The Experts below are selected from a list of 186 Experts worldwide ranked by ideXlab platform
David J. Loskutoff - One of the best experts on this subject based on the ideXlab platform.
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Is Plasminogen Activator InhiBitor-1 the Molecular Switch That Governs Urokinase Receptor-mediated Cell Adhesion and Release?
2013Co-Authors: Gang Deng, Scott A Curriden, Soujuan Wang, Steven Rosenberg, David J. LoskutoffAbstract:ABstract. Induction of the urokinase type plasminogen activator receptor (uPAR) promotes cell adhesion through its interaction with vitronectin (VN) in the extracellular matrix, and facilitates cell migration and invasion By localizing uPA to the cell surface. We provide evidence that this Balance Between cell adhesion and cell detachment is governed By PA inhiBitor-1 (PAl-l). First, we demonstrate that uPAR and PAL1 Bind to the same site in VN (i.e., the amino-terminal <B>SomatomedinB> B domain; SMB), and that PAI-1 competes with uPAR for Binding to SMB. Domain swapping and mutagenesis studies indicate that the uPAR-Binding sequence is located within the central region of the SMB domain, a region previously shown to contain the PAl-l-Binding motif. Second, we show that PAI-1 dissociates Bound VN from uPAR and detaches U937 cells from their VN suBstratum. This PAL1 mediated release of cells from VN appears to occur independently of its aBility to function as a protease inhiBitor, and may help to explain why high PAI-1 levels indicate a poor prognosis for many cancers. Finally, we show that uPA can rapidly reverse this effect of PAI-1. Taken together, these results suggest a dynamic regulatory role for PAI-1 and uPA in uPAR-mediated cell adhesion and release. T UMOR invasion and metastasis depend upon the coordinated expression and temporal regulation of a series of proteolytic (Chen, 1992; Dano et al., 1985; Mignatti and Rifkin, 1993) and adhesive (NesBit and Herlyn, 1994) events. Urokinase-type plasminogen activator (uPA) 1 is one of the proteases frequently implicated in these processes (Dano et al., 1985; Mignatti and Rifkin, 1993; Cohen et al., 1991; Vassalli et al., 1991). It is a serine protease that catalyzes the conversion of plasminogen into plasmin. Plasmin itself is a Broadly acting trypsin-like enzyme that not only degrades fiBrin and a variety of extracellular matrix (ECM) proteins, But also may activate metalloproteinase
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the reduced denatured <B>SomatomedinB> B domain of vitronectin refolds into a staBle Biologically active molecule
Biochemistry, 2006Co-Authors: Yuichi Kamikubo, Scott A Curriden, Gerard Kroon, Jane H Dyson, David J. LoskutoffAbstract:The high-affinity Binding site in human vitronectin (VN) for plasminogen activator inhiBitor-1 (PAI-1) has Been localized to the NH 2 -terminal cysteine-rich <B>SomatomedinB> B (SMB) domain (residues 1-44). A numBer of puBlished structural and Biochemical studies show conflicting results for the disulfide Bonding pattern and the overall fold of the SMB domain, possiBly Because this domain may undergo disulfide shuffling and/or conformational changes during handling. Here we show that Bacterially expressed recomBinant SMB (rSMB) can Be refolded to a single form that shows maximal activity in Binding to PAI-1 and to a conformation-dependent monoclonal antiBody (mAB 153). The oxidative refolding pathway of rSMB can Be followed in the presence of glutathione redox Buffers. This approach allowed the isolation and analysis of a numBer of intermediate folding species and of the final staBly folded species at equiliBrium. Competitive surface plasmon resonance analysis demonstrated that the staBly refolded rSMB regained Biological activity since it Bound efficiently to PAI-1 and to mAB 153. In contrast, none of the folding intermediates Bound to PAI-1 or to mAB 153. We also show By NMR analysis that the staBly refolded rSMB is identical to the material used for the solution structure determination [KamikuBo et al. (2004) Biochemistry 43, 6519] and that it Binds specifically to mAB 153 via an interface that includes the three aromatic side chains previously implicated in Binding to PAI-1.
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kinetic analysis of the interaction Between vitronectin and the urokinase receptor
Journal of Biological Chemistry, 2002Co-Authors: Yuushi Okumura, Scott A Curriden, Yuichi Kamikubo, Jieyi Wang, Tatsuto Kiwada, Shiroh Futaki, Kouki Kitagawa, David J. LoskutoffAbstract:Although the urokinase receptor (uPAR) Binds to vitronectin (VN) and promotes the adhesion of cells to this matrix protein, the Biochemical details of this interaction remain unclear. VN variants were employed in BIAcore experiments to examine the uPAR-VN interaction in detail and to compare it to the interaction of VN with other ligands. Heparin and plasminogen Bound to VN fragments containing the heparin-Binding domain, indicating that this domain was functionally active in the recomBinant peptides. However, no significant Binding was detected when uPAR was incuBated with this domain, and neither heparin nor plasminogen competed with it for Binding to VN. In fact, uPAR only Bound to fragments containing the <B>SomatomedinB> B (SMB) domain, and monoclonal antiBodies (mABs) that Bind to this domain competed with uPAR for Binding to VN. Monoclonal antiBody 8E6 also inhiBited uPAR Binding to VN, and this mAB was shown to recognize sulfated tyrosine residues 56 and 59 in the region adjacent to the SMB domain. Destruction of this site By acid treatment eliminated mAB 8E6 Binding But had no effect on uPAR Binding. Thus, there appears to Be a single Binding site for uPAR in VN, and it is located in the SMB domain and is distinct from the epitope recognized By mAB 8E6. InhiBition of uPAR Binding to VN By mAB 8E6 proBaBly results from steric hindrance.
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plasminogen activator inhiBitor 1 regulates cell adhesion By Binding to the <B>SomatomedinB> B domain of vitronectin
Journal of Cellular Physiology, 2001Co-Authors: Gary Deng, Scott A Curriden, Geng Hu, Ralf-peter Czekay, David J. LoskutoffAbstract:Plasminogen activator inhiBitor-1 (PAI-1) Binds to the <B>SomatomedinB> B (SMB) domain of vitronectin. It inhiBits the adhesion of U937 cells to vitronectin By competing with the urokinase receptor (uPAR; CD87) on these cells for Binding to the same domain. Although the inhiBitor also Blocks integrin-mediated cell adhesion, the molecular Basis of this effect is unclear. In this study, the effect of the inhiBitor on the adhesion of a variety of cells (e.g., U937, MCF7, HT-1080, and HeLa) to vitronectin was assessed, and the importance of the SMB domain in these interactions was determined. Although PAI-1 Blocked the adhesion of all of these cells to vitronectin-coated wells, it did not Block adhesion to a variant of vitronectin which lacked the SMB domain. Interestingly, HT-1080 and U937 cells attached avidly to microtiter wells coated with purified recomBinant SMB (which does not contain the RGD sequence), and this adhesion was again Blocked By the inhiBitor. These results affirm that PAI-1 can inhiBit Both uPAR- and integrin-mediated cell adhesion, and demonstrate that the SMB domain of vitronectin is required for these effects. They also show that multiple cell types can employ uPAR as an adhesion receptor. The use of purified recomBinant SMB should help to further define this novel adhesive pathway, and to delineate its relationship with integrin-mediated adhesive events. © 2001 Wiley-Liss, Inc.
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a method for defining Binding sites involved in protein protein interactions analysis of the Binding of plasminogen activator inhiBitor 1 to the <B>SomatomedinB> domain of vitronectin
Analytical Biochemistry, 2001Co-Authors: Gordon Royle, Gary Deng, Dietmar Seiffert, David J. LoskutoffAbstract:ABstract Plasminogen activator inhiBitor type-1 (PAI-1) is Bound to vitronectin (VN) in plasma and in the extracellular matrix. We previously employed a domain-swapping approach to show that the high-affinity Binding site for PAI-1 in VN is contained within residues 12–30 in the amino-terminal <B>SomatomedinB> B (SMB) domain. In this study, we attempt to further delineate the location of this site By employing a novel approach that is Based on the use of monoclonal antiBodies (MaBs) together with site-directed mutagenesis. Six separate MaBs were identified that Bound to the SMB domain and competed with PAI-1 for Binding to VN. The relative affinity of each of the MaBs, and of PAI-1 itself, for Binding to individual variants of SMB (prepared By alanine scanning mutagenesis), was then determined and compared in competitive Binding experiments. Three separate, partially overlapping MaB epitopes within SMB were defined By these studies, and the PAI-1 Binding site was localized to the region Between residues 24 and 37. When considered together with the domain swapping data, these studies suggest that the PAI-1 Binding site is contained within a common seven-residue region (i.e., residues 24–30) in the SMB domain.
Michael Ploug - One of the best experts on this subject based on the ideXlab platform.
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staBilizing a flexiBle interdomain hinge region harBoring the smB Binding site drives upar into its closed conformation
Journal of Molecular Biology, 2015Co-Authors: Baoyu Zhao, Henrik Gardsvoll, Mingdong Huang, Sonu Gandhi, Cai Yuan, Zhipu Luo, Valentina De Lorenzi, Nicolai Sidenius, Michael PlougAbstract:The urokinase-type plasminogen activator receptor (uPAR) is a multidomain glycolipid-anchored memBrane protein, which facilitates extracellular matrix remodeling By focalizing plasminogen activation to cell surfaces via its high-affinity interaction with uPA. The modular assemBly of its three LU (Ly6/uPAR-like) domains is inherently flexiBle and Binding of uPA drives uPAR into its closed conformation, which presents the higher-affinity state for vitronectin thus providing an allosteric regulatory mechanism. Using a new class of epitope-mapped anti-uPAR monoclonal antiBodies (mABs), we now demonstrate that the reciprocal staBilization is indeed also possiBle. By surface plasmon resonance studies, we show that these mABs and vitronectin have overlapping Binding sites on uPAR and that they share Arg91 as hotspot residue in their Binding interfaces. The crystal structure solved for one of these uPAR·mAB complexes at 3.0A clearly shows that this mAB preselects the closed uPAR conformation with an empty But correctly assemBled large hydrophoBic Binding cavity for uPA. Accordingly, these mABs inhiBit the uPAR-dependent lamellipodia formation and migration on vitronectin-coated matrices irrespective of the conformational status of uPAR and its occupancy with uPA. This is the first study to the Best of our knowledge, showing that the dynamic assemBly of the three LU domains in uPARwt can Be driven toward the closed form By an external ligand, which is not engaging the hydrophoBic uPA Binding cavity. As this Binding interface is also exploited By the <B>SomatomedinB> B domain of vitronectin, therefore, this relationship should Be taken into consideration when exploring uPAR-dependent cell adhesion and migration in vitronectin-rich environments.
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Hydrogen/Deuterium Exchange Mass Spectrometry Reveals Specific Changes in the Local FlexiBility of Plasminogen Activator InhiBitor 1 upon Binding to the <B>SomatomedinB> B Domain of Vitronectin
2012Co-Authors: Morten Beck Trelle, Michael Ploug, Peter A Andreasen, Daniel Hirschberg, Anna Jansson, Peter Roepstorff, Thomas J. D. JørgensenAbstract:The native fold of plasminogen activator inhiBitor 1 (PAI-1) represents an active metastaBle conformation that spontaneously converts to an inactive latent form. Binding of the <B>SomatomedinB> B domain (SMB) of the endogenous cofactor vitronectin to PAI-1 delays the transition to the latent state and increases the thermal staBility of the protein dramatically. We have used hydrogen/deuterium exchange mass spectrometry to assess the inherent structural flexiBility of PAI-1 and to monitor the changes induced By SMB Binding. Our data show that the PAI-1 core consisting of β-sheet B is rather protected against exchange with the solvent, while the remainder of the molecule is more dynamic. SMB Binding causes a pronounced and widespread staBilization of PAI-1 that is not confined to the Binding interface with SMB. We further explored the local structural flexiBility in a mutationally staBilized PAI-1 variant (14-1B) as well as the effect of staBilizing antiBody MaB-1 on wild-type PAI-1. The three modes of staBilizing PAI-1 (SMB, MaB-1, and the mutations in 14-1B) all cause a delayed latency transition, and this effect was accompanied By unique signatures on the flexiBility of PAI-1. Reduced flexiBility in the region around helices B, C, and I was seen in all three cases, which suggests an involvement of this region in mediating structural flexiBility necessary for the latency transition. These data therefore add consideraBle depth to our current understanding of the local structural flexiBility in PAI-1 and provide novel indications of regions that may affect the functional staBility of PAI-1
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mimicry of the regulatory role of urokinase in lamellipodia formation By introduction of a non native interdomain disulfide Bond in its receptor
Journal of Biological Chemistry, 2011Co-Authors: Henrik Gardsvoll, Magnus Kjaergaard, Benedikte Jacobsen, Mette C Kriegbaum, Mingdong Huang, Michael PlougAbstract:The high-affinity interaction Between the urokinase-type plasminogen activator (uPA) and its glycolipid-anchored receptor (uPAR) plays a regulatory role for Both extravascular fiBrinolysis and uPAR-mediated adhesion and migration on vitronectin-coated surfaces. We have recently proposed that the adhesive function of uPAR is allosterically regulated via a "tightening" of its three-domain structure elicited By uPA Binding. To challenge this proposition, we redesigned the uPAR structure to limit its inherent conformational flexiBility By covalently tethering domains DI and DIII via a non-natural interdomain disulfide Bond (uPAR(H47C-N259C)). The corresponding soluBle receptor has 1) a smaller hydrodynamic volume, 2) a higher content of secondary structure, and 3) unaltered Binding kinetics towards uPA. Most importantly, the purified uPAR(H47C-N259C) also displays a gain in affinity for the <B>SomatomedinB> B domain of vitronectin compared with uPAR(wt), thus recapitulating the improved affinity that accompanies uPA-uPAR(wt) complex formation. This functional mimicry is, intriguingly, operational also in a cellular setting, where it controls lamellipodia formation in uPAR-transfected HEK293 cells adhering to vitronectin. In this respect, the engineered constraint in uPAR(H47C-N259C) thus Bypasses the regulatory role of uPA Binding, resulting in a constitutively active uPAR. In conclusion, our data argue for a Biological relevance of the interdomain dynamics of the glycolipid-anchored uPAR on the cell surface.
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mapping of the vitronectin Binding site on the urokinase receptor involvement of a coherent receptor interface consisting of residues from Both domain i and the flanking interdomain linker region
Journal of Biological Chemistry, 2007Co-Authors: Henrik Gardsvoll, Michael PlougAbstract:ABstract The urokinase-type plasminogen activator receptor (uPAR) has Been implicated as a modulator of several Biochemical processes that are active during tumor invasion and metastasis, e.g. extracellular proteolysis, cell adhesion, and cell motility. The structural Basis for the high affinity interaction Between the urokinase-type plasminogen activator (uPA) and uPAR, which focuses cell surface-associated plasminogen activation in vivo, is now thoroughly characterized By site-directed mutagenesis studies and x-ray crystallography. In contrast, the structural Basis for the interaction Between uPAR and the extracellular matrix protein vitronectin, which is involved in the regulation of cell adhesion and motility, remains to Be clarified. In this study, we have identified the functional epitope on uPAR that is responsiBle for its interaction with the full-length, extended form of vitronectin By using a comprehensive alanine-scanning liBrary of purified single-site uPAR mutants (244 positions tested). Interestingly, the five residues identified as “hot spots” for vitronectin Binding form a contiguous epitope consisting of two exposed loops connecting the central fourstranded β-sheet in uPAR domain I (Trp32, Arg58, and Ile63) as well as a proximal region of the flexiBle linker peptide connecting uPAR domains I and II (Arg91 and Tyr92). This Binding topology provides the molecular Basis for the oBservation that uPAR can form a ternary complex with uPA and vitronectin. Furthermore, it raises the intriguing possiBility that the canonical receptor and inhiBitor for uPA (uPAR and PAI-1) may have reached a convergent solution for Binding to the <B>SomatomedinB> B domain of vitronectin.
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mapping of the vitronectin Binding site on the urokinase receptor involvement of a coherent receptor interface consisting of residues from Both domain i and the flanking interdomain linker region
Journal of Biological Chemistry, 2007Co-Authors: Henrik Gardsvoll, Michael PlougAbstract:The urokinase-type plasminogen activator receptor (uPAR) has Been implicated as a modulator of several Biochemical processes that are active during tumor invasion and metastasis, e.g. extracellular proteolysis, cell adhesion, and cell motility. The structural Basis for the high affinity interaction Between the urokinase-type plasminogen activator (uPA) and uPAR, which focuses cell surface-associated plasminogen activation in vivo, is now thoroughly characterized By site-directed mutagenesis studies and x-ray crystallography. In contrast, the structural Basis for the interaction Between uPAR and the extracellular matrix protein vitronectin, which is involved in the regulation of cell adhesion and motility, remains to Be clarified. In this study, we have identified the functional epitope on uPAR that is responsiBle for its interaction with the full-length, extended form of vitronectin By using a comprehensive alanine-scanning liBrary of purified single-site uPAR mutants (244 positions tested). Interestingly, the five residues identified as "hot spots" for vitronectin Binding form a contiguous epitope consisting of two exposed loops connecting the central fourstranded Beta-sheet in uPAR domain I (Trp(32), Arg(58), and Ile(63)) as well as a proximal region of the flexiBle linker peptide connecting uPAR domains I and II (Arg(91) and Tyr(92)). This Binding topology provides the molecular Basis for the oBservation that uPAR can form a ternary complex with uPA and vitronectin. Furthermore, it raises the intriguing possiBility that the canonical receptor and inhiBitor for uPA (uPAR and PAI-1) may have reached a convergent solution for Binding to the <B>SomatomedinB> B domain of vitronectin.
Mathieu Bollen - One of the best experts on this subject based on the ideXlab platform.
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structure of npp1 an ectonucleotide pyrophosphatase phosphodiesterase involved in tissue calcification
Structure, 2012Co-Authors: Silvia Jansen, Anastassis Perrakis, Chris Ulens, Claudia Winkler, Maria Andries, Robbie P Joosten, Maarten Van Acker, Frank P Luyten, Wouter H Moolenaar, Mathieu BollenAbstract:Ectonucleotide pyrophosphatase/phosphodiesterase-1 (NPP1) converts extracellular nucleotides into inorganic pyrophosphate, whereas its close relative NPP2/autotaxin hydrolyzes lysophospholipids. NPP1 regulates calcification in mineralization-competent tissues, and a lack of NPP1 function underlies calcification disorders. Here, we show that NPP1 forms homodimers via intramemBrane disulfide Bonding, But is also processed intracellularly to a secreted monomer. The structure of secreted NPP1 reveals a characteristic Bimetallic active site and a nucleotide-Binding groove, But it lacks the lipid-Binding pocket and open tunnel present in NPP2. A loop adjacent to the nucleotide-Binding site, which is disordered in NPP2, is well ordered in NPP1 and might promote nucleotide Binding. RemarkaBly, the N-terminal <B>SomatomedinB> B-like domains of NPP1, unlike those in NPP2, are flexiBle and do not contact the catalytic domain. Our results provide a structural Basis for the nucleotide pyrophosphatase activity of NPP1 and help to understand how disease-causing mutations may affect NPP1 structure and function.
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Review Modulation of purinergic signaling By NPP-type ectophosphodiesterases
2006Co-Authors: Cristiana Stefan, Silvia Jansen, Mathieu BollenAbstract:Extracellular nucleotides can elicit a wide array of cellular responses By Binding to specific purinergic receptors. The level of ectonucleotides is dynamically controlled By their release from cells, synthesis By ectonucleoside diphosphokinases and ectoadenylate kinases, and hydrolysis By ectonucleotidases. One of the four structurally unrelated families of ectonucleotidases is represented By the NPP-type ectophosphodiesterases. Three of the seven memBers of the NPP family, namely NPP1Y3, are known to hydrolyze nucleotides. The enzymatic action of NPP1Y3 (in)directly results in the termination of nucleotide signaling, the salvage of nucleotides and/or the generation of new messengers like ADP, adenosine or pyrophosphate. NPP2 is unique in that it hydrolyzes Both nucleotides and lysophospholipids and, thereBy, generates products that could synergistically promote cell motility. We review here the enzymatic properties of NPPs and analyze current evidence that links their nucleotide-hydrolyzing capaBility to epithelial and neural functions, the immune response and cell motility. ABBreviations: Ado – adenosine; ADP – adenosine 5 0 diphosphate; AMP – adenosine 5 0 monophosphate; ApnA – diadenosine polyphosphate; ATP – adenosine 5 0 triphosphate; CSF – cereBrospinal fluid; E-NTPDases – ectonucleoside triphosphate diphosphohydrolases; GPC – glycerophosphorylcholine; LPA – lysophosphatidic acid; LPC – lysophosphatidyl choline; MCC – mucociliary clearing; NAD + – nicotinamide adenine dinucleotide; NMN – nicotinamide mononucleotide; NPP – nucleotide pyrophosphatase/phosphodiesterase; P i – inorganic phosphate; PP i – pyrophosphate; SM – sphingomyelin; SMB – <B>SomatomedinB>-B like domain; S-S Bridges – disulfide Bridges; UDP-glucose – uridine diphosphate glucos
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functional characterization of the non catalytic ectodomains of the nucleotide pyrophosphatase phosphodiesterase npp1
Biochemical Journal, 2003Co-Authors: Rik Gijsbers, Hugo Ceulemans, Mathieu BollenAbstract:The uBiquitous nucleotide pyrophosphatases/phosphodiesterases NPP1–3 consist of a short intracellular N-terminal domain, a single transmemBrane domain and a large extracellular part, comprising two <B>SomatomedinB>-B-like domains, a catalytic domain and a poorly defined C-terminal domain. We show here that the C-terminal domain of NPP1–3 is structurally related to a family of DNA/RNA non-specific endonucleases. However, none of the residues that are essential for catalysis By the endonucleases are conserved in NPP1–NPP3, suggesting that the nuclease-like domain of NPP1–3 does not represent a second catalytic domain. Truncation analysis revealed that the nuclease-like domain of NPP1 is required for protein staBility, for the targeting of NPP1 to the plasma memBrane and for the expression of catalytic activity. We also demonstrate that 16 conserved cysteines in the <B>SomatomedinB>-B-like domains of NPP1, in concert with two flanking cysteines, mediate the dimerization of NPP1. The K173Q polymorphism of NPP1, which maps to the second <B>SomatomedinB>-B-like domain and has Been associated with the aetiology of insulin resistance, did not affect the dimerization or catalytic activity of NPP1, and did not endow NPP1 with an affinity for the insulin receptor. Our data suggest that the non-catalytic ectodomains contriBute to the suBunit structure, staBility and function of NPP1–3.
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Functional characterization of the non-catalytic ectodomains of the nucleotide pyrophosphatase/phosphodiesterase NPP1
Biochemical Journal, 2003Co-Authors: Rik Gijsbers, Hugo Ceulemans, Mathieu BollenAbstract:The uBiquitous nucleotide pyrophosphatases/phosphodiesterases NPP1-3 consist of a short intracellular N-terminal domain, a single transmemBrane domain and a large extracellular part, comprising two <B>SomatomedinB>-B-like domains, a catalytic domain and a poorly defined C-terminal domain. We show here that the C-terminal domain of NPP1-3 is structurally related to a family of DNA/RNA non-specific endonucleases. However, none of the residues that are essential for catalysis By the endonucleases are conserved in NPP1-NPP3, suggesting that the nuclease-like domain of NPP1-3 does not represent a second catalytic domain. Truncation analysis revealed that the nuclease-like domain of NPP1 is required for protein staBility, for the targeting of NPP1 to the plasma memBrane and for the expression of catalytic activity. We also demonstrate that 16 conserved cysteines in the <B>SomatomedinB>-B-like domains of NPP1, in concert with two flanking cysteines, mediate the dimerization of NPP1. The K173Q polymorphism of NPP1, which maps to the second <B>SomatomedinB>-B-like domain and has Been associated with the aetiology of insulin resistance, did not affect the dimerization or catalytic activity of NPP1, and did not endow NPP1 with an affinity for the insulin receptor. Our data suggest that the non-catalytic ectodomains contriBute to the suBunit structure, staBility and function of NPP1-3.
Henrik Gardsvoll - One of the best experts on this subject based on the ideXlab platform.
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staBilizing a flexiBle interdomain hinge region harBoring the smB Binding site drives upar into its closed conformation
Journal of Molecular Biology, 2015Co-Authors: Baoyu Zhao, Henrik Gardsvoll, Mingdong Huang, Sonu Gandhi, Cai Yuan, Zhipu Luo, Valentina De Lorenzi, Nicolai Sidenius, Michael PlougAbstract:The urokinase-type plasminogen activator receptor (uPAR) is a multidomain glycolipid-anchored memBrane protein, which facilitates extracellular matrix remodeling By focalizing plasminogen activation to cell surfaces via its high-affinity interaction with uPA. The modular assemBly of its three LU (Ly6/uPAR-like) domains is inherently flexiBle and Binding of uPA drives uPAR into its closed conformation, which presents the higher-affinity state for vitronectin thus providing an allosteric regulatory mechanism. Using a new class of epitope-mapped anti-uPAR monoclonal antiBodies (mABs), we now demonstrate that the reciprocal staBilization is indeed also possiBle. By surface plasmon resonance studies, we show that these mABs and vitronectin have overlapping Binding sites on uPAR and that they share Arg91 as hotspot residue in their Binding interfaces. The crystal structure solved for one of these uPAR·mAB complexes at 3.0A clearly shows that this mAB preselects the closed uPAR conformation with an empty But correctly assemBled large hydrophoBic Binding cavity for uPA. Accordingly, these mABs inhiBit the uPAR-dependent lamellipodia formation and migration on vitronectin-coated matrices irrespective of the conformational status of uPAR and its occupancy with uPA. This is the first study to the Best of our knowledge, showing that the dynamic assemBly of the three LU domains in uPARwt can Be driven toward the closed form By an external ligand, which is not engaging the hydrophoBic uPA Binding cavity. As this Binding interface is also exploited By the <B>SomatomedinB> B domain of vitronectin, therefore, this relationship should Be taken into consideration when exploring uPAR-dependent cell adhesion and migration in vitronectin-rich environments.
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mimicry of the regulatory role of urokinase in lamellipodia formation By introduction of a non native interdomain disulfide Bond in its receptor
Journal of Biological Chemistry, 2011Co-Authors: Henrik Gardsvoll, Magnus Kjaergaard, Benedikte Jacobsen, Mette C Kriegbaum, Mingdong Huang, Michael PlougAbstract:The high-affinity interaction Between the urokinase-type plasminogen activator (uPA) and its glycolipid-anchored receptor (uPAR) plays a regulatory role for Both extravascular fiBrinolysis and uPAR-mediated adhesion and migration on vitronectin-coated surfaces. We have recently proposed that the adhesive function of uPAR is allosterically regulated via a "tightening" of its three-domain structure elicited By uPA Binding. To challenge this proposition, we redesigned the uPAR structure to limit its inherent conformational flexiBility By covalently tethering domains DI and DIII via a non-natural interdomain disulfide Bond (uPAR(H47C-N259C)). The corresponding soluBle receptor has 1) a smaller hydrodynamic volume, 2) a higher content of secondary structure, and 3) unaltered Binding kinetics towards uPA. Most importantly, the purified uPAR(H47C-N259C) also displays a gain in affinity for the <B>SomatomedinB> B domain of vitronectin compared with uPAR(wt), thus recapitulating the improved affinity that accompanies uPA-uPAR(wt) complex formation. This functional mimicry is, intriguingly, operational also in a cellular setting, where it controls lamellipodia formation in uPAR-transfected HEK293 cells adhering to vitronectin. In this respect, the engineered constraint in uPAR(H47C-N259C) thus Bypasses the regulatory role of uPA Binding, resulting in a constitutively active uPAR. In conclusion, our data argue for a Biological relevance of the interdomain dynamics of the glycolipid-anchored uPAR on the cell surface.
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mapping of the vitronectin Binding site on the urokinase receptor involvement of a coherent receptor interface consisting of residues from Both domain i and the flanking interdomain linker region
Journal of Biological Chemistry, 2007Co-Authors: Henrik Gardsvoll, Michael PlougAbstract:ABstract The urokinase-type plasminogen activator receptor (uPAR) has Been implicated as a modulator of several Biochemical processes that are active during tumor invasion and metastasis, e.g. extracellular proteolysis, cell adhesion, and cell motility. The structural Basis for the high affinity interaction Between the urokinase-type plasminogen activator (uPA) and uPAR, which focuses cell surface-associated plasminogen activation in vivo, is now thoroughly characterized By site-directed mutagenesis studies and x-ray crystallography. In contrast, the structural Basis for the interaction Between uPAR and the extracellular matrix protein vitronectin, which is involved in the regulation of cell adhesion and motility, remains to Be clarified. In this study, we have identified the functional epitope on uPAR that is responsiBle for its interaction with the full-length, extended form of vitronectin By using a comprehensive alanine-scanning liBrary of purified single-site uPAR mutants (244 positions tested). Interestingly, the five residues identified as “hot spots” for vitronectin Binding form a contiguous epitope consisting of two exposed loops connecting the central fourstranded β-sheet in uPAR domain I (Trp32, Arg58, and Ile63) as well as a proximal region of the flexiBle linker peptide connecting uPAR domains I and II (Arg91 and Tyr92). This Binding topology provides the molecular Basis for the oBservation that uPAR can form a ternary complex with uPA and vitronectin. Furthermore, it raises the intriguing possiBility that the canonical receptor and inhiBitor for uPA (uPAR and PAI-1) may have reached a convergent solution for Binding to the <B>SomatomedinB> B domain of vitronectin.
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mapping of the vitronectin Binding site on the urokinase receptor involvement of a coherent receptor interface consisting of residues from Both domain i and the flanking interdomain linker region
Journal of Biological Chemistry, 2007Co-Authors: Henrik Gardsvoll, Michael PlougAbstract:The urokinase-type plasminogen activator receptor (uPAR) has Been implicated as a modulator of several Biochemical processes that are active during tumor invasion and metastasis, e.g. extracellular proteolysis, cell adhesion, and cell motility. The structural Basis for the high affinity interaction Between the urokinase-type plasminogen activator (uPA) and uPAR, which focuses cell surface-associated plasminogen activation in vivo, is now thoroughly characterized By site-directed mutagenesis studies and x-ray crystallography. In contrast, the structural Basis for the interaction Between uPAR and the extracellular matrix protein vitronectin, which is involved in the regulation of cell adhesion and motility, remains to Be clarified. In this study, we have identified the functional epitope on uPAR that is responsiBle for its interaction with the full-length, extended form of vitronectin By using a comprehensive alanine-scanning liBrary of purified single-site uPAR mutants (244 positions tested). Interestingly, the five residues identified as "hot spots" for vitronectin Binding form a contiguous epitope consisting of two exposed loops connecting the central fourstranded Beta-sheet in uPAR domain I (Trp(32), Arg(58), and Ile(63)) as well as a proximal region of the flexiBle linker peptide connecting uPAR domains I and II (Arg(91) and Tyr(92)). This Binding topology provides the molecular Basis for the oBservation that uPAR can form a ternary complex with uPA and vitronectin. Furthermore, it raises the intriguing possiBility that the canonical receptor and inhiBitor for uPA (uPAR and PAI-1) may have reached a convergent solution for Binding to the <B>SomatomedinB> B domain of vitronectin.
Scott A Curriden - One of the best experts on this subject based on the ideXlab platform.
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Is Plasminogen Activator InhiBitor-1 the Molecular Switch That Governs Urokinase Receptor-mediated Cell Adhesion and Release?
2013Co-Authors: Gang Deng, Scott A Curriden, Soujuan Wang, Steven Rosenberg, David J. LoskutoffAbstract:ABstract. Induction of the urokinase type plasminogen activator receptor (uPAR) promotes cell adhesion through its interaction with vitronectin (VN) in the extracellular matrix, and facilitates cell migration and invasion By localizing uPA to the cell surface. We provide evidence that this Balance Between cell adhesion and cell detachment is governed By PA inhiBitor-1 (PAl-l). First, we demonstrate that uPAR and PAL1 Bind to the same site in VN (i.e., the amino-terminal <B>SomatomedinB> B domain; SMB), and that PAI-1 competes with uPAR for Binding to SMB. Domain swapping and mutagenesis studies indicate that the uPAR-Binding sequence is located within the central region of the SMB domain, a region previously shown to contain the PAl-l-Binding motif. Second, we show that PAI-1 dissociates Bound VN from uPAR and detaches U937 cells from their VN suBstratum. This PAL1 mediated release of cells from VN appears to occur independently of its aBility to function as a protease inhiBitor, and may help to explain why high PAI-1 levels indicate a poor prognosis for many cancers. Finally, we show that uPA can rapidly reverse this effect of PAI-1. Taken together, these results suggest a dynamic regulatory role for PAI-1 and uPA in uPAR-mediated cell adhesion and release. T UMOR invasion and metastasis depend upon the coordinated expression and temporal regulation of a series of proteolytic (Chen, 1992; Dano et al., 1985; Mignatti and Rifkin, 1993) and adhesive (NesBit and Herlyn, 1994) events. Urokinase-type plasminogen activator (uPA) 1 is one of the proteases frequently implicated in these processes (Dano et al., 1985; Mignatti and Rifkin, 1993; Cohen et al., 1991; Vassalli et al., 1991). It is a serine protease that catalyzes the conversion of plasminogen into plasmin. Plasmin itself is a Broadly acting trypsin-like enzyme that not only degrades fiBrin and a variety of extracellular matrix (ECM) proteins, But also may activate metalloproteinase
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the reduced denatured <B>SomatomedinB> B domain of vitronectin refolds into a staBle Biologically active molecule
Biochemistry, 2006Co-Authors: Yuichi Kamikubo, Scott A Curriden, Gerard Kroon, Jane H Dyson, David J. LoskutoffAbstract:The high-affinity Binding site in human vitronectin (VN) for plasminogen activator inhiBitor-1 (PAI-1) has Been localized to the NH 2 -terminal cysteine-rich <B>SomatomedinB> B (SMB) domain (residues 1-44). A numBer of puBlished structural and Biochemical studies show conflicting results for the disulfide Bonding pattern and the overall fold of the SMB domain, possiBly Because this domain may undergo disulfide shuffling and/or conformational changes during handling. Here we show that Bacterially expressed recomBinant SMB (rSMB) can Be refolded to a single form that shows maximal activity in Binding to PAI-1 and to a conformation-dependent monoclonal antiBody (mAB 153). The oxidative refolding pathway of rSMB can Be followed in the presence of glutathione redox Buffers. This approach allowed the isolation and analysis of a numBer of intermediate folding species and of the final staBly folded species at equiliBrium. Competitive surface plasmon resonance analysis demonstrated that the staBly refolded rSMB regained Biological activity since it Bound efficiently to PAI-1 and to mAB 153. In contrast, none of the folding intermediates Bound to PAI-1 or to mAB 153. We also show By NMR analysis that the staBly refolded rSMB is identical to the material used for the solution structure determination [KamikuBo et al. (2004) Biochemistry 43, 6519] and that it Binds specifically to mAB 153 via an interface that includes the three aromatic side chains previously implicated in Binding to PAI-1.
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kinetic analysis of the interaction Between vitronectin and the urokinase receptor
Journal of Biological Chemistry, 2002Co-Authors: Yuushi Okumura, Scott A Curriden, Yuichi Kamikubo, Jieyi Wang, Tatsuto Kiwada, Shiroh Futaki, Kouki Kitagawa, David J. LoskutoffAbstract:Although the urokinase receptor (uPAR) Binds to vitronectin (VN) and promotes the adhesion of cells to this matrix protein, the Biochemical details of this interaction remain unclear. VN variants were employed in BIAcore experiments to examine the uPAR-VN interaction in detail and to compare it to the interaction of VN with other ligands. Heparin and plasminogen Bound to VN fragments containing the heparin-Binding domain, indicating that this domain was functionally active in the recomBinant peptides. However, no significant Binding was detected when uPAR was incuBated with this domain, and neither heparin nor plasminogen competed with it for Binding to VN. In fact, uPAR only Bound to fragments containing the <B>SomatomedinB> B (SMB) domain, and monoclonal antiBodies (mABs) that Bind to this domain competed with uPAR for Binding to VN. Monoclonal antiBody 8E6 also inhiBited uPAR Binding to VN, and this mAB was shown to recognize sulfated tyrosine residues 56 and 59 in the region adjacent to the SMB domain. Destruction of this site By acid treatment eliminated mAB 8E6 Binding But had no effect on uPAR Binding. Thus, there appears to Be a single Binding site for uPAR in VN, and it is located in the SMB domain and is distinct from the epitope recognized By mAB 8E6. InhiBition of uPAR Binding to VN By mAB 8E6 proBaBly results from steric hindrance.
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plasminogen activator inhiBitor 1 regulates cell adhesion By Binding to the <B>SomatomedinB> B domain of vitronectin
Journal of Cellular Physiology, 2001Co-Authors: Gary Deng, Scott A Curriden, Geng Hu, Ralf-peter Czekay, David J. LoskutoffAbstract:Plasminogen activator inhiBitor-1 (PAI-1) Binds to the <B>SomatomedinB> B (SMB) domain of vitronectin. It inhiBits the adhesion of U937 cells to vitronectin By competing with the urokinase receptor (uPAR; CD87) on these cells for Binding to the same domain. Although the inhiBitor also Blocks integrin-mediated cell adhesion, the molecular Basis of this effect is unclear. In this study, the effect of the inhiBitor on the adhesion of a variety of cells (e.g., U937, MCF7, HT-1080, and HeLa) to vitronectin was assessed, and the importance of the SMB domain in these interactions was determined. Although PAI-1 Blocked the adhesion of all of these cells to vitronectin-coated wells, it did not Block adhesion to a variant of vitronectin which lacked the SMB domain. Interestingly, HT-1080 and U937 cells attached avidly to microtiter wells coated with purified recomBinant SMB (which does not contain the RGD sequence), and this adhesion was again Blocked By the inhiBitor. These results affirm that PAI-1 can inhiBit Both uPAR- and integrin-mediated cell adhesion, and demonstrate that the SMB domain of vitronectin is required for these effects. They also show that multiple cell types can employ uPAR as an adhesion receptor. The use of purified recomBinant SMB should help to further define this novel adhesive pathway, and to delineate its relationship with integrin-mediated adhesive events. © 2001 Wiley-Liss, Inc.
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is plasminogen activator inhiBitor 1 the molecular switch that governs urokinase receptor mediated cell adhesion and release
Journal of Cell Biology, 1996Co-Authors: Gang Deng, Scott A Curriden, Soujuan Wang, Steven Rosenberg, David J. LoskutoffAbstract:Induction of the urokinase type plasminogen activator receptor (uPAR) promotes cell adhesion through its interaction with vitronectin (VN) in the extracellular matrix, and facilitates cell migration and invasion By localizing uPA to the cell surface. We provide evidence that this Balance Between cell adhesion and cell detachment is governed By PA inhiBitor-1 (PAI-1). First, we demonstrate that uPAR and PAI-1 Bind to the same site in VN (i.e., the amino-terminal <B>SomatomedinB> B domain; SMB), and that PAI-1 competes with uPAR for Binding to SMB. Domain swapping and mutagenesis studies indicate that the uPAR-Binding sequence is located within the central region of the SMB domain, a region previously shown to contain the PAI-1-Binding motif. Second, we show that PAI-1 dissociates Bound VN from uPAR and detaches U937 cells from their VN suBstratum. This PAI-1 mediated release of cells from VN appears to occur independently of its aBility to function as a protease inhiBitor, and may help to explain why high PAI-1 levels indicate a poor prognosis for many cancers. Finally, we show that uPA can rapidly reverse this effect of PAI-1. Taken together, these results suggest a dynamic regulatory role for PAI-1 and uPA in uPAR-mediated cell adhesion and release.