SMART MODE:

NORMAL GENOMIC

• Liang OD, Chavakis T, Kanse SM, Preissner KT
• Ligand binding regions in the receptor for urokinase-type plasminogen activator.
• J Biol Chem. 2001; 276: 28946-53
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• 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.

• Hapke S et al.
• beta(3)A-integrin downregulates the urokinase-type plasminogen activator receptor (u-PAR) through a PEA3/ets transcriptional silencing element in the u-PAR promoter.
• Mol Cell Biol. 2001; 21: 2118-32
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• Migration of cells requires interactions with the extracellular matrix mediated, in part, by integrins, proteases, and their receptors. Previous studies have shown that beta(3)-integrin interacts with the urokinase-type plasminogen activator receptor (u-PAR) at the cell surface. Since integrins mediate signaling into the cell, the current study was undertaken to determine if in addition beta(3)-integrin regulates u-PAR expression. Overexpression of beta(3)-integrin in CHO cells, which are avid expressers of the receptor, downregulated u-PAR protein and mRNA expression. The u-PAR promoter (-1,469 bp) that is normally constitutively active in CHO cells was downregulated by induced beta(3)-integrin expression. A region between -398 and -197 bp of the u-PAR promoter was critical for beta(3)-integrin-induced downregulation of u-PAR promoter activity. Deletion of the PEA3/ets motif at -248 bp substantially impaired the ability of beta(3)-integrin to downregulate the u-PAR promoter, suggesting that the PEA3/ets site acts as a silencing element. An expression vector encoding the transcription factor PEA3 caused inhibition of the wild-type but not the PEA3/ets-deleted u-PAR promoter. The PEA3/ets site bound nuclear factors from CHO cells specifically, but binding was enhanced when beta(3)-integrin was overexpressed. A PEA3 antibody inhibited DNA-protein complex formation, indicating the presence of PEA3. Downregulation of the u-PAR promoter was achieved by the beta(3)A-integrin isoform but not by other beta(3)-integrin isoforms and required the cytoplasmic membrane NITY(759) motif. Moreover, overexpression of the short but not the long isoform of the beta(3)-integrin adapter protein beta(3)-endonexin blocked u-PAR promoter activity through the PEA3/ets binding site. Thus, besides the physical interaction of beta(3)-integrin and u-PAR at the cell surface, beta(3) signaling is implicated in the regulation of u-PAR gene transcription, suggesting a mutual regulation of adhesion and proteolysis receptors.

• Zhou HM, Nichols A, Meda P, Vassalli JD
• Urokinase-type plasminogen activator and its receptor synergize to promote pathogenic proteolysis.
• EMBO J. 2000; 19: 4817-26
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• Urokinase-type plasminogen activator (uPA) is a potent catalyst of extracellular proteolysis, which also binds to a high-affinity plasma membrane receptor (uPAR). Binding of uPA may influence pericellular proteolysis and/or activate intracellular signal transduction. Transgenic mice overexpressing either uPA or uPAR in basal epidermis and hair follicles had no detectable cutaneous alterations. In contrast, bi-transgenic mice overexpressing both uPA and uPAR, obtained by crossing the two transgenic lines, developed extensive alopecia induced by involution of hair follicles, epidermal thickening and sub-epidermal blisters. The phenotype was due to uPA catalytic activity since combined overexpression of uPAR and uPAR-binding but catalytically inactive uPA in the same tissue was not detrimental in another bi-transgenic line. It was accompanied by increased plasmin-generating capacity, up-regulation and activation of matrix metalloproteinases type-2 and -9, and cleavage of uPAR. Thus, combined overexpression of uPA and uPAR acts in synergy to promote pathogenic extracellular proteolysis.

• Dubuisson L, Monvoisin A, Nielsen BS, Le Bail B, Bioulac-Sage P, Rosenbaum J
• Expression and cellular localization of the urokinase-type plasminogen activator and its receptor in human hepatocellular carcinoma.
• J Pathol. 2000; 190: 190-5
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• The urokinase-type plasminogen activator (uPA) and its receptor (uPAR) play an important role in tumour invasion. Previous studies have shown by RT-PCR that uPA and uPAR mRNAs are expressed in human hepatocellular carcinoma (HCC). Here, in situ hybridization, immunohistochemistry, and double immunofluorescence were used to identify the cells expressing uPA and uPAR in 26 HCCs. The results indicate that uPA and uPAR were expressed in every case, almost exclusively in stromal cells, mostly myofibroblasts and macrophages, except for rare tumoural hepatocytes expressing cytokeratin 7. These results show the important role of stromal cells of HCC in the pericellular proteolysis which facilitates cancer cell invasion.

• Montuori N, Salzano S, Rossi G, Ragno P
• Urokinase-type plasminogen activator up-regulates the expression of its cellular receptor.
• FEBS Lett. 2000; 476: 166-70
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• The expression of the receptor for the urokinase-type plasminogen activator (uPAR) can be regulated by several hormones, cytokines, tumor promoters, etc. Recently, it has been reported that uPAR is capable of transducing signals, even though it is lacking a transmembrane domain and a cytoplasmatic tail. We now report that uPAR cell surface expression can be positively regulated by its ligand, uPA, in thyroid cells. The effect of uPA is independent of its proteolytic activity, since inactivated uPA or its aminoterminal fragment have the same effects of the active enzyme. The increase of uPAR on the cell surface correlates with an increase of specific uPAR mRNA. Finally, uPA up-regulates uPAR expression also in other cell lines of different type and origin, thus suggesting that the regulatory role of uPA on uPAR expression is not restricted to thyroid cells, but it occurs in different tissues, both normal and tumoral.

• Sidenius N, Sier CF, Blasi F
• Shedding and cleavage of the urokinase receptor (uPAR): identification and characterisation of uPAR fragments in vitro and in vivo.
• FEBS Lett. 2000; 475: 52-6
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• Applying a novel, highly specific and sensitive immunoabsorption/Western blotting technique we have identified in vitro in conditioned cell culture medium and in vivo in human urine different soluble forms of the urokinase-type plasminogen activator receptor (uPAR/CD87). These include the uPAR fragment D2D3 and the never before identified domain 1 (D1) fragment. These forms correspond to fragments previously characterised as biologically active as inducers of chemotaxis and cell adhesion. We find that stimulation of U937 cells is associated with increased uPAR expression, cleavage of surface uPAR, and release of soluble fragments to the culture medium suggesting that monocytes are a source of the circulating and urinary soluble uPAR fragments found in vivo. Our study demonstrates that potentially biologically active uPAR fragments are produced in the human body, indicating a possible function in the regulation of not only proteolysis but also signal transduction related processes.

• Longstaff C, Merton RE, Fabregas P, Felez J
• Characterization of cell-associated plasminogen activation catalyzed by urokinase-type plasminogen activator, but independent of urokinase receptor (uPAR, CD87).
• Blood. 1999; 93: 3839-46
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• The 55-kD urokinase (uPA) receptor (uPAR, CD87) is capable of binding uPA and may be involved in regulating cell-associated plasminogen activation and pericellular proteolysis. While investigating the relationship between uPAR levels and plasmin generation, we found that uPA-catalyzed plasminogen activation is stimulated by cells which do not express uPAR. This uPAR-independent mechanism appears to be at least as effective in vitro as uPAR-dependent stimulation, such that stimulation on the order of 30-fold was observed, resulting from improvements in both apparent kcat and apparent Km. The mechanism depends on simultaneous binding of both uPA and plasminogen to the cell and requires the presence of the amino-terminal fragment (ATF), available in single chain and two chain high-molecular-weight uPA, but not low-molecular-weight uPA. Stimulation was observed in all leukemic cell lines investigated at similar optimum concentrations of 10(6) to 10(7) cells/mL and may be more general. A mechanism is proposed whereby uPA can associate with binding sites on the cell surface of lower affinity, but higher capacity than uPAR, but these are sufficient to stimulate plasmin generation even at subphysiologic uPA concentrations. This mechanism is likely to operate under conditions commonly used for in vitro studies and may have some significance in vivo.

• Mohanam S, Gladson CL, Rao CN, Rao JS
• Biological significance of the expression of urokinase-type plasminogen activator receptors (uPARs) in brain tumors.
• Front Biosci. 1999; 4: 17887-17887
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• The urokinase-type plasminogen activator receptor (uPAR) plays a critical role in the regulation of cell-surface plasminogen activation in several physiological and pathological conditions. Recent evidence suggests that the uPAR is also involved in processes that are not related to plasminogen activation, including cell adhesion and transmission of extracellular signals across the plasma membrane. The uPAR influences cell migration and spreading both in vivo and in vitro through the cell-surface activation of plasminogen. The uPAR can bind to vitronectin, an adhesive extracellular matrix protein that contains the Arg-gly-Asp (RGD) cell adhesion domain and that serves as a ligand for several integrin receptors. uPAR also forms complexes with (1, (2, and (3 integrins, thereby allowing mutual interactions and regulation between cell adhesion and proteolysis. Recently, uPAR has been shown to have strong prognostic value for predicting disease recurrence and overall survival in certain types of cancer. We discuss here the biological significance of uPAR in the glioblastoma invasion process. Strong correlations found between elevated uPAR levels in glioblastoma cells and tumor invasiveness have led to uPAR being selected as a target for therapy in experimental animal models. Using antisense vectors to down regulate uPAR expression at the level of the mRNA and protein in glioblastoma cells, has been shown to inhibit tumor formation in nude mice. These results provide a potential basis from which to develop novel therapeutic strategies to direct the expression of antisense uPAR and to evaluate the efficiency of this technique for cancer gene therapy in patients with brain tumor.

• Gardsvoll H, Dano K, Ploug M
• Mapping part of the functional epitope for ligand binding on the receptor for urokinase-type plasminogen activator by site-directed mutagenesis.
• J Biol Chem. 1999; 274: 37995-8003
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• The urokinase-type plasminogen activator receptor (uPAR) is a glycolipid anchored multidomain member of the Ly-6/uPAR protein domain superfamily. Studies by site-directed photoaffinity labeling, chemical cross-linking, and ligand-induced protection against chemical modification have highlighted the possible involvement of uPAR domain I and particularly loop 3 thereof in ligand binding (Ploug, M. (1998) Biochemistry 37, 16494-16505). Guided by these results we have now performed an alanine scanning analysis of this region in uPAR by site-directed mutagenesis and subsequently measured the effects thereof on the kinetics of uPA binding in real-time by surface plasmon resonance. Only four positions in loop 3 of uPAR domain I exhibited significant changes in the contribution to the free energy of uPA binding (DeltaDeltaG >/= 1.3 kcal mol(-1)) upon single-site substitutions to alanine (i.e. Arg(53), Leu(55), Tyr(57), and Leu(66)). The energetic impact of these four alanine substitutions was not caused by gross structural perturbations, since all monoclonal antibodies tested having conformation-dependent epitopes on this domain exhibited unaltered binding kinetics. These sites together with a three-dimensional structure for uPAR may provide an appropriate target for rational drug design aimed at developing new receptor binding antagonists with potential application in cancer therapy.

• Aceto J, Kieber-Emmons T, Cines DB
• Carboxy-terminal processing of the urokinase receptor: implications for substrate recognition and glycosylphosphatidylinositol anchor addition.
• Biochemistry. 1999; 38: 992-1001
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• Proteins linked to cell membranes by a glycosylphosphatidylinositol (GPI) anchor must first undergo cleavage by a putative transamidase between the omega and omega + 1 positions within a proposed small amino acid (SAD) domain in the carboxy terminus of the nascent polypeptide. The requirements for such processing, defined in an engineered placental alkaline phosphatase construct (miniPLAP), suggest the SAD domain functions as an autonomous unit within the context of an otherwise permissive carboxy-terminal sequence with only certain amino acids tolerated at the omega, omega + 1, and omega + 2 positions. To test whether this hypothesis could be generalized, we engineered a chimeric molecule containing the extracellular domain of miniPLAP and the carboxy-terminal portion of the urokinase receptor (MP/uPAR) into which various amino acid substitutions were introduced. The variant proteins were translated and metabolically labeled in vitro using a cell-free translation system that contains the enzymatic machinery required for carboxy-terminal processing and GPI anchor addition. The results of this study indicate that the SAD domain functions as an independent, but not an autonomous, unit. The requirements for processing in miniPLAP and MP/uPAR differed markedly in some respects, in part due to the influence of the amino acid at the omega + 4 position which both modified cleavage between the omega and omega + 1 positions and permitted a second cleavage site to be generated in some cases. In addition, substitution of bulky hydrophobic amino acids in series at the omega + 2 and omega + 3 positions inhibited carboxy-terminal processing in a dose-dependent manner, suggesting the presence of a critical docking site adjacent to the cleavage site. These results suggest the carboxy-terminal transamidase recognizes a more extended structure similar to the mechanism proposed for serine proteases. Further, the data provide a potential means for isolating the transamidase.

• Durko M, Brodt P
• Suppression of type I collagenase expression by antisense RNA in melanoma cells results in reduced synthesis of the urokinase-type plasminogen activator receptor.
• Biochem Biophys Res Commun. 1998; 247: 342-8
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• Previously we reported that suppression of type I collagenase synthesis in human melanoma cells with antisense RNA significantly reduced proteolysis of type I and type IV collagen matrices (Durko et al., 1997, Biochim. Biophys. Acta 1356, 271). Because plasmin is a major activator of the type I collagenase, we assessed the impact of type I collagenase suppression on the urokinase/plasmin system of proteolysis. Gel zymography revealed the appearance of two new caseinolytic bands of Mr 81-83000 in conditioned media of type I collagenase-depleted, but not of wild-type cells and these were identified as plasmin bands. This increased extracellular plasmin activity coincided with reduced membrane-associated plasminogen levels and decreased expression of the urokinase-type plasminogen activator receptor at both the mRNA (up to 83% reduction) and cell-surface (up to 48% reduction) levels, while urokinase mRNA levels remained unchanged. The results indicate that in these cells the urokinase/plasmin system is regulated by type I collagenase levels.

• Ploug M
• Identification of specific sites involved in ligand binding by photoaffinity labeling of the receptor for the urokinase-type plasminogen activator. Residues located at equivalent positions in uPAR domains I and III participate in the assembly of a composite ligand-binding site.
• Biochemistry. 1998; 37: 16494-505
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• Plasminogen activation by the urokinase-type plasminogen activator (uPA) is facilitated in the presence of cells expressing the glycolipid-anchored high-affinity receptor for uPA (denoted uPAR). Structures involved in the interaction between human uPAR and a decamer peptide antagonist of uPA binding (SLNFSQYLWS) were previously tagged by specific site-directed photoaffinity labeling [Ploug, M., Ostergaard, S., Hansen, L. B. L., Holm, A., and Dano, K. (1998) Biochemistry 37, 3612-3622]. Replacement of the key functional residues Phe4 and Trp9 with either benzophenone or (trifluoromethyl)aryldiazirine rendered this peptide antagonist photoactivatable, and as a consequence, it incorporated covalently upon photolysis into either uPAR domain I or domain III depending on the actual position of the photophore in the sequence. The residues of uPAR specifically targeted by photoaffinity labeling were identified by matrix-assisted laser desorption mass spectrometry, NH2-terminal sequence analysis, and amino acid composition analysis after enzymatic fragmentation and HPLC purification. According to these data, the formation of the receptor-ligand complex positions Phe4 of the peptide antagonist very close to Arg53 and Leu66 in uPAR domain I and Trp9 of the antagonist in the vicinity of His251 in uPAR domain III. The gross molecular arrangement of the deduced receptor-ligand interface provides a rational structural basis for the observed requirement for the intact multidomain state of uPAR for achieving high-affinity ligand binding, since according to this model ligand binding must rely on a close spatial proximity of uPAR domains I and III. In addition, these data suggest that the assembly of the composite ligand binding site in uPAR may resemble the homophilic interdomain dimerization of kappa-bungarotoxin, a structural homologue of the Ly-6/uPAR domain family.

• Ploug M, Ostergaard S, Hansen LB, Holm A, Dano K
• Photoaffinity labeling of the human receptor for urokinase-type plasminogen activator using a decapeptide antagonist. Evidence for a composite ligand-binding site and a short interdomain separation.
• Biochemistry. 1998; 37: 3612-22
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• Binding of urokinase-type plasminogen activator (uPA) to its cellular receptor (uPAR) renders the cell surface a favored site for plasminogen activation. Recently, a 15-mer peptide antagonist of the uPA-uPAR interaction, with an IC50 value of 10 nM, was identified using phage display technology [Goodson, R. J., Doyle, M. V., Kaufman, S. E., and Rosenberg, S. (1994) Proc. Natl. Acad. Sci. 91, 7129-7133]. In the present study, the molecular aspects of the interaction between this peptide and uPAR have been investigated. We have characterized the real-time receptor binding kinetics for the antagonist using surface plasmon resonance and identified critical residues by alanine replacements. The minimal peptide antagonist thus derived (SLNFSQYLWS) was rendered photoactivatable by replacing residues important for uPAR binding with photochemically active derivatives of phenylalanine containing either (trifluoromethyl)diazirine or benzophenone. These peptides incorporated covalently into purified soluble uPAR upon photoactivation, and this was inhibited by preincubation with receptor binding derivatives of uPA. The intact three-domain structure of uPAR was essential for efficient photoaffinity labeling. Proteolytic domain mapping using chymotrypsin revealed a specific labeling of both uPAR domain I and domains II + III dependent on the position of the photoprobe in the antagonist. On the basis of these studies, we propose the existence of a composite ligand binding site in uPAR combined of residues located in distinct structural domains. According to this model, a close spatial proximity between uPAR domain I and either domains II or III in intact uPAR is required for the assembly of this composite binding site. Since the receptor binding properties of the peptide antagonist closely mimic those of uPA itself, these two ligands presumably share coincident binding site in uPAR.

• Quax PH et al.
• Binding of human urokinase-type plasminogen activator to its receptor: residues involved in species specificity and binding.
• Arterioscler Thromb Vasc Biol. 1998; 18: 693-701
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• Urokinase-type plasminogen activator (UPA), particularly when bound to its receptor (UPAR), is thought to play a major role in local proteolytic processes, thus facilitating cell migration as may occur during angiogenesis, neointima and atherosclerotic plaque formation, and tumor cell invasion. To facilitate understanding of the need and function of the UPA/UPAR interaction in cell migration and vascular remodeling, we changed several amino acid residues in UPA so as to interfere with its interaction with its receptor. The receptor-binding domain of UPA has been localized to a region in the growth factor domain between residues 20 and 32. Since the binding of UPA to UPAR appears to be species specific, we used the differences in amino acid sequences in the growth factor domain of UPA between various species to construct a human UPA variant that does not bind to the human UPAR. We substituted Asn22 for its mouse equivalent Tyr by site-directed mutagenesis. This mutant UPA had similar plasminogen activator characteristics as wild-type UPA, including its specific activity and interaction with plasminogen activator inhibitor-1. However, no UPA/UPAR complexes could be observed in cross-linking experiments using DFP-treated 125I-labeled mutant UPA and lysates of various cells, including U937 histiocytic lymphoma cells, phorbol myristate acetate-treated human ECs, and mouse LB6 cells transfected with human UPAR cDNA. In direct binding experiments, DFP-treated 125I-labeled mutant UPA could not bind to phorbol myristate acetate-treated ECs, whereas wild-type UPA did bind. Furthermore, a 25-fold excess of wild-type UPA completely prevented the binding of DFP-treated 125I-labeled wild-type UPA to the human receptor on transfected LB6 cells, whereas an equal amount of mutant UPA had only a very small effect. In ligand blotting assays, very weak binding of mutant UPA to human UPAR could be observed. Changing Asn22 into the other amino acid residues alanine or glutamine had no effect on binding to UPAR on human ECs. The functional integrity of the growth factor domain in the non-receptor binding Asn22Tyr mutant is suggested by the fact that binding of this mutant to a murine UPAR can be restored after additional mutations in the growth factor domain, Asn27,His29,Trp30 to Arg27,Arg29,Arg30. We conclude that Asn22 and Asn27,His29,Trp30 in human UPA are key determinants in the species-specific binding of the enzyme to its receptor and that changing Asn22 into Tyr results in a UPA mutant with strongly reduced binding to UPAR.

• Ploug M, Rahbek-Nielsen H, Nielsen PF, Roepstorff P, Dano K
• Glycosylation profile of a recombinant urokinase-type plasminogen activator receptor expressed in Chinese hamster ovary cells.
• J Biol Chem. 1998; 273: 13933-43
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• Association of urokinase-type plasminogen activator (uPA) to cells via binding to its specific cellular receptor (uPAR) augments the potential of these cells to support plasminogen activation, a process that has been implicated in the degradation of extracellular matrix proteins during cell migration and tissue remodeling. The uPA receptor is a glycolipid-anchored membrane protein belonging to the Ly-6/uPAR superfamily and is the only multidomain member identified so far. We have now purified the three individual domains of a recombinant soluble uPAR variant, expressed in Chinese hamster ovary cells, after limited proteolysis using chymotrypsin and pepsin. The glycosylation patterns of these domains have been determined by matrix assisted laser desorption ionization and electrospray ionization mass spectrometry. Of the five potential attachment sites for asparagine-linked carbohydrate in uPAR only four are utilized, as the tryptic peptide derived from domain III containing Asn233 was quantitatively recovered without carbohydrate. The remaining four attachment sites were shown to exhibit site-specific microheterogeneity of the asparagine-linked carbohydrate. The glycosylation on Asn52 (domain I) and Asn172 (domain II) is dominated by the smaller biantennary complex-type oligosaccharides, while Asn162 (domain II) and Asn200 (domain III) predominantly carry tri- and tetraantennary complex-type oligosaccharides. The carbohydrate moiety on Asn52 in uPAR domain I could be selectively removed by N-glycanase treatment under nondenaturing conditions. This susceptibility was abrogated when uPAR participitated in a bimolecular complex with pro-uPA or smaller receptor binding derivatives thereof, demonstrating the proximity of the ligand-binding site to this particular carbohydrate moiety. uPAR preparations devoid of carbohydrate on domain I exhibited altered binding kinetics toward uPA (a 4-6-fold increase in Kd) as assessed by real time biomolecular interaction analysis.

• Hasui Y, Osada Y
• Urokinase-type plasminogen activator and its receptor in bladder cancer.
• J Natl Cancer Inst. 1997; 89: 678-9

• Conne B, Berczy M, Belin D
• Detection of polymorphisms in the human urokinase-type plasminogen activator gene.
• Thromb Haemost. 1997; 77: 434-5
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• Expressed polymorphisms in the genes encoding components of the fibrinolytic cascade could have implications for the predisposition to thrombolytic disorders and/or for tumor metastasis. The occurrence of published two amino acid sequences at position 194 of the human urokinase-type plasminogen activator prompted us to search by SSCP for frequent polymorphisms in several exons of the gene. Surprisingly, only one sequence was detected in codon 194 (> 200 alleles). Two polymorphisms were observed in this study: the most frequent one, a C to T change near the beginning of exon 8, is probably silent; a less frequent polymorphism results in the replacement of a Leu residue by a Pro, in the kringle domain.

• Behrendt N, Dano K
• Reply to comment on 'Effect of purified, soluble urokinase receptor on the plasminogen-prourokinase activation system' (A. A-R. Higazi)
• FEBS Lett. 1997; 402: 293-4

• Higazi AA
• Commentary on: 'Effect of purified soluble urokinase receptor on the plasminogen prourokinase activation system' by N. Behrendt and K. Dano, FEBS Letters, 393 (1996) 31-36.
• FEBS Lett. 1997; 402: 291-2

• Ragno P, Montuori N, Rossi G
• Urokinase-type plasminogen-activator receptor associates to a cell surface molecule in monocytic cells.
• Biochem Biophys Res Commun. 1996; 224: 252-7
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• The monocyte-like THP-1 cells express on their surface the urokinase-type plasminogen activator receptor (uPA-R). This receptor, chemically cross-linked to its possible ligands, migrates, in SDS PAGE, slower than the uPA-R expressed on the epithelial thyroid cell line TAD-2, cross-linked to the same ligands. The different migration corresponds to a difference in molecular weight of 15 kDa. Similar results were obtained with peripheral monocytes and primary cultures of thyroid cells. The molecular weight of the native receptor is about 50 kDa and appears to be identical in these two cell types. Such results suggest that, in monocytic cells, uPA-R associates to a 15 kDa molecule. This molecule is probably linked to the cell surface by a glyco-phospho-inositol anchor since, by phospholipase-C treatment, it is co-eluted with the urokinase-type plasminogen activator receptor from THP-1 cells.

• Tkachuk V, Stepanova V, Little PJ, Bobik A
• Regulation and role of urokinase plasminogen activator in vascular remodelling.
• Clin Exp Pharmacol Physiol. 1996; 23: 759-65
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• 1. Urokinase plasminogen activator (uPA) is produced and secreted by multiple vascular cell types, thus influencing the processes and the extent to which the vasculature is remodelled during the development of the intima or a neointima and during hypertrophy and angiogenesis. 2. Urokinase plasminogen activator mRNA expression is up- and down-regulated by growth factors, cytokines and steroids. Urokinase plasminogen activator is secreted as a single chain inactive form that may be proteolytically converted to active or inactive forms. Targeting of proteolytic activity may occur via focalized expression of uPA and its cell surface receptors (uPAR). Proteolytic activity is also controlled through the often co-ordinated expression of specific inhibitors. 3. A proteolytic cascade involving uPA provides its major role in tissue remodelling through the primary degradation of extracellular matrix and secondarily through the activation of transforming growth factor-beta or release from the matrix of basic fibroblast growth factor. In addition, uPA secreted by growth factor-stimulated vascular cells may contribute to the chemotactic and mitogenic responses ascribed to the growth factor and recent evidence strongly suggests that uPA has direct biological actions on vascular cells. 4. The cell surface binding of uPA via its growth factor-like domain to uPAR localizes and activates the protease, but may also initiate transmembrane signalling of biological responses, including migration/invasion and proliferation. As the uPAR lacks intracellular signalling domains, the signals may be transduced via interactions between uPA/uPAR and more classical signalling receptors. The mechanism by which uPA may be involved in cell signalling is yet to be elucidated.

• Kindzelskii AL, Laska ZO, Todd RF 3rd, Petty HR
• Urokinase-type plasminogen activator receptor reversibly dissociates from complement receptor type 3 (alpha M beta 2' CD11b/CD18) during neutrophil polarization.
• J Immunol. 1996; 156: 297-309
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• Previous studies have shown that the leukocyte integrin CR3 (CD11b/CD18) is physically associated with the urokinase-type plasminogen activator receptor (uPAR;CD87), a glycosyl-phosphatidylinositol (GPI)-linked protein, in resting neutrophil membranes. We now show that uPAR-to-CR3 interactions are reversible, correlating with cell shape. Neutrophils were first labeled with fluorescein conjugates of anti-CR3 F(ab')2 fragments followed by capping using a second-step F(ab')2 directed against murine F(ab')2s. Cells were then probed using rhodamine-conjugated anti-uPAR F(ab')2s. Although uPAR co-caps with CR3 on resting cells, uPAR was found to dissociate or "uncap" coincident with spontaneous cell polarization for migration. CR3 caps transformed into uropods while uPAR accumulated at lamellipodia of polarized cells. Capping was unnecessary for the observed distribution of CR3 and uPAR since the anti-CR3 and anti-uPAR F(ab')2s traffic to the uropod and lamellipodium, respectively, during polarization of uncapped cells. These receptors reassociate when cells return to a spherical morphology. In contrast to uPAR, Fc gamma RIIIB did not dissociate from CR3 caps during cell polarization. Resonance energy transfer (RET) microscopy was used to image the spatial distribution of RET and to follow the kinetics of association and dissociation. Initial levels of RET dramatically fell during cell polarization, but did not change on cells fixed with paraformaldehyde. Receptor reassociation was a biphasic process with initial reassociation about the perimeter of a cap, followed by a plateau and a slower rise in RET within a cap. We suggest that cells regulate receptor-receptor associations depending upon their physiologic activities.

• Bugge TH et al.
• Urokinase-type plasminogen activator is effective in fibrin clearance in the absence of its receptor or tissue-type plasminogen activator.
• Proc Natl Acad Sci U S A. 1996; 93: 5899-904
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• The availability of gene-targeted mice deficient in the urokinase-type plasminogen activator (uPA), urokinase receptor (uPAR), tissue-type plasminogen activator (tPA), and plasminogen permits a critical, genetic-based analysis of the physiological and pathological roles of the two mammalian plasminogen activators. We report a comparative study of animals with individual and combined deficits in uPAR and tPA and show that these proteins are complementary fibrinolytic factors in mice. Sinusoidal fibrin deposits are found within the livers of nearly all adult mice examined with a dual deficiency in uPAR and tPA, whereas fibrin deposits are never found in livers collected from animals lacking uPAR and rarely detected in animals lacking tPA alone. This is the first demonstration that uPAR has a physiological role in fibrinolysis. However, uPAR-/-/tPA-/- mice do not develop the pervasive, multi-organ fibrin deposits, severe tissue damage, reduced fertility, and high morbidity and mortality observed in mice with a combined deficiency in tPA and the uPAR ligand, uPA. Furthermore, uPAR-/-/tPA-/- mice do not exhibit the profound impairment in wound repair seen in uPA-/-/tPA-/- mice when they are challenged with a full-thickness skin incision. These results indicate that plasminogen activation focused at the cell surface by uPAR is important in fibrin surveillance in the liver, but that uPA supplies sufficient fibrinolytic potential to clear fibrin deposits from most tissues and support wound healing without the benefit of either uPAR or tPA.

• Higazi AA et al.
• Single-chain urokinase-type plasminogen activator bound to its receptor is relatively resistant to plasminogen activator inhibitor type 1.
• Blood. 1996; 87: 3545-9
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• Urokinase-type plasminogen activator (uPA) is synthesized as single-chain protein (scuPA) with little intrinsic activity. scuPA is activated when it is converted to two-chain urokinase (tcuPA) by plasmin or when it binds as a single-chain molecule to its cellular receptor (uPAR). Previous data indicate that complexes between scuPA and its receptor have somewhat higher affinity for plasminogen than does tcuPA. The current study indicates that plasminogen activator activity of scuPA bound to recombinant, soluble uPAR (suPAR) is also fivefold less sensitive to inhibition by plasminogen activator type 1 (PAI-1) than is soluble or receptor-bound tcuPA. Binding of PaI-1 to suPAR/scuPA complexes is totally reversible and can be overcome by increasing the concentration of plasminogen, suggesting a competitive mechanism of inhibition (Ki = 18 nmol/L). Binding of scuPA to suPAR also retards its cleavage by plasmin. These results indicates that binding of single-chain urokinase to its receptor promotes its activity, retards its inhibition, and protects it from conversion to a two-chain form of the enzyme, a step that may precede its inactivation and clearance from cell surfaces. These results are consistent with a physiologic role for receptor-bound single-chain urokinase as a cellular plasminogen activator.

• Tang H, Zhu Y, Song H
• [The structure and function of urokinase receptor and its expression in neoplastic tissues]
• Zhonghua Yi Xue Za Zhi. 1995; 75: 315-7

• Bugge TH et al.
• The receptor for urokinase-type plasminogen activator is not essential for mouse development or fertility.
• J Biol Chem. 1995; 270: 16886-94
• Display abstract

• The urokinase-type plasminogen activator receptor (uPAR) gene was disrupted in mice in order to explore the role of cell surface-associated plasminogen activation in development and hemostasis. Homozygous, uPAR-/- mice were born and survived to adulthood with no overt phenotypic abnormalities. There was no indication of loss of fetal animals based on the Mendelian pattern of transmission of the mutant uPAR gene. uPAR-/- mice carried no detectable uPAR in lung, spleen, and other tissues when measured both immunologically by Western blot analysis and functionally by ligand cross-linking analyses. In addition, activated peritoneal macrophages collected from uPAR-/- mice failed to promote plasminogen activation in vitro. The loss of the receptor also resulted in a redistribution of uPA in some tissues but had no impact on pro-uPA activation in the urogenital tract. Thus, in the absence of other challenging factors such as infection, injury, or other functional deficits, uPAR deficiency does not compromise fertility, development, or hemostasis. These mice provide a means to test the proposed function of uPA/uPAR in wound repair, atherogenesis, and tumor cell invasion in vivo.

• Spraggon G et al.
• The crystal structure of the catalytic domain of human urokinase-type plasminogen activator.
• Structure. 1995; 3: 681-91
• Display abstract

• BACKGROUND: Urokinase-type plasminogen activator (u-PA) promotes fibrinolysis by catalyzing the conversion of plasminogen to the active protease plasmin via the cleavage of a peptide bond. When localized to the external cell surface it contributes to tissue remodelling and cellular migration; inhibition of its activity impedes the spread of cancer. u-PA has three domains: an N-terminal receptor-binding growth factor domain, a central kringle domain and a C-terminal catalytic protease domain. The biological roles of the fibrinolytic enzymes render them therapeutic targets, however, until now no structure of the protease domain has been available. Solution of the structure of the u-PA serine protease was undertaken to provide such data. RESULTS: The crystal structure of the catalytic domain of recombinant, non-glycosylated human u-PA, complexed with the inhibitor Glu-Gly-Arg chloromethyl ketone (EGRcmk), has been determined at a nominal resolution of 2.5 A and refined to a crystallographic R-factor of 22.4% on all data (20.4% on data > 3 sigma). The enzyme has the expected topology of a trypsin-like serine protease. CONCLUSIONS: The enzyme has an S1 specificity pocket similar to that of trypsin, a restricted, less accessible, hydrophobic S2 pocket and a solvent-accessible S3 pocket which is capable of accommodating a wide range of residues. The EGRcmk inhibitor binds covalently at the active site to form a tetrahedral hemiketal structure. Although the overall structure is similar to that of homologous serine proteases, at six positions insertions of extra residues in loop regions create unique surface areas. One of these loop regions is highly mobile despite being anchored by the disulphide bridge which is characteristic of a small subset of serine proteases namely tissuetype plasminogen activator, Factor XII and Complement Factor I.

• Rettenberger P et al.
• A competitive chromogenic assay to study the functional interaction of urokinase-type plasminogen activator with its receptor.
• Biol Chem Hoppe Seyler. 1995; 376: 587-94
• Display abstract

• Urokinase-type plasminogen activator (uPA) converts plasminogen to plasmin which degrades various extracellular matrix components. uPA is focused to the cell surface via binding to a specific receptor (uPAR, also termed CD87). uPAR-bound uPA mediates pericellular proteolysis in a variety of biological processes, e.g. cell migration, tissue remodeling and tumor invasion. We have developed a competitive microtiter plate-based chromogenic assay which allows the analysis of uPA/uPAR interaction. The plates are coated with recombinant uPAR expressed in Chinese hamster ovary (CHO) cells. Proteolytically active uPA (HMW-uPA) is added to the microtiter plate-attached uPAR. The amount of receptor-bound uPA is then determined indirectly via addition of plasminogen, which is activated to plasmin, followed by cleavage of a plasmin-specific chromogenic substrate. Substances interfering with binding of HMW-uPA to uPAR diminish the generation of plasmin, as indicated by a reduction of cleaved chromogenic substrate. This assay was used to analyze the inhibitory capacity of a variety of proteins and peptides, respectively, on the uPA/uPAR interaction: i) uPAR and uPAR-variants expressed in CHO cells, yeast or E. coli, ii) the aminoterminal fragment (ATF) of human uPA or yeast recombinant pro-uPA, iii) synthetic peptides derived from the sequence of the uPAR-binding region of uPA, and iv) antibodies directed against uPAR. This assay may be helpful in identifying uPA and uPAR analogues or antagonists which efficiently block uPA/uPAR interaction.

• Ragno P, Montuori N, Rossi G
• Urokinase-type plasminogen activator/type-2 plasminogen-activator inhibitor complexes are not internalized upon binding to the urokinase-type-plasminogen-activator receptor in THP-1 cells. Interaction of urokinase-type plasminogen activator/type-2 plasminogen-activator inhibitor complexes with the cell surface.
• Eur J Biochem. 1995; 233: 514-9
• Display abstract

• The urokinase-type plasminogen activator (uPA) and its inhibitor PAI-2 form a covalent complex that, upon binding to the uPA receptor (uPA-R), is cleaved into two fragments of molecular masses 70 kDa and 22 kDa. The 70-kDa fragment results from the interaction of the B chain of uPA and PAI-2 whereas the 22-kDa fragment is the A chain of the enzyme [13]. We prove that, at 37 degrees C, the 70-kDa fragment is released into the medium, whereas the 22-kDa fragment remains bound to the cell surface. uPA complexed with its other specific inhibitor, PAI-1, is cleaved into fragments of identical sizes, but the 70-kDa component is internalized via the alpha 2-macroglobulin receptor. At 4 degrees C, both uPA/PAI-2 complex degradation products remain bound to the uPA-R. We propose that the 70-kDa molecule, which lacks the uPA binding region for uPA-R, is bound to uPA-R via a new binding site, unmasked only when uPA-R is occupied by uPA/PAI-2 complexes.

• Rabbani SA, Rajwans N, Achbarou A, Murthy KK, Goltzman D
• Isolation and characterization of multiple isoforms of the rat urokinase receptor in osteoblasts.
• FEBS Lett. 1994; 338: 69-74
• Display abstract

• A rat urokinase receptor (uPAR) cDNA fragment was amplified by RT-PCR from RNA of the rat osteoblastic cell line CFK-1. Using this DNA species as a hybridization probe two rat uPAR cDNAs were isolated from a CFK-1 cDNA library. These two cDNAs encode an identical uPAR protein except for a single base mutation which results in the substitution of cysteine to serine at amino acid 71 in one variant. PCR analysis of rat genomic DNA revealed the presence of an additional uPAR arising from alternate splicing which is expressed in a variety of tissues. These studies provide the tools for examining uPAR function in fibrinolysis, tumor invasion and metastasis in the rat and for identifying the mechanism of species specificity in uPA actions.

• Suh TT, Nerlov C, Dano K, Degen JL
• The murine urokinase-type plasminogen activator receptor gene.
• J Biol Chem. 1994; 269: 25992-8
• Display abstract

• The murine urokinase-type plasminogen activator receptor (uPAR) gene has been isolated and its complete nucleotide sequence established. The gene is organized into seven exons comprising 9.5% of the 13,207-base pair region that spans the interval between the transcription initiation and polyadenylation sites. The region upstream of the transcription initiation site lacks TATA- or CCAAT-like elements but is flanked by a G+C-rich region, which contains a number of potential regulatory elements including Sp1 and AP1 binding motifs. The close association of both Sp1 and AP1 sites within the proximal promoter region is consistent with the observation that the murine uPAR gene is inducible by phorbol esters. The major functional domains of the encoded protein, including the signal peptide, three cysteine-rich internal repeats, and the glycolipid anchor attachment motif, are encoded by separate exons. Based on the organization of the murine uPAR gene and the distribution of protein domains within the exons in the Ly-6 family of genes, it appears that the uPAR gene evolved secondarily to two internal duplication events within a Ly-6-like ancestral gene. The cloned and sequenced murine uPAR gene will be a valuable tool in understanding the regulation and biological roles of uPAR in that it will permit detailed studies of gene expression and uPAR-dependent processes in vitro, as well as the generation of both gain-of-function and loss-of-function mutants in transgenic mice.

• Casey JR, Petranka JG, Kottra J, Fleenor DE, Rosse WF
• The structure of the urokinase-type plasminogen activator receptor gene.
• Blood. 1994; 84: 1151-6
• Display abstract

• The cellular receptor for urokinase-type plasminogen activator (uPAR) is a glycosylphosphatidylinositol (GPI)-anchored membrane protein that plays a central role in pericellular plasminogen activation. It contains 313 amino acid residues, including 28 cysteine residues in a pattern of three homologous repeats. The cysteine residue pattern suggests that uPAR belongs to a superfamily of proteins including CD59, murine Ly-6, and a variety of elapid snake venom toxins. A novel 1.7-kb uPAR cDNA was isolated that is missing exon 5 and that contains 380 bp not previously reported at the 5' end. This cDNA was used to probe a human genomic library from which three clones were isolated and analyzed. The uPAR gene consists of 7 exons spread over 23 kb of genomic DNA. Exons 2, 4, and 6 code for homologous domains within the mature protein, as do exons 3, 5, and 7; CD59-like homologous pairs are encoded by exons 2-3, 4-5, and 6-7, respectively. The structure of the gene for uPAR further confirms the relationship of this molecule to the superfamily containing CD59, Ly-6, and the elapid snake venom toxins.

• Fazioli F, Blasi F
• Urokinase-type plasminogen activator and its receptor: new targets for anti-metastatic therapy?
• Trends Pharmacol Sci. 1994; 15: 25-9
• Display abstract

• Urokinase-type plasminogen activator (uPA) and its receptor are instrumental in cell invasion and metastasis; their high levels of expression in human tumours correlates with a high risk of recurrence. uPA has a pleiotropic effect on cell migration and spreading in vivo and in vitro through the activation of plasminogen or other protein factors at the cell surface or in the extracellular matrix. Three specific inhibitors, with different tissue-specificities and regulatory properties, modulate cell-surface exposure of uPA activity. Overall, uPA is at the centre of a complex system affecting cell movement and invasiveness, and inhibition of uPA is now a goal of anti-metastatic therapy. The role of uPA and its inhibition are discussed in this review by Francesca Fazioli and Francesco Blasi.

• Lijnen HR, De Cock F, Collen D
• Characterization of the binding of urokinase-type plasminogen activator (u-PA) to plasminogen, to plasminogen-activator inhibitor-1 and to the u-PA receptor.
• Eur J Biochem. 1994; 224: 567-74
• Display abstract

• Binding parameters [association-rate (kass) and dissociation-rate (kdiss) constants, and affinity constants (KA = kass/kdiss)] for the interaction between urokinase-type plasminogen activator (u-PA) and its substrate plasminogen, its inhibitor plasminogen activator inhibitor-1 (PAI-1) and its receptor (u-PAR), were determined by real-time biospecific interaction analysis (BIA). The KA values for the binding of [S741A]recombinant plasminogen (plasminogen with N-terminal Glu and with the active site Ser741 mutagenized to Ala) or of active site-blocked plasmin (D-ValPheLysCH2-plasmin) to the 54-kDa or 32-kDa molecular forms of recombinant single-chain u-PA (rscu-PA) ranged between 0.57 x 10(6) M-1 and 1.7 x 10(6) M-1, compared to 14-22 x 10(6) M-1 for binding to the corresponding active site-blocked recombinant two-chain u-PA (rtcu-PA) moieties. KA values for binding of these plasmin(ogen) moieties to [Ser356deHAla]rtcu-PA (rtcu-PA with the active site Ser356 converted to dehydroAla) were 81 x 10(6) M-1 and 670 x 10(6) M-1, respectively. Binding of active site-blocked LMM-plasmin (a low-molecular-mass plasmin derivative lacking kringles 1-4) and of the plasmin B chain to [Ser356deHAla]rtcu-PA occurred with KA values of 3.7 x 10(6) M-1 and 0.33 x 10(6) M-1, compared to 670 x 10(6) M-1 for the binding of intact D-ValPheLysCH2-plasmin to [Ser356deHAla]rtcu-PA. The KA values for binding of latent PAI-1 to 54-kDa or 32-kDa molecular forms of rscu-PA and rtcu-PA were in the range 0.34-2.1 x 10(6) M-1. Reactivated PAI-1 bound to 54-kDa and 32-kDa rtcu-PA moieties with KA values of 26 x 10(6) M-1 and 28 x 10(6) M-1, compared to 0.77 x 10(6) M-1 and 3.2 x 10(6) M-1 for binding to the corresponding single-chain u-PA species, and 450 x 10(6) M-1 for binding to [Ser356deHAla]rtcu-PA. KA values for binding of plasmin(ogen) to the covalent rtcu-PA/PAI-1 complex were similar or somewhat higher than those for binding to uncomplexed rtcu-PA. Single-chain and two-chain 54-kDa u-PA moieties bound with a 1:1 stoichiometry and with very high affinity to u-PAR (KA of 4.6-8.5 x 10(9) M-1), whereas no significant binding of 32-kDa u-PA moieties was observed (KA < or = 0.2 x 10(6) M-1).(ABSTRACT TRUNCATED AT 400 WORDS)

• Ploug M, Ellis V, Dano K
• Ligand interaction between urokinase-type plasminogen activator and its receptor probed with 8-anilino-1-naphthalenesulfonate. Evidence for a hydrophobic binding site exposed only on the intact receptor.
• Biochemistry. 1994; 33: 8991-7
• Display abstract

• The cellular receptor for urokinase-type plasminogen activator (uPAR) is a glycolipid-anchored membrane protein thought to play a primary role in the generation of pericellular proteolytic activity, and to be involved in cancer cell invasion and metastasis. This protein is composed of three homologous domains, the NH2-terminal of which is involved in the high-affinity binding (Kd approximately 0.1-1.0 nM) to the epidermal growth factor-like module of urokinase-type plasminogen activator (uPA). Here we report that intact uPAR binds the low molecular weight fluorophore 8-anilino-1-naphthalenesulfonate (ANS) to form a 1:1 stoichiometric complex and that the resulting enhancement of the ANS fluorescence probes the functional state of uPAR as judged by several independent criteria. First, the uPAR-mediated increase in ANS fluorescence can be titrated by uPA as well as by its receptor binding derivatives (the amino-terminal fragment and the growth factor-like module). Second, an anti-uPAR monoclonal antibody, capable of preventing uPA binding, can also titrate the uPAR-dependent ANS fluorescence whereas other antibodies not interfering with uPA binding are unable to exert this effect. Third, the dissociation profile of uPA-uPAR complexes as a function of increasing concentrations of guanidine hydrochloride closely parallels the loss of the ANS binding site in uPAR. Finally, liberation of the NH2-terminal domain from uPAR by limited chymotrypsin cleavage after Tyr87 leads to a loss of both enhanced ANS fluorescence and high-affinity uPA binding.(ABSTRACT TRUNCATED AT 250 WORDS)

• Ragno P, Montuori N, Vassalli JD, Rossi G
• Processing of complex between urokinase and its type-2 inhibitor on the cell surface. A possible regulatory mechanism of urokinase activity.
• FEBS Lett. 1993; 323: 279-84
• Display abstract

• Complexes between the urokinase-type plasminogen activator (uPA) and its type-2 inhibitor (PAI-2) are bound by a cell-surface receptor for uPA and rapidly cleaved into two fragments of 70 and 22 kDa. The 70-kDa fragment contains the active site of uPA and PAI-2, while the 22-kDa species was identified as the amino terminal fragment of uPA, that binds specifically to the receptor. When the experiment is performed at 4 degrees C, both fragments remain bound to the cell surface and can be eluted by acid treatment. We therefore postulate that after the binding of the uPA-PAI-2 complex, a new binding site for the 70-kDa species becomes available. This additional binding favours the cleavage of the complex into the 70-and 22-kDa fragments; the 70-kDa species is endocytosed or released, while the 22-kDa fragment remains on the cell surface to prevent the binding of intact uPA.

• Behrendt N, Ploug M, Ronne E, Hoyer-Hansen G, Dano K
• Cellular receptor for urokinase-type plasminogen activator: protein structure.
• Methods Enzymol. 1993; 223: 207-22

• de Munk GA, Molinari A, Rijken DC
• Inactivation of high and low molecular weight single-chain urokinase-type plasminogen activator (pro-urokinase) by thrombin in the presence of thrombomodulin.
• Thromb Haemost. 1993; 69: 88-88

• Bayraktutan U, Jones P
• A novel urokinase receptor on monocyte-like macrophage cell line.
• Biochem Soc Trans. 1993; 21: 395-395

• Behrendt N, Ronne E, Dano K
• Binding of the urokinase-type plasminogen activator to its cell surface receptor is inhibited by low doses of suramin.
• J Biol Chem. 1993; 268: 5985-9
• Display abstract

• The multipotent drug suramin, which is currently being studied as an anticancer agent, was found to inhibit the interaction between the urokinase-type plasminogen activator (u-PA) and its cellular receptor. 50% inhibition of binding was obtained with a suramin concentration between 30 and 60 micrograms/ml when using U937 cells and a ligand concentration of 0.3 nM. This concentration of the drug is well below the serum levels found in suramin-treated patients. Inhibition of binding was also demonstrated at the molecular level, using chemical cross-linking or an enzyme-linked immunosorbent assay-type technique based on the ligand interaction. The inhibition was not caused by a mere polyanion effect since polysulfates such as heparin, heparan sulfate, and pentosan polysulfate were non-inhibitory or showed only a very weak inhibition. However, polysulfonated compounds with structures resembling suramin (i.e. trypan blue and Evans blue) did prove inhibitory. The inhibition found with suramin showed a concentration dependence consistent with a mixed competitive and noncompetitive mechanism. The off-rate of prebound ligand was accelerated by the drug. It is speculated that the present effect may contribute to the anti-invasive properties of suramin by destroying the cellular potential for localized plasminogen activation and proteolytic matrix degradation.

• Kratzschmar J, Haendler B, Kojima S, Rifkin DB, Schleuning WD
• Bovine urokinase-type plasminogen activator and its receptor: cloning and induction by retinoic acid.
• Gene. 1993; 125: 177-83
• Display abstract

• Full-length cDNAs encoding bovine urokinase-type plasminogen activator (u-PA) and urokinase receptor (u-PAR) were cloned from an aortic endothelial cell cDNA library using PCR-amplified cDNA fragments as probes. Bovine u-PA amino acid identity ranges from 79.5 to 70.9% when compared to its pig, human, baboon and mouse analogues, while bovine u-PAR is 61.8 and 59.6% identical to its human and mouse counterparts, respectively. All Cys residues previously found in mature u-PA and u-PAR from these different species are also conserved in the bovine molecules. Bovine u-PA and its cell-surface receptor display one and six potential sites of N-linked glycosylation, respectively. Northern blot hybridization demonstrated a moderate induction of u-PA and u-PAR mRNA in bovine aortic endothelial cells after treatment with 10 nM and 1 microM retinoic acid for 8 hours.

• Ploug M, Kjalke M, Ronne E, Weidle U, Hoyer-Hansen G, Dano K
• Localization of the disulfide bonds in the NH2-terminal domain of the cellular receptor for human urokinase-type plasminogen activator. A domain structure belonging to a novel superfamily of glycolipid-anchored membrane proteins.
• J Biol Chem. 1993; 268: 17539-46
• Display abstract

• The receptor for human urokinase-type plasminogen activator (uPAR) is synthesized as a 313-residue-long polypeptide containing 28 cysteine residues, the pattern of which defines three homologous repeats within the protein. These entities are believed to represent a novel protein domain structure, of which the NH2-terminal domain of uPAR can be covalently cross-linked to the epidermal growth factor-like module of urokinase after receptor-ligand interaction. The NH2-terminal domain of a recombinant, soluble uPAR derivative, labeled with [35S]cysteine, was isolated after limited proteolysis with chymotrypsin. The four disulfide bonds present within this domain were assigned by a combination of plasma desorption mass spectrometry, amino acid composition, and sequence analyses of peptides generated by trypsin, endoproteinase Asp-N, and thermolysin. The following disulfide bond structure was determined: Cys3-Cys24, Cys6-Cys12, Cys17-Cys45, and Cys71-Cys76. Similar cysteine pairing is likely to be found within other members of this protein superfamily, i.e. the membrane inhibitor of reactive lysis, Ly-6, and the remaining two domains of uPAR. However, an additional pair of cysteines present within these domains probably forms a fifth disulfide bond.

• Ellis V, Behrendt N, Dano K
• Cellular receptor for urokinase-type plasminogen activator: function in cell-surface proteolysis.
• Methods Enzymol. 1993; 223: 223-33

• Ploug M, Stoffer B, Jensen AL
• In situ alkylation of cysteine residues in a hydrophobic membrane protein immobilized on polyvinylidene difluoride membranes by electroblotting prior to microsequence and amino acid analysis.
• Electrophoresis. 1992; 13: 148-53
• Display abstract

• For identification of cysteine residues on microsequence analysis it is crucial to derivatize the sulfhydryl groups. This reaction requires a desalting step which often represents a major obstacle, especially if the sample consists of limited amounts of a hydrophobic membrane protein. An alkylation procedure is described, allowing efficient derivatization (greater than 90%) of cysteines and cystines even in low microgram quantities, as revealed by test analyses with lysozyme and a hydrophobic membrane protein. The modified protein is recovered in high yields in a form suitable for both microsequence analysis and amino acid analysis. The method involves electrophoretic desalting by miniaturized Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis and in situ alkylation after electro-transfer onto polyvinylidene difluoride membranes. Precautions against NH2-terminal blocking during sample preparations are provided. The general applicability of the method is illustrated by the structural characterization of the low abundance membrane receptor for human urokinase plasminogen activator.

• Ragno P, Cassano S, Degen J, Kessler C, Blasi F, Rossi G
• The receptor for the plasminogen activator of urokinase type is up-regulated in transformed rat thyroid cells.
• FEBS Lett. 1992; 306: 193-8
• Display abstract

• Five rat thyroid cell lines were tested for the expression of the cell surface receptor for urokinase type plasminogen activator (uPA). All tested lines were found to bind uPA, but transformed 1-5G and Ki-Mol cells, which are also high uPA producers, bound at least ten times more uPA, as compared to non-producers, 'normal' TL5 cells. Moreover, it was possible to remove membrane-bound uPA by treating the cells with phosphatidylinositol-specific phospholipase C, suggesting that rat uPAR, like its human counterpart, is linked to the membrane by a glucosyl-phosphatidylinositol anchor. The specificity of the binding was tested by competition with three different synthetic peptides corresponding to amino acids 14-37 of human, rat and mouse uPA. The results indicate also that the receptor binding region of rat uPA is located within the growth factor domain of the molecule and that its expression may be dependent on the transformed state of the cells.

• Borglum AD, Byskov A, Roldan AL, Kruse TA
• Two PstI polymorphisms for the urokinase-type plasminogen activator receptor gene (PLAUR).
• Hum Genet. 1992; 89: 584-584
• Display abstract

• The cDNA probe puPAR-2 detects two PstI polymorphisms in the urokinase-type plasminogen activator receptor gene (PLAUR). This probe and the polymorphic system are described.

• Hoyer-Hansen G et al.
• Urokinase plasminogen activator cleaves its cell surface receptor releasing the ligand-binding domain.
• J Biol Chem. 1992; 267: 18224-9
• Display abstract

• The cellular receptor for urokinase-type plasminogen activator (uPAR) is a glycolipid-anchored three-domain membrane protein playing a central role in pericellular plasminogen activation. We have found that urokinase (uPA) can cleave its receptor between domains 1 and 2 generating a cell-associated uPAR variant without ligand-binding properties. In extracts of U937 cells there are two uPAR variants which after complete deglycosylation have apparent molecular masses of 35,000 and 27,000. Analysis with monoclonal antibodies showed that these variants represented the intact uPAR and a two-domain form, uPAR(2+3), lacking ligand-binding domain 1. Trypsin treatment showed that both variants are present on the outside of the cells. Addition to the culture medium of an anticatalytic monoclonal antibody to uPA inhibited the formation of the uPAR(2+3), indicating that uPA is involved in its generation. Purified uPAR can be cleaved directly by uPA as well as by plasmin. The uPA-catalyzed cleavage does not require binding of the protease to the receptor through its epidermal growth factor-like receptor-binding domain, since low molecular weight uPA that lacks this domain also cleaves uPAR. This unusual reaction in which a specific binding protein is proteolytically inactivated by its own ligand may represent a regulatory step in the plasminogen activation cascade.

• Solberg H et al.
• Identification and characterization of the murine cell surface receptor for the urokinase-type plasminogen activator.
• Eur J Biochem. 1992; 205: 451-8
• Display abstract

• Cell-binding experiments have indicated that murine cells on their surface have specific binding sites for mouse urokinase-type plasminogen activator (u-PA). In contrast to the human system, chemical cross-linking studies with an iodinated ligand did not yield any covalent adducts in the murine system, but in ligand-blotting analysis, two mouse u-PA-binding proteins could be visualized. To confirm that these proteins are the murine counterpart of the human u-PA receptor (u-PAR), a peptide was derived from the murine cDNA clone assigned to represent the murine u-PAR due to cross-hybridization and pronounced sequence similarity with human u-PAR cDNA [Kristensen, P., Eriksen, J., Blasi, F. & Dano, K. (1991) J. Cell Biol. 115, 1763-1771]. A rabbit antiserum raised against this peptide specifically recognized two polypeptide bands with electrophoretic mobilities identical to those identified by ligand-blotting analysis. Binding of mouse u-PA to its receptor showed species specificity in ligand-blotting analysis, since mouse u-PA did not bind to human u-PAR and human u-PA did not bind to mouse u-PAR. The apparent M(r) of mouse u-PAR varied between different mouse cell lines and ranged over M(r) 45,000-60,000. In four of the cell lines, mouse u-PA bound to two mouse u-PAR variant proteins, whereas in the other two cell lines studied, there was only one mouse u-PA-binding protein. In the monocyte macrophage cell line P388D.1, trypsin-treatment of intact cells could remove only the large mouse u-PAR variant (M(r) 60,000) indicating that only this type was a cell-surface-exposed molecule. The smaller mouse u-PAR variant (M(r) 45,000), was deglycosylated by the enzyme endo-beta-N-acetylglucosaminidase H and is probably an intracellular precursor form carrying only high-mannose carbohydrate. Deglycosylation of this variant yielded a polypeptide with an apparent M(r) of about 30,000, which corresponds to the Mr calculated from the cDNA derived protein sequence of mouse u-PAR. Receptor-bound mouse u-PA could be released by phosphatidylinositol-specific phospholipase C treatment, indicating that mouse u-PAR is attached to the cell surface by glycosylphosphatidylinositol. Purification of the two mouse u-PAR variant proteins by diisopropylfluorophosphate-inactivated mouse u-PA-Sepharose affinity chromatography yielded two silver-stained bands when analysed by SDS/PAGE, corresponding in electrophoretic mobility to those seen by ligand-blotting analysis.(ABSTRACT TRUNCATED AT 400 WORDS)

• Ploug M et al.
• The receptor for urokinase-type plasminogen activator is deficient on peripheral blood leukocytes in patients with paroxysmal nocturnal hemoglobinuria.
• Blood. 1992; 79: 1447-55
• Display abstract

• Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired clonal defect in bone marrow-derived cells and is clinically associated with intravascular hemolysis, hemoglobinuria, and an increased frequency of venous thrombosis. The common denominator of PNH-affected blood cells appears to be a defect in the membrane attachment of proteins normally anchored by glycosyl-phosphatidylinositol (GPI). We report here that the cellular receptor for urokinase-type plasminogen activator (u-PAR) is deficient on affected peripheral blood monocytes and granulocytes from four individuals with PNH as evidenced by chemical cross-linking analysis as well as by immunofluorescence flow cytometry using a monoclonal anti-u-PAR antibody. In contrast, on normal blood monocytes and granulocytes we find significant amounts of u-PAR, which is attached to the plasma membrane by a GPI-anchor as defined by its sensitivity towards a specific phospholipase treatment. By two-color flow cytometry it was shown that deficiency of u-PAR expression paralleled that of another GPI-anchored protein. As u-PAR is involved in the initiation of pericellular proteolysis, the reduced expression of u-PAR on PNH-affected leukocytes led to an overall reduction in the capacity for plasminogen activation by cell-surface-bound urokinase. Whereas the abnormal susceptibility of PNH-affected erythrocytes to lysis by autologous complement has been related to the low expression of three GPI-anchored complement regulatory proteins on the cell surface, we now propose that lack of u-PAR expression on the surface of peripheral blood leukocytes may be causally related to the high incidence of venous thrombosis observed in PNH patients.

• Ploug M, Eriksen J, Plesner T, Hansen NE, Dano K
• A soluble form of the glycolipid-anchored receptor for urokinase-type plasminogen activator is secreted from peripheral blood leukocytes from patients with paroxysmal nocturnal hemoglobinuria.
• Eur J Biochem. 1992; 208: 397-404
• Display abstract

• The cellular urokinase-type plasminogen-activator (uPA) receptor (uPAR) is a glycolipid-anchored membrane protein thought to be involved in pericellular proteolysis during cell migration and tumor invasion. In the present study, we have identified and characterized two soluble forms of uPAR which have retained their ligand-binding capability. One variant was generated in vitro by treatment of intact normal cells with either a phosphatidylinositol-specific phospholipase C (PLC) or endoproteinase Asp-N. The other soluble uPAR variant was secreted in vivo from peripheral blood leukocytes affected by the stem-cell disorder paroxysmal nocturnal hemoglobinuria (PNH), and was found in the plasma from these PNH patients as well as in the conditioned medium from cultured PNH leukocytes. Under normal conditions, we find no evidence for any shedding or secretion of a soluble uPA-binding counterpart to human uPAR in plasma. Unlike normal leukocytes, the PNH-affected cells do not express uPAR on the cell surface, although they do contain apparently normal levels of uPAR-specific mRNA. The secreted uPAR derived from PNH cells has a mobility in SDS/PAGE that is slightly higher than that of uPAR solubilized by PtdIns-specific PLC or detergent, but resembles that of a truncated, recombinant uPAR variant, which has its C-terminus close to the proposed glycolipid-attachment site, suggesting that the secreted protein has been proteolytically processed for glycolipid attachment. The presence in plasma from PNH patients of such a secreted, hydrophilic form of uPAR lends support to the hypothesis that the lesion underlying the PNH disorder resides either in glycolipid biosynthesis or in the function of an as-yet-unidentified transamidating enzyme assumed to cleave and assemble the truncated uPAR with the preformed glycolipid moiety.

• Moller LB, Ploug M, Blasi F
• Structural requirements for glycosyl-phosphatidylinositol-anchor attachment in the cellular receptor for urokinase plasminogen activator.
• Eur J Biochem. 1992; 208: 493-500
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• The urokinase-plasminogen-activator receptor (u-PAR) is a glycosyl-phosphatidylinositol(glycosyl-PtdIns)-anchored membrane protein. Using site-directed mutagenesis, we have studied features in the u-PAR sequence important for successful glycosyl-PtdIns attachment. Two critical sequence elements were identified. In the sequence Ser282-Gly283-Ala284, simultaneous substitution of all of these residues prevented membrane anchoring. Individual substitution of each of the residues indicated that Gly283 is the more critical residue and the likely attachment site. However, it was unexpectedly found that mutation of this residue gave rise only to a partial impairment of glycosyl-PtdIns attachment. We therefore propose that more than one residue within this sequence can be utilized as glycosyl-PtdIns-attachment site. In the last eight COOH-terminal amino acids encoded in u-PAR cDNA, deletion of this sequence (residues 306-313) completely prevented glycosyl-PtdIns attachment. However, the remaining COOH-terminal region proved still to possess a potential glycosyl-PtdIns signal activity; it could be converted to a new functional glycosyl-PtdIns signal by substitution of a single positively charged residue (Arg304). Substitution of Arg304 by Leu converted this truntaced u-PAR to a glycosyl-PtdIns-anchored protein, indistinguishable from the wild type. Substitution of Arg304 by a negatively charged residue (Glu) led to a partial acquisition of the glycosyl-PtdIns-anchoring ability. These findings show that charged amino acids placed in the COOH-terminus interfere negatively with glycosyl-PtdIns-anchoring, and, furthermore, that this effect is more pronounced for positively charged than for negatively charged amino acid residues.

• Borglum AD et al.
• Assignment of the urokinase-type plasminogen activator receptor gene (PLAUR) to chromosome 19q13.1-q13.2.
• Am J Hum Genet. 1992; 50: 492-7
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• The urokinase-type plasminogen activator receptor (uPAR) is a key molecule in the regulation of cell-surface plasminogen activation and, as such, plays an important role in many normal as well as pathological processes. We applied a cDNA probe from the corresponding gene (PLAUR) in a location analysis using a panel of human/rodent cell hybrids and in a multipoint linkage analysis of 40 CEPH families. These two independent studies both found PLAUR to be located on chromosome 19. The cell hybrid study suggested that PLAUR is located at chromosome 19q13-qter, and the multipoint analysis indicated that PLAUR is located at chromosome 19q13.1-q13.2 and surrounded by DNA markers in the following way (with distances given in recombination fractions): D19S27-.11-CYP2A-.06-PLAUR-.03-D19S8-.04-APOC 2-.24-PRKCG. Further, a ligand-binding study performed on cell hybrids verified the species specificity of the uPAR and confirmed the chromosome assignment.

• Min HY, Semnani R, Mizukami IF, Watt K, Todd RF 3rd, Liu DY
• cDNA for Mo3, a monocyte activation antigen, encodes the human receptor for urokinase plasminogen activator.
• J Immunol. 1992; 148: 3636-42
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• We have cloned the cDNA for Mo3, an activation Ag expressed by human monocytes and myelomonocytic cell lines after stimulation by PMA, LPS, muramyl dipeptide, certain cytokines, and cAMP agonists. We have previously shown that Mo3 expression in vivo is associated predominantly with macrophages in inflammatory sites. Mo3 is a highly glycosylated protein of about 50 kDa in monocytes and U-937 cells and is anchored to the plasma membrane by glycosyl-phosphatidylinositol linkage. We purified Mo3 protein by cleavage from the U-937 cell surface with phosphatidylinositol-specific phospholipase C, followed by affinity chromatography using a mAb. An internal peptide sequence was determined and used to design oligonucleotide probes for screening an expression cDNA library. Nucleotide sequencing indicated that the complete coding sequence encodes 335 amino acids, including a predicted signal peptide of 22 residues and a hydrophobic C-terminal portion that is probably cleaved during formation of the GPI linkage. The resulting mature protein of about 290 amino acids is consistent with the 29-kDa molecular mass of deglycosylated Mo3. A Northern blot of RNA from U-937 cells revealed a 1.5-kb band that was induced by PMA treatment. Mo3 cDNA was transfected into Cos cells and surface expression of Mo3 was detected by ELISA using various anti-Mo3 mAb. We performed a computer search of the National Biomedical Research Foundation database and found that Mo3 is identical to the human receptor for the urokinase plasminogen activator (uPA-R). Purified soluble Mo3, as well as anti-Mo3 antibodies, were able to block uPA binding to its receptor on U-937 cells, indicating that Mo3 is indeed uPA-R. The use of these anti-Mo3 antibodies may be helpful in assessing the role of uPA-R in processes such as inflammation and tumor invasion.

• Pyke C et al.
• Urokinase-type plasminogen activator is expressed in stromal cells and its receptor in cancer cells at invasive foci in human colon adenocarcinomas.
• Am J Pathol. 1991; 138: 1059-67
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• In this study in situ hybridization methods were used to examine biopsy samples from 13 adenocarcinomas of the colon for the presence of mRNA for the urokinase-type plasminogen activator (u-PA) and its specific cell-surface receptor (u-PAR). In all cases, u-PA mRNA was present in fibroblastlike cells in the stroma adjacent to the invasive tumor nodules. Urokinase-type plasminogen activator mRNA was not detected in the malignant cells. All specimens also contained u-PAR mRNA in cells located at the tumoral-stromal interface of invasive foci, but in contrast at least some of these cells were in all but one case identified as being of malignant origin. Stromal cells, probably tumor-infiltrating macrophages and neutrophils, also were positive in these areas. These results support the view that components of the plasminogen activation system may act to influence proteolytic events occurring at the interface between stroma and malignant cells in adenocarcinomas of the colon in humans.

• Kristensen P, Eriksen J, Blasi F, Dano K
• Two alternatively spliced mouse urokinase receptor mRNAs with different histological localization in the gastrointestinal tract.
• J Cell Biol. 1991; 115: 1763-71
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• Two mouse urokinase-type plasminogen activator receptor (muPAR) cDNAs were isolated: muPAR1 is homologous to the human urokinase-type plasminogen activator receptor while muPAR2 codes for a 199 residue protein sharing the first 133 residues with muPAR1. Mouse genomic DNA sequencing indicates that the two different mRNAs arise by alternative splicing. In situ hybridization showed differential expression of the two mRNAs in mouse gastric mucosa. muPAR1 mRNA is located in luminal epithelial cells situated close to urokinase-type plasminogen activator-producing connective tissue cells of the lamina propria, pointing to plasmin generation controlled by the cooperation of different cells that may play a role in the release of gastric epithelial cells. muPAR2 mRNA is expressed in the basal epithelial cells, and the deduced protein sequence includes the receptor ligand binding domain, but omits the region involved in glycolipid-mediated membrane anchoring, suggesting that muPAR2 may code for a secreted uPA binding protein.

• Hollas W, Boyd D
• Regulation of the urokinase receptor by its plasminogen activator.
• Thromb Haemost. 1991; 66: 678-83
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• There is now ample evidence that the proteolytic action of urokinase (UK) is potentiated by a specific cell surface receptor. The present study was undertaken to assess the role of UK as a modulator of its receptor. GEO colonic cells, which secrete relatively low levels of UK (congruent to 0.1 nM/72 h per 10(6) cells) and display approximately 10(4) receptors per cell, 10% of which are "tagged" with the endogenous plasminogen activator (PA), was selected for the study. A 90% reduction in the specific binding of radioactive DFP-UK was observed for cells cultivated in the presence of two-chain (TC) UK (Mr 55,000). This only partly reflected occupation of the receptors with UK supplied in the culture medium, since the specific binding of the radioligand was still reduced by 60% after an acid pretreatment, which dissociates receptor-bound UK. The reduction in radioactive DFP-UK binding to cells treated with high molecular weight UK, either in the single or two-chain form, was both concentration and time dependent. Maximum reductions (70%) were achieved by treatment of the cells for 24 h with 1 nM of the plasminogen activator. In contrast, low molecular weight UK, which lacks part of the UK A chain, had no effect on ligand binding. Attenuation of radioactive DFP-UK binding to UK treated GEO cells was a consequence of a 60% reduction in the number of binding sites. Treatment of GEO cells with an antibody, which blocks the binding of endogenous UK to its receptor, augmented radioactive DFP-UK binding by two-fold. These data indicate that for one colonic cell line, at least, UK down-regulates its own binding site subsequent to it being bound to the receptor.

• Palfree RG
• The urokinase-type plasminogen activator receptor is a member of the Ly-6 superfamily.
• Immunol Today. 1991; 12: 170-170

• Fibbi G, Magnelli L, Pucci M, Del Rosso M
• Interaction of urokinase A chain with the receptor of human keratinocytes stimulates release of urokinase-like plasminogen activator.
• Exp Cell Res. 1990; 187: 33-8
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• On the basis of a fibrinolytic assay with 125I-fibrin, zymography, and immunoprobing with anti-human urokinase antibody, we have observed that the in vitro established NCTC human keratinocyte cell line releases into the culture medium a 54,000-Da plasminogen activator which is indistinguishable from human urokinase. Only the early release following the washing of keratinocyte monolayers is accounted for by secretion of preformed enzyme, while late secretory events require the de novo synthesis of urokinase. The released enzyme can interact by autocriny with its own receptor present on keratinocytes. The addition to the keratinocyte culture medium of the urokinase A chain can stimulate a concentration-dependent urokinase oversecretion, which is not paralleled by oversecretion of plasminogen activator inhibitor-1. Since stimulation of urokinase production can be obtained by an A chain concentration (5 ng/ml) which was previously shown to be efficient in inducing keratinocyte mobilization in an in vitro migration model system, we hypothesize that this mechanism may be important in vivo during the process of wound repair.

• Estreicher A, Muhlhauser J, Carpentier JL, Orci L, Vassalli JD
• The receptor for urokinase type plasminogen activator polarizes expression of the protease to the leading edge of migrating monocytes and promotes degradation of enzyme inhibitor complexes.
• J Cell Biol. 1990; 111: 783-92
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• Receptor-bound urokinase-type plasminogen activator (uPA) remains associated to the surface of human monocytes for many hours. Monocytes induced to migrate in a chemotactic gradient of f-Met-Leu-Phe rapidly polarize their uPA receptors to the leading front of the cells. Receptor-bound enzyme can be inhibited by plasminogen activator inhibitor 2 (PAI-2), with a kinetics comparable to that determined for the free enzyme, and uPA/PAI-2 complexes can bind to the uPA receptor. In contrast to the active enzyme, the uPA/PAI-2 complex is rapidly cleared from the monocyte cell surface; this involves an initial cleavage of the complex at the cell surface, followed by endocytosis and degradation. These results indicate that the uPA receptor can function both to focus plasmin-mediated extracellular matrix degradation in front of migrating cells, and to target uPA/PAI-2 enzyme/inhibitor complexes for degradation; they suggest that this receptor is a key determinant in the control of uPA-catalyzed extracellular proteolysis.

• Shi W, Zhang CY
• [Structure and characteristics of singer chain urokinase-type plasminogen activator]
• Sheng Li Ke Xue Jin Zhan. 1990; 21: 314-7

• Tozser J, Berta A
• Urokinase-type plasminogen activator in rabbit tears. Comparison with human tears.
• Exp Eye Res. 1990; 51: 33-7
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• Plasminogen activator activity was determined in stimulated normal rabbit tears, and was identified as being due to urokinase using specific antibodies. The activator activity is about 100-fold higher in rabbit tears than in human tears. The molecular weight of the activator was determined by fibrin zymography to be 40-50 kDa. However, a multiband pattern of urokinase-type antigen ranging in molecular weight from 60-70 kDa to 200 kDa was detected by immunoblotting. The 60-70 kDa protein was resistant to reduction. The antigen concentration is more than 100-fold higher than the concentration expected from the activity measurement, indicating that most of the urokinase in rabbit tears is in inactive form, such as in inhibitor or receptor complexes.

• Stief TW, Martin E, Jimenez JM, Rodriguez JM
• Protection of urokinase from plasminogen activator inhibitors in human plasma by hydrophobic agents.
• Thromb Res. 1990; 58: 199-203

• Mangel WF
• Enzyme systems. Better reception for urokinase.
• Nature. 1990; 344: 488-9

• Au YP, Wang TW, Clowes AW
• Nucleotide and deduced amino acid sequences of baboon urokinase-type plasminogen activator.
• Nucleic Acids Res. 1990; 18: 3411-3411

• Nielsen LS, Kellerman GM, Behrendt N, Picone R, Dano K, Blasi F
• A 55,000-60,000 Mr receptor protein for urokinase-type plasminogen activator. Identification in human tumor cell lines and partial purification.
• J Biol Chem. 1988; 263: 2358-63
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• The iodinated Mr approximately equal to 15,000 amino-terminal fragment (ATF) of the urokinase-type plasminogen activator (u-PA) molecule bound specifically to the cell surface of all of seven cultured human tumor cell lines studied. Cross-linking of iodinated ATF to the cell surface using a bifunctional amino-reactive reagent followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography revealed with the four cell lines studied the occurrence of a single band migrating with an Mr of 70,000-75,000, indicating complex formation with an Mr of 55,000-60,000 u-PA receptor protein (u-PA-R). In the human monocyte cell line U937 cultivated in the presence of phorbol ester, the amount of complex was strongly increased, and a fraction of the complex had a slower electrophoretic mobility. Comparison between autoradiograms of reduced and unreduced samples suggests that u-PA-R consists of one polypeptide chain. Two forms of u-PA-R, which differed with respect to affinity to concanavalin A, were identified. u-PA-R retained its ability to bind to ATF after cell lysis, and it was purified approximately 2,200-fold from biosynthetically labeled U937 cells by affinity chromatography with proenzyme u-PA coupled to Sepharose. The purified Mr 55,000-60,000 protein was specifically bound and cross-linked to u-PA, proenzyme u-PA, and ATF, but not to tissue-type plasminogen activator or other unrelated proteins.

• Appella E, Blasi F
• The growth factor module of urokinase is the binding sequence for its receptor.
• Ann N Y Acad Sci. 1987; 511: 192-5

• Blasi F, Vassalli JD, Dano K
• Urokinase-type plasminogen activator: proenzyme, receptor, and inhibitors.
• J Cell Biol. 1987; 104: 801-4

• Medcalf RL, Hamilton JA
• Human synovial fibroblasts produce urokinase-type plasminogen activator.
• Arthritis Rheum. 1986; 29: 1397-401
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• The plasminogen activator produced by cultured human synovial fibroblasts was investigated both biochemically and immunologically. Stimulated either by all-trans-retinoic acid or by monocyte-conditioned medium, these fibroblasts elaborated a plasminogen activator with electrophoretic mobility similar to that of urokinase (Mr = 52 kilodaltons), and which also had immunologic cross-reactivity with urokinase. The plasminogen activator found in rheumatoid synovial fluids has been shown to be of the urokinase type. The findings reported here are consistent with the notion that synovial fibroblasts are a source of this proteinase.

• Lijnen HR, Collen D
• Stimulation by heparin of the plasmin-mediated conversion of single-chain to two-chain urokinase-type plasminogen activator.
• Thromb Res. 1986; 43: 687-90

• Blasi F, Stoppelli MP, Cubellis MV
• The receptor for urokinase-plasminogen activator.
• J Cell Biochem. 1986; 32: 179-86
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• Many human cells and cell lines possess a specific receptor that binds urokinase plasminogen activator (uPA) with an affinity of about 10(-10) M. Bound enzyme is not internalized, is slowly dissociated, and retains its enzymatic activity. The amino acid sequence of uPA responsible for receptor binding is located within the first 35 aminoterminal residues, ie, in the growth factor domain. Binding, however, is not competed for by other proteins that contain the growth factor domain (including epidermal growth factor). Cells that produce uPA secrete the pro-uPA form, which subsequently binds to the receptor. A431 cells, in fact, have their receptors completely saturated with pro-uPA. It is proposed that uPA:uPA-receptor interaction plays a direct role in physiological and pathological processes that require cell migration.

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