1

“…Interestingly, heparin has been shown to directly inhibit interaction of other ligands, including factor IXa (49), apolipoprotein A-V (50), C4b-binding protein (51) and PAI-1 (52), to LRP-1. In the case of PAI-1, the heparin binding and LRP-1 binding regions have been found to overlap (53,54). Binding of proteins to heparin is commonly mediated by clusters of positively charged residues (55).…”

The Urokinase/PAI-2 Complex

“…Our results suggest that the high affinity binding sites for LRP lie solely within the uPA moiety of the uPA-PAI-2 complex as we could find no evidence of a corresponding site within either the relaxed or the stressed PAI-2 molecule. This indicates a clear distinction with PAI-1, in which a high affinity site within the PAI-1 moiety of uPA-PAI-1 complex contributes significantly to LRP binding (38). Furthermore, the uPA-PAI-2 complex is still able to bind LRP in the presence of uPAR, indicating the validity of this interaction at the cell surface.…”

“…It is thought that the high affinity binding of uPA-PAI-1 to LRP results from either the combination of low affinity sites in each molecule (36) or the unveiling of a cryptic high affinity binding site within PAI-1 (38). Using synthetic reactive center loop peptides to induce the relaxed conformation of PAI-2 in the absence of uPA (thus mimicking that found in uPA-PAI-2 complex) (46), we found no evidence for an LRP binding site within PAI-2.…”

Plasminogen Activator Inhibitor-1 Regulates Tumor Growth and Angiogenesis

“…On a molecular level, this suggests that the association of integrins with the uPA⅐uPAR complex (27,28,32,35) may serve to correctly position this complex in close proximity to the cryptic vitronectin RGD adhesion site that becomes accessible upon binding of uPA to PAI-1 and subsequent dissociation of the PAI-1⅐uPA complex from vitronectin (36). Thus, uPA/uPAR may serve to escort the integrin to the appropriate high affinity binding site within the matrix.…”