Urokinase induces expression of its own receptor in Beas2B lung epithelial cells

Interaction between the urokinase-type plasminogen activator (uPA) and its receptor (uPAR) localizes cellular proteolysis and promotes cellular proliferation and migration. The interaction between uPA and uPAR at the surface of epithelial cells thereby contributes to the pathogenesis of lung inflammation and neoplasia. In this study, we sought to determine if uPA itself alters uPAR expression by lung epithelial cells. uPA enhanced uPAR expression as well as (125)I-uPA binding in Beas2B lung epithelial cells in a time- and concentration-dependent manner. The uPA-mediated induction of uPAR is not accomplished through its receptor and requires enzymatic activity. The low molecular weight fragment of uPA, lacking the receptor binding domain, was as potent as intact two-chain uPA in inducing expression of uPAR at the cell surface. Plasmin, the end product of plasminogen activation, did not alter uPA-mediated uPAR expression. Induction of uPAR by uPA represents a novel pathway by which epithelial cells can regulate uPAR-dependent cellular responses that may contribute to stromal remodeling in lung injury or neoplasia.     

Interplay of human tissue kallikrein 4 (hK4) with the plasminogen activation system: hK4 regulates the structure and functions of the urokinase-type plasminogen activator receptor (uPAR)

The plasminogen activation system is involved in cancer progression and metastasis. Among other proteolytic factors, it includes the serine protease urokinase-type plasminogen activator (uPA) and its three-domain (D1D2D3) receptor uPAR (CD87), which focuses plasminogen activation to the cell surface. The function of uPAR is regulated in part through shedding of domain D1 by proteases, e.g., uPA itself or plasmin. Human tissue kallikrein 4 (hK4), which is highly expressed in prostate and ovarian tumor tissue, was previously shown to cleave and activate the pro-enzyme forms of prostate-specific antigen (PSA, tissue kallikrein hK3) and uPA. Here we demonstrate that uPAR is also a target for hK4, being cleaved in the D1-D2 linker sequence and, to a lesser extent, in its D3 juxtamembrane domain. hK4 may thus modulate the tumor-associated uPA/uPAR-system activity by either activating the pro-enzyme form of uPA or cleaving the cell surface-associated uPA receptor.     

Proteolytic cleavage of the urokinase receptor substitutes for the agonist-induced chemotactic effect.

Physiological concentrations of urokinase plasminogen activator (uPA) stimulated a chemotactic response in human monocytic THP-1 through binding to the urokinase receptor (uPAR). The effect did not require the protease moiety of uPA, as stimulation was achieved also with the N-terminal fragment (ATF), while the 33 kDa low molecular weight uPA was ineffective. Co-immunoprecipitation experiments showed association of uPAR with intracellular kinase(s), as demonstrated by in vitro kinase assays. Use of specific antibodies identified p56/p59hck as a kinase associated with uPAR in THP-1 cell extracts. Upon addition of ATF, p56/p59hck activity was stimulated within 2 min and returned to normal after 30 min. Since uPAR lacks an intracellular domain capable of interacting with intracellular kinase, activation of p56/p59hck must require a transmembrane adaptor. Evidence for this was strongly supported by the finding that a soluble form of uPAR (suPAR) was capable of inducing chemotaxis not only in THP-1 cells but also in cells lacking endogenous uPAR (IC50, 5 pM). However, activity of suPAR require chymotrypsin cleavage between the N-terminal domain D1 and D2 + D3. Chymotrypsin-cleaved suPAR also induced activation of p56/p59hck in THP-1 cells, with a time course comparable with ATF. Our data show that uPA-induced signal transduction takes place via uPAR, involves activation of intracellular tyrosine kinase(s) and requires an as yet undefined adaptor capable of connecting the extracellular ligand binding uPAR to intracellular transducer(s).     

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.     

Urokinase-type Plasminogen Activator-like Proteases in Teleosts Lack Genuine Receptor-binding Epidermal Growth Factor-like Domains

Plasminogen activation catalyzed by urokinase-type plasminogen activator (uPA) plays an important role in normal and pathological tissue remodeling processes. Since its discovery in the mid-1980s, the cell membrane-anchored urokinase-type plasminogen activator receptor (uPAR) has been believed to be central to the functions of uPA, as uPA-catalyzed plasminogen activation activity appeared to be confined to cell surfaces through the binding of uPA to uPAR. However, a functional uPAR has so far only been identified in mammals. We have now cloned, recombinantly produced, and characterized two zebrafish proteases, zfuPA-a and zfuPA-b, which by several criteria are the fish orthologs of mammalian uPA. Thus, both proteases catalyze the activation of fish plasminogen efficiently and both proteases are inhibited rapidly by plasminogen activator inhibitor-1 (PAI-1). But zfuPA-a differs from mammalian uPA by lacking the exon encoding the uPAR-binding epidermal growth factor-like domain; zfuPA-b differs from mammalian uPA by lacking two cysteines of the epidermal growth factor-like domain and a uPAR-binding sequence comparable with that found in mammalian uPA. Accordingly, no zfuPA-b binding activity could be found in fish white blood cells or fish cell lines. We therefore propose that the current consensus of uPA-catalyzed plasminogen activation taking place on cell surfaces, derived from observations with mammals, is too narrow. Fish uPAs appear incapable of receptor binding in the manner known from mammals and uPA-catalyzed plasminogen activation in fish may occur mainly in solution. Studies with nonmammalian vertebrate species are needed to obtain a comprehensive understanding of the mechanism of plasminogen activation.     

Urinary-type plasminogen activator (uPA) expression and uPA receptor localization are regulated by alpha 3beta 1 integrin in oral keratinocytes

Expression of urinary-type plasminogen activator (uPA) and its receptor (uPAR) is correlated with matrix proteolysis, cell adhesion, motility, and invasion. To evaluate the functional link between adhesion and proteolysis in gingival keratinocytes (pp126), cells were treated with immobilized integrin antibodies to induce integrin clustering. Clustering of alpha(3) and beta(1) integrin subunits, but not alpha(2), alpha(5), alpha(6), or beta(4), enhanced uPA secretion. Bead-immobilized laminin-5 and collagen I, two major alpha(3)beta(1) ligands, also induced uPA expression. Coordinate regulation of the serpin plasminogen activator inhibitor 1 was also apparent; however, a net increase in uPA activity was predominant. alpha(3)beta(1) integrin clustering induced extracellular signal-regulated kinase 1/2 phosphorylation, and both uPA induction and extracellular signal-regulated kinase activation were blocked by the mitogen-activated protein kinase/extracellular signal-regulated kinase kinase inhibitor PD98059. Integrin aggregation also promoted a dramatic redistribution of uPAR on the cell surface to sites of clustered alpha(3)beta(1) integrins. Co-immunoprecipitation of beta(1) integrin with uPAR provided further evidence that protein-protein interactions between uPAR and beta(1) integrin control uPAR distribution. As a functional consequence of uPA up-regulation and uPA-mediated plasminogen activation, the globular domain of the laminin-5 alpha(3) subunit, a major pp126 matrix protein, was proteolytically processed from a 190-kDa form to a 160-kDa species. Laminin-5 containing the 160-kDa alpha(3) subunit efficiently nucleates hemidesmosome formation and reduces cell motility. Together, these data suggest that multivalent aggregation of the alpha(3)beta(1) integrin regulates proteinase expression, matrix proteolysis, and subsequent cellular behavior.     

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.     

Structural analysis of the interaction between urokinase-type plasminogen activator and its receptor: a potential target for anti-invasive cancer therapy

The ability to degrade the extracellular matrix by controlled proteolysis is an important property of malignant cancer cells, which enables them to invade the surrounding tissue and to gain access to the circulation by intravasation. One proteolytic system thought to be involved in these processes is urokinase-mediated plasminogen activation. Expression of a glycolipid-anchored receptor for urokinase-type plasminogen activator (uPA) targets this system to the cell surface. This receptor (uPAR) is composed of three homologous modules belonging to the Ly-6/uPAR/alpha-neurotoxin protein domain family. Integrity of the three-domain structure of uPAR is required for maintenance of its sub-nanomolar affinity for uPA, but the functional epitope for this interaction is primarily located in uPAR domain I. Using affinity maturation by combinatorial chemistry, we have recently identified a potent 9-mer peptide antagonist of the uPA-uPAR interaction having a high affinity for uPAR (K(d)< 1 nM). Photoaffinity labelling suggests that this peptide interacts with a composite binding site in uPAR involving both domains I and III. When tested in a chicken chorioallantoic membrane assay that was developed to quantify intravasation of human cells, this antagonist was able to reduce the intravasation of HEp-3 cancer cells by approx. 60%.     

Critical role of integrin alpha 5 beta 1 in urokinase (uPA)/urokinase receptor (uPAR,CD87) signaling

Urokinase-type plasminogen activator (uPA) induces cell adhesion and chemotactic movement. uPA signaling requires its binding to uPA receptor (uPAR/CD87), but how glycosylphosphatidylinositol-anchored uPAR mediates signaling is unclear. uPAR is a ligand for several integrins (e.g. alpha 5 beta 1) and supports cell-cell interaction by binding to integrins on apposing cells (in trans). We studied whether binding of uPAR to alpha 5 beta 1 in cis is involved in adhesion and migration of Chinese hamster ovary cells in response to immobilized uPA. This process was temperature-sensitive and required mitogen-activated protein kinase activation. Anti-uPAR antibody or depletion of uPAR blocked, whereas overexpression of uPAR enhanced, cell adhesion to uPA. Adhesion to uPA was also blocked by deletion of the growth factor domain (GFD) of uPA and by anti-GFD antibody, whereas neither the isolated uPA kringle nor serine protease domain supported adhesion directly. Interestingly, anti-alpha 5 antibody, RGD peptide, and function-blocking mutations in alpha 5 beta 1 blocked adhesion to uPA. uPA-induced cell migration also required GFD, uPAR, and alpha 5 beta 1, but alpha 5 beta 1 alone did not support uPA-induced adhesion and migration. Thus, binding of uPA causes uPAR to act as a ligand for alpha 5 beta 1 to induce cell adhesion, intracellular signaling, and cell migration. We demonstrated that uPA induced RGD-dependent binding of uPAR to alpha 5 beta 1 in solution. These results suggest that uPA-induced adhesion and migration of Chinese hamster ovary cells occurs as a consequence of (a) uPA binding to uPAR through GFD, (b) the subsequent binding of a uPA.uPAR complex to alpha 5 beta 1 via uPAR, and (c) signal transduction through alpha 5 beta 1.     

Increased expression of the urokinase plasminogen activator system by Helicobacter pylori in gastric epithelial cells

The gastric pathogen Helicobacter pylori (H. pylori) is linked to peptic ulcer and gastric cancer, but the relevant pathophysiological mechanisms are unclear. We now report that H. pylori stimulates the expression of plasminogen activator inhibitor (PAI)-1, urokinase plasminogen activator (uPA), and its receptor (uPAR) in gastric epithelial cells and the consequences for epithelial cell proliferation. Real-time PCR of biopsies from gastric corpus, but not antrum, showed significantly increased PAI-1, uPA, and uPAR in H. pylori-positive patients. Transfection of primary human gastric epithelial cells with uPA, PAI-1, or uPAR promoters in luciferase reporter constructs revealed expression of all three in H+/K+ATPase- and vesicular monoamine transporter 2-expressing cells; uPA was also expressed in pepsinogen- and uPAR-containing trefoil peptide-1-expressing cells. In each case expression was increased in response to H. pylori and for uPA, but not PAI-1 or uPAR, required the virulence factor CagE. H. pylori also stimulated soluble and cell surface-bound uPA activity, and both were further increased by PAI-1 knockdown, consistent with PAI-1 inhibition of endogenous uPA. H. pylori stimulated epithelial cell proliferation, which was inhibited by uPA immunoneutralization and uPAR knockdown; exogenous uPA also stimulated proliferation that was further increased after PAI-1 knockdown. The proliferative effects of uPA were inhibited by immunoneutralization of the EGF receptor and of heparin-binding EGF (HB-EGF) by the mutant diphtheria toxin CRM197 and an EGF receptor tyrosine kinase inhibitor. H. pylori induction of uPA therefore leads to epithelial proliferation through activation of HB-EGF and is normally inhibited by concomitant induction of PAI-1; treatments directed at inhibition of uPA may slow the progression to gastric cancer.     

Peptide-derived antagonists of the urokinase receptor. affinity maturation by combinatorial chemistry, identification of functional epitopes, and inhibitory effect on cancer cell intravasation

The high-affinity interaction between urokinase-type plasminogen activator (uPA) and its glycolipid-anchored receptor (uPAR) plays an important role in pericellular plasminogen activation. Since proteolytic degradation of the extracellular matrix has an established role in tumor invasion and metastasis, the uPA-uPAR interaction represents a potential target for therapeutic intervention. By affinity maturation using combinatorial chemistry we have now developed and characterized a 9-mer, linear peptide antagonist of the uPA-uPAR interaction demonstrating specific, high-affinity binding to human uPAR (K(d) approximately 0.4 nM). Studies by surface plasmon resonance reveal that the off-rate for this receptor-peptide complex is comparable to that measured for the natural protein ligand, uPA. The functional epitope on human uPAR for this antagonist has been delineated by site-directed mutagenesis, and its assignment to loop 3 of uPAR domain III (Met(246), His(249), His(251), and Phe(256)) corroborates data previously obtained by photoaffinity labeling and provides a molecular explanation for the extreme selectivity observed for the antagonist toward human compared to mouse, monkey, and hamster uPAR. When human HEp-3 cancer cells were inoculated in the presence of this peptide antagonist, a specific inhibition of cancer cell intravasation was observed in a chicken chorioallantoic membrane assay. These data imply that design of small organic molecules mimicking the binding determinants of this 9-mer peptide antagonist may have a potential application in combination therapy for certain types of cancer.     

The urokinase-type plasminogen activator receptor, a GPI-linked protein, is localized in caveolae

The urokinase plasminogen activator receptor (uPAR), a glycosylphosphatidylinositol-linked glycoprotein, plays a central role in the regulation of pericellular proteolysis and participates in events leading to cell activation. Here, we demonstrate that uPAR, on a human melanoma cell line, is localized in caveolae, flask-shaped microinvaginations of the plasma membrane found in a variety of cell types. Indirect immunofluorescence with anti-uPAR antibodies on the melanoma cells showed a punctated staining pattern that accumulated to stretches along sides of cell-cell contact and membrane ruffles. uPAR colocalized with caveolin, a characteristic protein in the coat of caveolae, as demonstrated by double staining with specific antibodies. Further, uPAR could be directly localized in caveolae by in vivo immunoelectron microscopy. Both uPAR and its ligand, uPA, were present in caveolae enriched low density Triton X-100 insoluble complexes, as shown by immunoblotting. From such complexes, caveolin could be coprecipitated with uPAR-specific antibodies suggesting a close spatial association between uPAR and caveolin that might have implications for the signal transduction mediated by uPAR. Further, functional studies indicated that the localization of uPAR and its ligand in caveolae enhances pericellular plasminogen activation, since treatment of the cells with drugs that interfere with the structural integrity of caveolae, such as nystatin, markedly reduced cell surface plasmin generation. Thus, caveolae promote efficient cell surface plasminogen activation by clustering uPAR, uPA, and possibly other protease receptors in one membrane compartment.     

Targeting of tumor cells by cell surface urokinase plasminogen activator-dependent anthrax toxin

Urokinase plasminogen activator receptor (uPAR) binds pro-urokinase plasminogen activator (pro-uPA) and thereby localizes it near plasminogen, causing the generation of active uPA and plasmin on the cell surface. uPAR and uPA are overexpressed in a variety of human tumors and tumor cell lines, and expression of uPAR and uPA is highly correlated to tumor invasion and metastasis. To exploit these characteristics in the design of tumor cell-selective cytotoxins, we constructed mutated anthrax toxin-protective antigen (PrAg) proteins in which the furin cleavage site is replaced by sequences cleaved specifically by uPA. These uPA-targeted PrAg proteins were activated selectively on the surface of uPAR-expressing tumor cells in the presence of pro-uPA and plasminogen. The activated PrAg proteins caused internalization of a recombinant cytotoxin, FP59, consisting of anthrax toxin lethal factor residues 1-254 fused to the ADP-ribosylation domain of Pseudomonas exotoxin A, thereby killing the uPAR-expressing tumor cells. The activation and cytotoxicity of these uPA-targeted PrAg proteins were strictly dependent on the integrity of the tumor cell surface-associated plasminogen activation system. We also constructed a mutated PrAg protein that selectively killed tissue plasminogen activator-expressing cells. These mutated PrAg proteins may be useful as new therapeutic agents for cancer treatment.     

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.     

Differential proteome expression associated with urokinase plasminogen activator receptor (uPAR) suppression in malignant epithelial cancer

Dysregulation of the plasminogen activation cascade is a prototypic feature in many malignant epithelial cancers. Principally, this is thought to occur through activation of overexpressed urokinase plasminogen activator (uPA) concomitant with binding to its high specificity cell surface receptor urokinase plasminogen activator receptor (uPAR). Up-regulation of uPA and uPAR in cancer appears to potentiate the malignant phenotype, either (i) directly by triggering plasmin-mediated degradation or activation of uPA's or plasmin's proteolytic targets (e.g., extracellular matrix zymogen proteases or nascent growth factors) or indirectly by simultaneously altering a range of downstream functions including signal transduction pathways ( Romer, J. ; Nielsen, B. S. ; Ploug, M. The urokinase receptor as a potential target in cancer therapy Curr. Pharm. Des. 2004, 10 ( 19), 235976 ). Because many malignant epithelial cancers express high levels of uPAR, uPA or other components of the plasminogen activation cascade and because these are often associated with poor prognosis, characterizing how uPAR changes the downstream cellular "proteome" is fundamental to understanding any role in cancer. This study describes a carefully designed proteomic study of the effects of antisense uPAR suppression in a previously studied colon cancer cell line (HCT116). The study utilized replicate 2DE gels and two independent gel image analysis software packages to confidently identify 64 proteins whose expression levels changed (by > or =2 fold) coincident with a moderate ( approximately 40%) suppression of cell-surface uPAR. Not surprisingly, many of the altered proteins have previously been implicated in the regulation of tumor progression (e.g., p53 tumor suppressor protein and c-myc oncogene protein among many others). In addition, through a combination of proteomics and immunological methods, this study demonstrates that stathmin 1alpha, a cytoskeletal protein implicated in tumor progression, undergoes a basic isoelectric point shift (p I) following uPAR suppression, suggesting that post-translational modification of stathmin occur secondary to uPAR suppression. Overall, these results shed new light on the molecular mechanisms involved in uPAR signaling and how it may promulgate the malignant phenotype.     

Direct binding of occupied urokinase receptor (uPAR) to LDL receptor-related protein is required for endocytosis of uPAR and regulation of cell surface urokinase activity

Low-density lipoprotein receptor-related protein (LRP) mediates internalization of urokinase:plasminogen activator inhibitor complexes (uPA:PAI-1) and the urokinase receptor (uPAR). Here we investigated whether direct interaction between uPAR, a glycosyl-phosphatidylinositol-anchored protein, and LRP, a transmembrane receptor, is required for clearance of uPA:PAI-1, regeneration of unoccupied uPAR, activation of plasminogen, and the ability of HT1080 cells to invade extracellular matrix. We found that in the absence of uPA:PAI-1, uPAR is randomly distributed along the plasma membrane, whereas uPA:PAI-1 promotes formation of uPAR-LRP complexes and initiates redistribution of occupied uPAR to clathrin-coated pits. uPAR-LRP complexes are endocytosed via clathrin-coated vesicles and traffic together to early endosomes (EE) because they can be coimmunoprecipitated from immunoisolated EE, and internalization is blocked by depletion of intracellular K(+). Direct binding of domain 3 (D3) of uPAR to LRP is required for clearance of uPA-PAI-1-occupied uPAR because internalization is blocked by incubation with recombinant D3. Moreover, uPA-dependent plasmin generation and the ability of HT1080 cells to migrate through Matrigel-coated invasion chambers are also inhibited in the presence of D3. These results demonstrate that GPI-anchored uPAR is endocytosed by piggybacking on LRP and that direct binding of occupied uPAR to LRP is essential for internalization of occupied uPAR, regeneration of unoccupied uPAR, plasmin generation, and invasion and migration through extracellular matrix.     

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.     

Differential binding of urokinase and peptide antagonists to the urokinase receptor: evidence from characterization of the receptor in four primate species

The urokinase plasminogen activator receptor (uPAR) is a membrane protein active in localizing the plasminogen activation cascade system on the cell surface. The resulting pericellular proteolytic activity is responsible for degradation reactions in the extracellular matrix that are needed for the invasion of cancer cells, thus making uPAR a potential target for anti-invasive therapy based on binding antagonists. A remarkable property of the uPA-uPAR system is a pronounced species specificity in ligand recognition. We have now cloned and studied uPAR from four primate species and show that even though these sequences contain very few substitutions relative to the human uPAR, the receptor protein products differ markedly in terms of ligand selectivity. Thus, a well described competitive peptide antagonist directed against the human uPAR reacts with only one of the monkey receptors (chimpanzee uPAR), in spite of the fact that uPAR from all of the four species cross-reacts with human uPA. Notably, uPAR from African green monkey, which is completely devoid of reactivity with the peptide, contains only three substitutions relative to chimpanzee uPAR in the molecular regions critical for binding. These findings aid the elucidation of the structure/function relationship of uPAR and, unexpectedly, identify a structural distinction governing the binding of uPA and a very similar peptide antagonist.