Analysis of a two-domain binding site for the urokinase-type plasminogen activator–plasminogen activator inhibitor-1 complex in low-density-lipoprotein-receptor-related protein

2001 ◽  
Vol 357 (1) ◽  
pp. 289-296 ◽  
Author(s):  
Olav M. ANDERSEN ◽  
Helle H. PETERSEN ◽  
Christian JACOBSEN ◽  
S⊘ren K. MOESTRUP ◽  
Michael ETZERODT ◽  
...  
Keyword(s):  
Binding Site ◽  
Plasminogen Activator ◽  
Low Density Lipoprotein ◽  
Density Lipoprotein ◽  
Plasminogen Activator Inhibitor ◽  
Plasminogen Activator Inhibitor 1 ◽  
Type Plasminogen Activator ◽  
Urokinase Type Plasminogen Activator ◽  
Density Lipoprotein Receptor ◽  
Pai 1

The low-density-lipoprotein-receptor (LDLR)-related protein (LRP) is composed of several classes of domains, including complement-type repeats (CR), which occur in clusters that contain binding sites for a multitude of different ligands. Each ≈ 40-residue CR domain contains three conserved disulphide linkages and an octahedral Ca2+ cage. LRP is a scavenging receptor for ligands from extracellular fluids, e.g. α2-macroglobulin (α2M)–proteinase complexes, lipoprotein-containing particles and serine proteinase–inhibitor complexes, like the complex between urokinase-type plasminogen activator (uPA) and the plasminogen activator inhibitor-1 (PAI-1). In the present study we analysed the interaction of the uPA–PAI-1 complex with an ensemble of fragments representing a complete overlapping set of two-domain fragments accounting for the ligand-binding cluster II (CR3–CR10) of LRP. By ligand blotting, solid-state competition analysis and surface-plasmon-resonance analysis, we demonstrate binding to multiple CR domains, but show a preferential interaction between the uPA–PAI-1 complex and a two-domain fragment comprising CR domains 5 and 6 of LRP. We demonstrate that surface-exposed aspartic acid and tryptophan residues at identical positions in the two homologous domains, CR5 and CR6 (Asp958,CR5, Asp999,CR6, Trp953,CR5 and Trp994,CR6), are critical for the binding of the complex as well as for the binding of the receptor-associated protein (RAP)–the folding chaperone/escort protein required for transport of LRP to the cell surface. Accordingly, the present work provides (1) an identification of a preferred binding site within LRP CR cluster II; (2) evidence that the uPA–PAI-1 binding site involves residues from two adjacent protein domains; and (3) direct evidence identifying specific residues as important for the binding of uPA–PAI-1 as well as for the binding of RAP.

Cellular Degradation of Free and Inhibitor-bound Tissue-type Plasminogen Activator

2000 ◽  
Vol 83 (02) ◽  
pp. 290-296 ◽  
Author(s):  
Chantal Camani ◽  
Olivier Gavin ◽  
Egbert Kruithof
Keyword(s):  
Plasminogen Activator ◽  
Low Density Lipoprotein ◽  
Density Lipoprotein ◽  
Plasminogen Activator Inhibitor ◽  
Tissue Type ◽  
Tissue Type Plasminogen Activator ◽  
Type Plasminogen Activator ◽  
Density Lipoprotein Receptor ◽  
Activator Inhibitor Type ◽  
Pai 1

SummaryThe low density lipoprotein receptor-related protein (LRP) is a multiligand clearance receptor that removes free tissue-type plasminogen activator (t-PA) or complexes of t-PA with plasminogen activator inhibitor type 1 (PAI-1) from the blood circulation or the pericellular space. Co-receptors are essential for LRP-mediated clearance of several ligands (e.g. glycosaminoglycans for thrombin/protease nexin and lipoprotein lipase, and the urokinase receptor for urokinase/PAI-1 complexes). The present study was undertaken to investigate whether LRP-mediated t-PA clearance requires a co-receptor as well.In five cell lines from different organs and species degradation of t-PA and t-PA/PAI-1 was mediated by LRP (or LRP-like receptors). No degradation of t-PA and t-PA/PAI-1 occurred in THP-1 or U-937 human monocyte-like cells, despite the presence of functional LRP. As glycosaminoglycans can bind t-PA and PAI-1 we investigated whether they are involved in t-PA/PAI-1 degradation. Pre-treatment of COS cells or HT1080 cells with chlorate, an inhibitor of glycosaminoglycan sulfation, did not decrease t-PA/PAI-1 degradation. Furthermore, CHO cells genetically deficient in glycosaminoglycans efficiently degraded t-PA/PAI-1. Thus it is unlikely that glycosaminoglycans are co-receptors for degradation of t-PA or t-PA/PAI-1.This study indicates that THP-1 and U-937 cells lack a critical component (co-receptor?) for the LRP-mediated degradation of t-PA. Abbreviations: LRP, low density lipoprotein receptor-related protein; PAI-1, plasminogen activator inhibitor type 1; RAP, receptor-associated protein; t-PA, tissue-type plasminogen activator; u-PA, urokinase; u-PAR, urokinase receptor.

scholarly journals Inhibition of plasminogen activator inhibitor-1 binding to endocytosis receptors of the low-density-lipoprotein receptor family by a peptide isolated from a phage display library

2006 ◽  
Vol 399 (3) ◽  
pp. 387-396 ◽  
Author(s):  
Jan K. Jensen ◽  
Anders Malmendal ◽  
Birgit Schiøtt ◽  
Sune Skeldal ◽  
Katrine E. Pedersen ◽  
...  
Keyword(s):  
Plasminogen Activator ◽  
Low Density Lipoprotein ◽  
Density Lipoprotein ◽  
Plasminogen Activator Inhibitor ◽  
Low Density ◽  
Lipoprotein Receptor ◽  
Plasminogen Activator Inhibitor 1 ◽  
Low Density Lipoprotein Receptor ◽  
Density Lipoprotein Receptor ◽  
Pai 1

The functions of the serpin PAI-1 (plasminogen activator inhibitor-1) are based on molecular interactions with its target proteases uPA and tPA (urokinase-type and tissue-type plasminogen activator respectively), with vitronectin and with endocytosis receptors of the low-density-lipoprotein family. Understanding the significance of these interactions would be facilitated by the ability to block them individually. Using phage display, we have identified the disulfide-constrained peptide motif CFGWC with affinity for natural human PAI-1. The three-dimensional structure of a peptide containing this motif (DVPCFGWCQDA) was determined by liquid-state NMR spectroscopy. A binding site in the so-called flexible joint region of PAI-1 was suggested by molecular modelling and validated through binding studies with various competitors and site-directed mutagenesis of PAI-1. The peptide with an N-terminal biotin inhibited the binding of the uPA–PAI-1 complex to the endocytosis receptors low-density-lipoprotein-receptor-related protein 1A (LRP-1A) and very-low-density-lipoprotein receptor (VLDLR) in vitro and inhibited endocytosis of the uPA–PAI-1 complex in U937 cells. We conclude that the isolated peptide represents a novel approach to pharmacological interference with the functions of PAI-1 based on inhibition of one specific molecular interaction.

Plasminogen Activator Inhibitor 1: Physiological and Pathophysiological Roles

Physiology ◽  
2002 ◽  
Vol 17 (2) ◽  
pp. 56-61 ◽  
Author(s):  
Bernd R. Binder ◽  
Günter Christ ◽  
Florian Gruber ◽  
Nelly Grubic ◽  
Peter Hufnagl ◽  
...  
Keyword(s):  
Plasminogen Activator ◽  
Low Density Lipoprotein ◽  
Density Lipoprotein ◽  
Plasminogen Activator Inhibitor ◽  
Plasminogen Activator Inhibitor 1 ◽  
Density Lipoprotein Receptor ◽  
The Matrix ◽  
Activator Inhibitor ◽  
Pai 1 ◽  
Dependent Mechanism

Plasminogen activator inhibitor 1 (PAI-1) inhibits plasminogen activators (u-PA and t-PA) by forming stable complexes endocytosed via a low-density lipoprotein receptor superfamily member-dependent mechanism. PAI-1 circulates actively in plasma and latently in platelets but is also secreted and deposited into the matrix by several cells, where it participates in tissue repair processes.

The Role of the Low-Density Lipoprotein Receptor-Related Protein (LRP) in the Plasma Clearance and Liver Uptake of Recombinant Single-chain Urokinase-type Plasminogen Activator in Rats

1997 ◽  
Vol 77 (04) ◽  
pp. 710-717 ◽  
Author(s):  
Marieke E van der Kaaden ◽  
Dingeman C Rijken ◽  
J Kar Kruijt ◽  
Theo J C van Berkel ◽  
Johan Kuiper
Keyword(s):  
Plasminogen Activator ◽  
Low Density Lipoprotein ◽  
Density Lipoprotein ◽  
Single Chain ◽  
Liver Uptake ◽  
Type Plasminogen Activator ◽  
Parenchymal Liver ◽  
Urokinase Type Plasminogen Activator ◽  
Density Lipoprotein Receptor

SummaryUrokinase-type plasminogen activator (u-PA) is used as a thrombolytic agent in the treatment of acute myocardial infarction. In vitro, recombinant single-chain u-PA (rscu-PA) expressed in E.coli is recognized by the Low-Density Lipoprotein Receptor-related Protein (LRP) on rat parenchymal liver cells. In this study we investigated the role of LRP in the liver uptake and plasma clearance of rscu-PA in rats. A preinjection of the LRP inhibitor GST-RAP reduced the maximal liver uptake of 125I-rscu-PA at 5 min after injection from 50 to 30% of the injected dose and decreased the clearance of rscu-PA from 2.37 ml/min to 1.58 ml/min. Parenchymal, Kupffer and endothelial cells were responsible for 40, 50 and 10% of the liver uptake, respectively. The reduction in liver uptake of rscu-PA by the preinjection of GST-RAP was caused by a 91 % and 62% reduction in the uptake by parenchymal and Kupffer cells, respectively. In order to investigate the part of rscu-PA that accounted for the interaction with LRP, experiments were performed with a mutant of rscu-PA lacking residues 11-135 (= deltal25- rscu-PA). Deletion of residues 11-135 resulted in a 80% reduction in liver uptake and a 2.4 times slower clearance (0.97 ml/min). The parenchymal, Kupffer and endothelial cells were responsible for respectively 60, 33 and 7% of the liver uptake of 125I-deltal25-rscu-PA. Preinjection of GST-RAP completely reduced the liver uptake of delta 125-rscu-PA and reduced its clearance to 0.79 ml/min. Treatment of isolated Kupffer cells with PI-PLC reduced the binding of rscu-PA by 40%, suggesting the involvement of the urokinase-type Plasminogen Activator Receptor (u-PAR) in the recognition of rscu-PA. Our results demonstrate that in vivo LRP is responsible for more than 90% of the parenchymal liver cell mediated uptake of rscu-PA and for 60% of the Kupffer cell interaction. It is also suggested that u-PAR is involved in the Kupffer cell recognition of rscu-PA.

scholarly journals Deep mutational scanning of the plasminogen activator inhibitor-1 functional landscape

2021 ◽  
Author(s):  
Zachary M Huttinger ◽  
Laura M Haynes ◽  
Andrew Yee ◽  
Colin A Kretz ◽  
David R Siemieniak ◽  
...  
Keyword(s):  
Amino Acid ◽  
Plasminogen Activator ◽  
Plasminogen Activator Inhibitor ◽  
Single Amino Acid ◽  
Plasminogen Activator Inhibitor 1 ◽  
Missense Variants ◽  
Type Plasminogen Activator ◽  
Urokinase Type Plasminogen Activator ◽  
Activator Inhibitor ◽  
Pai 1

The serine protease inhibitor (SERPIN) plasminogen activator inhibitor-1 (PAI-1) is a key regulator of the fibrinolytic system, inhibiting the serine proteases tissue- and urokinase-type plasminogen activator (tPA and uPA, respectively). Missense variants may render PAI-1 non-functional through misfolding, leading to its turnover as a protease substrate, or to a more rapid transition to the latent/inactive state. Deep mutational scanning was performed to evaluate the impact of amino acid sequence variation on PAI-1 inhibition of uPA using an M13 filamentous phage display system. The effects of single amino acid substitutions on PAI-1's functional inhibition of its canonical target proteases, tPA and uPA , have been determined for only a small fraction of potential mutations. To construct a more comprehensive dataset, a mutagenized PAI-1 library, encompassing ~70% of potential single amino acid substitutions, was displayed on M13 filamentous phage. From this library, the relative effects of 27% of all possible missense variants on PAI-1 inhibition of urokinase-type plasminogen activator were determined using high-throughput DNA sequencing with 826 missense variants demonstrating conserved inhibitory activity and 1137 resulting in loss of PAI-1 function. Comparison of these deep mutational scanning results to predictions from PolyPhen-2 and SIFT demonstrate the limitations of these algorithms, consistent with similar reports for other proteins. Comparison to common human PAI-1 gene variants present in the gnomAD database is consistent with evolutionary selection against loss of PAI-1 function. These findings provide insight into structure-function relationships for PAI-1 and other members of the SERPIN superfamily.

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