Urokinase links plasminogen activation and cell adhesion by cleavage of the RGD motif in vitronectin

Components of the plasminogen activation system including urokinase (uPA), its inhibitor (PAI‐1) and its cell surface receptor (uPAR) have been implicated in a wide variety of biological processes related to tissue homoeostasis. Firstly, the binding of uPA to uPAR favours extracellular proteolysis by enhancing cell surface plasminogen activation. Secondly, it promotes cell adhesion and signalling through binding of the provisional matrix protein vitronectin. We now report that uPA and plasmin induces a potent negative feedback on cell adhesion through specific cleavage of the RGD motif in vitronectin. Cleavage of vitronectin by uPA displays a remarkable receptor dependence and requires concomitant binding of both uPA and vitronectin to uPAR. Moreover, we show that PAI‐1 counteracts the negative feedback and behaves as a proteolysis‐triggered stabilizer of uPAR‐mediated cell adhesion to vitronectin. These findings identify a novel and highly specific function for the plasminogen activation system in the regulation of cell adhesion to vitronectin. The cleavage of vitronectin by uPA and plasmin results in the release of N‐terminal vitronectin fragments that can be detected in vivo, underscoring the potential physiological relevance of the process.     

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

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 somatomedin B domain of vitronectin.     

Conformational regulation of urokinase receptor function: impact of receptor occupancy and epitope-mapped monoclonal antibodies on lamellipodia induction

The urokinase-type plasminogen activator receptor (uPAR) is a glycolipid-anchored membrane protein with an established role in focalizing uPA-mediated plasminogen activation on cell surfaces. Distinct from this function, uPAR also modulates cell adhesion and migration on vitronectin-rich matrices. Although uPA and vitronectin engage structurally distinct binding sites on uPAR, they nonetheless cooperate functionally, as uPA binding potentiates uPAR-dependent induction of lamellipodia on vitronectin matrices. We now present data advancing the possibility that it is the burial of the β-hairpin in uPA per se into the hydrophobic ligand binding cavity of uPAR that modulates the function of this receptor. Based on these data, we now propose a model in which the inherent interdomain mobility in uPAR plays a major role in modulating its function. Particularly one uPAR conformation, which is stabilized by engagement of the β-hairpin in uPA, favors the proper assembly of an active, compact receptor structure that stimulates lamellipodia induction on vitronectin. This molecular model has wide implications for drug development targeting uPAR function.     

The uPA receptor and the somatomedin B region of vitronectin direct the localization of uPA to focal adhesions in microvessel endothelial cells.

Vitronectin is a plasma protein which can deposit into the extracellular matrix where it supports integrin and uPA dependent cell migration. In earlier studies, we have shown that the plasma protein, vitronectin, stimulates focal adhesion remodeling by recruiting urokinase-type plasminogen activator (uPA) to focal adhesion sites [Wilcox-Adelman, S. A., Wilkins-Port, C. E., McKeown-Longo, P. J., 2000. Localization of urokinase-type plasminogen activator to focal adhesions requires ligation of vitronectin integrin receptors. Cell. Adhes. Commun.7, 477-490]. In the present study, we used a variety of vitronectin constructs to demonstrate that the localization of uPA to adhesion sites requires the binding of both vitronectin integrin receptors and the uPA receptor (uPAR) to vitronectin. A recombinant fragment of vitronectin containing the connecting sequence (VN(CS)) was able to support integrin-dependent adhesion, spreading and focal adhesion assembly by human microvessel endothelial cells. Cells adherent to this fragment were not able to localize uPA to focal adhesions. A second recombinant fragment containing both the amino-terminal SMB domain and the CS domain was able to restore the localization of uPA to adhesion sites. This fragment, which contains a uPAR binding site, also resulted in the localization of uPAR to adhesion sites. uPAR blocking antibodies as well as phospholipase C treatment of cells inhibited uPA localization to adhesion sites confirming a role for uPAR in this process. The SMB domain alone was unable to direct either uPAR or uPA to adhesion sites in the absence of the CS domain. Our results indicate that vitronectin-dependent localization of uPA to adhesion sites requires the sequential binding of vitronectin integrins and uPAR to vitronectin.     

A composite role of vitronectin and urokinase in the modulation of cell morphology upon expression of the urokinase receptor

The urokinase receptor, urokinase receptor (uPAR), is a glycosylphosphatidylinositol-anchored membrane protein engaged in pericellular proteolysis and cellular adhesion, migration, and modulation of cell morphology. A direct matrix adhesion is mediated through the binding of uPAR to vitronectin, and this event is followed by downstream effects including changes in the cytoskeletal organization. However, it remains unclear whether the adhesion through uPAR-vitronectin is the only event capable of initiating these morphological rearrangements or whether lateral interactions between uPAR and integrins can induce the same response. In this report, we show that both of these triggering mechanisms can be operative and that uPAR-dependent modulation of cell morphology can indeed occur independently of a direct vitronectin binding. Expression of wild-type uPAR on HEK293 cells led to pronounced vitronectin adhesion and cytoskeletal rearrangements, whereas a mutant uPAR, uPAR(W32A) with defective vitronectin binding, failed to induce both phenomena. However, upon saturation of uPAR(W32A) with the protease ligand, pro-uPA, or its receptor-binding domain, the ability to induce cytoskeletal rearrangements was restored, although this did not rescue the uPAR-vitronectin binding and adhesion capability. On the other hand, using other uPAR variants, we could show that uPAR-vitronectin adhesion is indeed capable and sufficient to induce the same morphological rearrangements. This was shown with cells expressing a different single-site mutant, uPAR(Y57A), in the presence of a synthetic uPAR-binding peptide, as well as with wild-type uPAR, which underwent cytoskeletal rearrangements even when cultivated in uPA-deficient serum. Blocking of integrins with an Arg-Gly-Asp-containing peptide counteracted the matrix contacts necessary to initiate the uPAR-dependent cytoskeletal rearrangements, whereas inactivation of the Rac signaling pathway in all cases suppressed the occurrence of the same events.     

Ligand binding regions in the receptor for urokinase-type plasminogen activator

The interaction between urokinase plasminogen activator (uPA) and its cellular receptor (uPAR) is a key event in cell surface-associated plasminogen activation, relevant for cell migration and invasion. In order to define receptor recognition sites for uPA, we have expressed uPAR fragments as fusion products with the minor coat protein on the surface of M13 bacteriophages. Sequence analysis of cDNA fragments encoding uPA-binding peptides indicated the existence of a composite uPA-binding structure including all three uPAR domains. This finding was confirmed by experiments using an overlapping 15-mer peptide array covering the entire uPAR molecule. Four regions within the uPAR sequence were found to directly bind to uPA: two distinct regions containing amino acids 13--20 and amino acids 74--84 of the uPAR domain I, and regions in the putative loop 3 of the domains II and III. All the uPA-binding fragments from the three domains were shown to have an agonistic effect on uPA binding to immobilized uPAR. Furthermore, uPAR-(154--176) increased uPAR-transfected BAF3-cell adhesion on vitronectin in the presence of uPA, whereas uPAR-(247--276) stimulated the cell adhesion both in the absence or presence of uPA. The latter fragment was also able to augment the binding of vitronectin to uPAR in a purified system, thereby mimicking the effect of uPA on this interaction. These results indicate that uPA binding can take place to particular part(s) on several uPAR molecules and that direct uPAR-uPAR contacts may contribute to receptor activation and ligand binding.     

Single Amino Acid Substitutions in the Chemotactic Sequence of Urokinase Receptor Modulate Cell Migration and Invasion

The receptor for urokinase-type plasminogen activator (uPAR) plays an important role in controlling cell migration. uPAR binds urokinase and vitronectin extracellular ligands, and signals in complex with transmembrane receptors such as Formyl-peptide Receptors (FPR)s and integrins. Previous work from this laboratory has shown that synthetic peptides, corresponding to the uPAR_88–92 chemotactic sequence, when carrying the S90P or S90E substitutions, up- or down-regulate cell migration, respectively. To gain mechanistic insights into these opposite cell responses, the functional consequences of S90P and S90E mutations in full-length uPAR were evaluated. First, (HEK)-293 embryonic kidney cells expressing uPAR^S90P exhibit enhanced FPR activation, increased random and directional cell migration, long-lasting Akt phosphorylation, and increased adhesion to vitronectin, as well as uPAR/vitronectin receptor association. In contrast, the S90E substitution prevents agonist-triggered FPR activation and internalization, decreases binding and adhesion to vitronectin, and inhibits uPAR/vitronectin receptor association. Also, 293/uPAR^S90P cells appear quite elongated and their cytoskeleton well organized, whereas 293/uPAR^S90E cells assume a large flattened morphology, with random orientation of actin filaments. Interestingly, when HT1080 cells co-express wild type uPAR with uPAR S90E, the latter behaves as a dominant-negative, impairing uPAR-mediated signaling and reducing cell wound repair as well as lung metastasis in nude mice. In contrast, signaling, wound repair and in vivo lung metastasis of HT1080 cells bearing wild type uPAR are enhanced when they co-express uPAR^S90P. In conclusion, our findings indicate that Ser⁹⁰ is a critical residue for uPAR signaling and that the S90P and S90E exert opposite effects on uPAR activities. These findings may be accommodated in a molecular model, in which uPAR^S90E and uPAR^S90P are forced into inactive and active forms, respectively, suggesting important implications for the development of novel drugs targeting uPAR function.     

Plasminogen activator inhibitor-1 regulates cell adhesion by binding to the somatomedin B domain of vitronectin

Plasminogen activator inhibitor-1 (PAI-1) binds to the somatomedin 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.     

Mimicry of the regulatory role of urokinase in lamellipodia formation by introduction of a non-native interdomain disulfide bond in its receptor

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 somatomedin 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.     

Variability in the expression of urokinase receptor (CD87) mutants on cells: relevance to cell adhesion

The urokinase-type plasminogen activator receptor (uPAR, CD87) is a glycosylphosphatidylinositol (GPI)-anchored protein, containing three homologous Ly-6 domains, that mediates integrin-independent cell adhesion by directly binding to extracellular matrix protein vitronectin (VN). To elucidate the structural requirements for the uPAR-dependent cell adhesion on VN, several glycolipid-anchored variants of uPAR were expressed in BAF3 cells, (mouse pre B-lymphocytes) followed by functional analysis. The individual domains of uPAR were expressed at very low levels, the two domain mutants were expressed to a higher level and the wild type uPAR was expressed highly. Point mutations in domain 2 of uPAR have been shown to diminish cellular binding of the ligand urokinase and we observed a lack of VN binding to this mutant. Flow cytometry with a number of monoclonal antibodies indicated that the domain-specific antigenic determinants in these mutants were well preserved. Only the cells expressing the intact uPAR with all three domains adhered strongly to a VN substrate, whereas none of the other transfected cells showed significant cell adhesion. Hence, any alterations in the domain structure of uPAR reduce its expression and only the intact receptor can sustain the direct cell adhesion on VN-rich matrices found at sites of inflammation and injury.     

uPAR-induced cell adhesion and migration: vitronectin provides the key

Expression of the membrane receptor uPAR induces profound changes in cell morphology and migration, and its expression correlates with the malignant phenotype of cancers. To identify the molecular interactions essential for uPAR function in these processes, we carried out a complete functional alanine scan of uPAR in HEK293 cells. Of the 255 mutant receptors characterized, 34 failed to induce changes in cell morphology. Remarkably, the molecular defect of all of these mutants was a specific reduction in integrin-independent cell binding to vitronectin. A membrane-tethered plasminogen activator inhibitor-1, which has the same binding site in vitronectin as uPAR, replicated uPAR-induced changes. A direct uPAR-vitronectin interaction is thus both required and sufficient to initiate downstream changes in cell morphology, migration, and signal transduction. Collectively these data demonstrate a novel mechanism by which a cell adhesion molecule lacking inherent signaling capability evokes complex cellular responses by modulating the contact between the cell and the matrix without the requirement for direct lateral protein-protein interactions.     

The interaction between uPAR and vitronectin triggers ligand‐independent adhesion signalling by integrins

The urokinase‐type plasminogen activator receptor (uPAR) is a non‐integrin vitronectin (VN) cell adhesion receptor linked to the plasma membrane by a glycolipid anchor. Through structure–function analyses of uPAR, VN and integrins, we document that uPAR‐mediated cell adhesion to VN triggers a novel type of integrin signalling that is independent of integrin–matrix engagement. The signalling is fully active on VN mutants deficient in integrin binding site and is also efficiently transduced by integrins deficient in ligand binding. Although integrin ligation is dispensable, signalling is crucially dependent upon an active conformation of the integrin and its association with intracellular adaptors such as talin. This non‐canonical integrin signalling is not restricted to uPAR as it poses no structural constraints to the receptor mediating cell attachment. In contrast to canonical integrin signalling, where integrins form direct mechanical links between the ECM and the cytoskeleton, the molecular mechanism enabling the crosstalk between non‐integrin adhesion receptors and integrins is dependent upon membrane tension. This suggests that for this type of signalling, the membrane represents a critical component of the molecular clutch.     

Effect of hypoxia on cellular adhesion to vitronectin and fibronectin

Cellular invasion of extracellular matrix (ECM) occurs during normal and pathological settings. For cells to invade, they must adhere to the underlying substratum, break down barrier molecules, and detach from the substratum prior to migrating through the ECM. We previously demonstrated that incubation under reduced oxygen levels increases the in vitro invasiveness of trophoblast and breast carcinoma cells, an effect linked to elevated expression of the cell surface receptor for urokinase-type plasminogen activator (uPAR). This study examined the role of oxygen, integrins and the urokinase-type plasminogen activator (uPA) system on the adhesion of trophoblast and breast carcinoma cells to the ECM molecules vitronectin and fibronectin. Compared to exposure to 20 and 5% oxygen, exposure to 1% oxygen decreased adhesion of these cells to vitronectin and fibronectin, an effect that was reversible by re-exposure to 20% oxygen. Incubation in 1% oxygen also resulted in reduced expression of surface alpha(5) integrin. Furthermore, adhesion to vitronectin and fibronectin was reduced by compounds that interfere with integrin function, such as EDTA, anti-integrin antibodies, or by antibodies that interfere with the binding of pro-uPA to uPAR, soluble uPAR, soluble vitronectin, phosphatidylinositol-specific phospholipase C, as well as plasminogen activator inhibitor-1. These findings suggest an important role for oxygen in the regulation of cellular invasion, possibly in part through its effects on integrin and uPAR-mediated mechanisms of adhesion.     

Crystal structure of the urokinase receptor in a ligand-free form

The urokinase receptor urokinase-type plasminogen activator receptor (uPAR) is a surface receptor capable of not only focalizing urokinase-type plasminogen activator (uPA)-mediated fibrinolysis to the pericellular micro-environment but also promoting cell migration and chemotaxis. Consistent with this multifunctional role, uPAR binds several extracellular ligands, including uPA and vitronectin. Structural studies suggest that uPAR possesses structural flexibility. It is, however, not clear whether this flexibility is an inherent property of the uPAR structure per se or whether it is induced upon ligand binding. The crystal structure of human uPAR in its ligand-free state would clarify this issue, but such information remains unfortunately elusive. We now report the crystal structures of a stabilized, human uPAR (H47C/N259C) in its ligand-free form to 2.4 Å and in complex with amino-terminal fragment (ATF) to 3.2 Å. The structure of uPAR^H47C/N259C in complex with ATF resembles the wild-type uPAR·ATF complex, demonstrating that these mutations do not perturb the uPA binding properties of uPAR. The present structure of uPAR^H47C/N259C provides the first structural definition of uPAR in its ligand-free form, which represents one of the biologically active conformations of uPAR as defined by extensive biochemical studies. The domain boundary between uPAR DI–DII domains is more flexible than the DII–DIII domain boundary. Two important structural features are highlighted by the present uPAR structure. First, the DI–DIII domain boundary may face the cell membrane. Second, loop 130–140 of uPAR plays a dynamic role during ligand loading/unloading. Together, these studies provide new insights into uPAR structure–function relationships, emphasizing the importance of the inter-domain dynamics of this modular receptor.     

Urokinase regulates vitronectin binding by controlling urokinase receptor oligomerization

Adhesion of monocytes to the extracellular matrix is mediated by a direct high affinity interaction between cell-surface urokinase-type plasminogen activator (uPA) receptor (uPAR) and the extracellular matrix protein vitronectin. We demonstrate a tight connection between uPA-regulated uPAR oligomerization and high affinity binding to immobilized vitronectin. We find that binding of soluble uPAR (suPAR) to immobilized vitronectin is strictly ligand-dependent with a linear relationship between the observed binding and the concentration of ligand added. Nevertheless, a comparison of experimentally obtained binding curves to those generated using a simple equilibrium model suggests that the high affinity vitronectin-binding pro-uPA.suPAR complex contains two molecules of suPAR. In co-immunoprecipitation experiments, using different epitope-tagged suPAR molecules, suPAR/suPAR co-immunoprecipitation displayed a similar uPA dose dependence as that observed for vitronectin binding, demonstrating that the high affinity vitronectin-binding complex indeed contains oligomeric suPAR. Structurally, the kringle domain of uPA was found to be critical for the formation of the vitronectin-binding competent complex because the amino-terminal fragment, but not the growth factor-like domain, behaved as a full-length uPA. Our data represent the first demonstration of functional, ligand-induced uPAR oligomerization having extensive implications for glycosylphosphatidylinositol-anchored receptors in general, and for the biology of the uPA/uPAR system in particular.     

Antagonistic Anti-urokinase Plasminogen Activator Receptor (uPAR) Antibodies Significantly Inhibit uPAR-mediated Cellular Signaling and Migration

Interactions between urokinase plasminogen activator receptor (uPAR) and its various ligands regulate tumor growth, invasion, and metastasis. Antibodies that bind specific uPAR epitopes may disrupt these interactions, thereby inhibiting these processes. Using a highly diverse and naïve human fragment of the antigen binding (Fab) phage display library, we identified 12 unique human Fabs that bind uPAR. Two of these antibodies compete against urokinase plasminogen activator (uPA) for uPAR binding, whereas a third competes with β1 integrins for uPAR binding. These competitive antibodies inhibit uPAR-dependent cell signaling and invasion in the non-small cell lung cancer cell line, H1299. Additionally, the integrin-blocking antibody abrogates uPAR/β1 integrin-mediated H1299 cell adhesion to fibronectin and vitronectin. This antibody and one of the uPAR/uPA antagonist antibodies shows a significant combined effect in inhibiting cell invasion through Matrigel/Collagen I or Collagen I matrices. Our results indicate that these antagonistic antibodies have potential for the detection and treatment of uPAR-expressing tumors.     

Haemostatic factors occupy new territory: the role of the urokinase receptor system and kininogen in inflammation

Vascular cell adhesion and migration, proliferation or differentiation are cellular responses that are induced by haemostatic factors of the urokinase/plasminogen activation complex, but the respective underlying mechanisms are largely undefined. The direct and indirect contributions of the urokinase-type plasminogen activator receptor (uPAR) system in inflammatory processes, as they relate to recruitment of leukocytes, define novel functions and could serve as therapeutic targets for related vasculopathies. The presence of uPAR plays a crucial role in beta2-integrin-mediated adhesion of leukocytes; uPAR also directly mediates leukocyte adhesion to vitronectin, a multifunctional adhesion protein that is associated with the extracellular matrix. The latter process is inhibited by plasminogen activator inhibitor-1. Both beta2-integrin- and uPAR-dependent processes are activated by Zn2+ and are blocked by high-molecular-mass kininogen. Domain 5 of kininogen was identified, in particular, as an anti-adhesive component with a potent anti-inflammatory action in a peritonitis mouse model. In patients with acute myocardial infarction, elevated expression of uPAR on monocytes resulted in their increased adherence to the endothelium, which indicates a possible role of the uPAR system in monocyte recruitment to the infarcted area. Urokinase-type plasminogen activator was identified as a potent mitogen for vascular smooth muscle cells, an observation that was independent of the presence of uPAR and its proteolytic activity. Taken together, these results strongly suggest an essential role for the uPAR system in acute inflammation as well as in chronic degenerative vascular processes such as atherosclerosis. Targeting the uPAR system may allow specific therapeutic intervention in vascular pathologies.     

Crystal structure of the human urokinase plasminogen activator receptor bound to an antagonist peptide

We report the crystal structure of a soluble form of human urokinase-type plasminogen activator receptor (uPAR/CD87), which is expressed at the invasive areas of the tumor-stromal microenvironment in many human cancers. The structure was solved at 2.7 A in association with a competitive peptide inhibitor of the urokinase-type plasminogen activator (uPA)-uPAR interaction. uPAR is composed of three consecutive three-finger domains organized in an almost circular manner, which generates both a deep internal cavity where the peptide binds in a helical conformation, and a large external surface. This knowledge combined with the discovery of a convergent binding motif shared by the antagonist peptide and uPA allowed us to build a model of the human uPA-uPAR complex. This model reveals that the receptor-binding module of uPA engages the uPAR central cavity, thus leaving the external receptor surface accessible for other protein interactions (vitronectin and integrins). By this unique structural assembly, uPAR can orchestrate the fine interplay with the partners that are required to guide uPA-focalized proteolysis on the cell surface and control cell adhesion and migration.     

Mannose 6-Phosphate/Insulin-like Growth Factor 2 Receptor Limits Cell Invasion by Controlling αVβ3 Integrin Expression and Proteolytic Processing of Urokinase-type Plasminogen Activator Receptor

The multifunctional mannose 6-phosphate/insulin-like growth factor 2 receptor (M6P/IGF2R) is considered a tumor suppressor. We report here that RNA interference with M6P/IGF2R expression in urokinase-type plasminogen activator (uPA)/urokinase-type plasminogen activator receptor (uPAR) expressing human cancer and endothelial cells resulted in increased pericellular plasminogen activation, cell adhesion, and higher invasive potential through matrigel. M6P/IGF2R silencing led also to the cell surface accumulation of urokinase and plasminogen and enhanced expression of αV integrins. Genetic rescue experiments and inhibitor studies revealed that the enhanced plasminogen activation was due to a direct effect of M6P/IGF2R on uPAR, whereas increased cell adhesion to vitronectin was dependent on αV integrin expression and not uPAR. Increased cell invasion of M6P/IGF2R knockdown cells was rescued by cosilencing both uPAR and αV integrin. Furthermore, we found that M6P/IGF2R expression accelerates the cleavage of uPAR. M6P/IGF2R silencing resulted in an increased ratio of full-length uPAR to the truncated D2D3 fragment, incapable of binding most uPAR ligands. We conclude that M6P/IGF2R controls cell invasion by regulating αV integrin expression and by accelerating uPAR cleavage, leading to the loss of the urokinase/vitronectin/integrin-binding site on uPAR.     

Is plasminogen activator inhibitor-1 the molecular switch that governs urokinase receptor-mediated cell adhesion and release?

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 somatomedin 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.