The Mr 17500 region of the A chain of urokinase is required for interaction with a specific receptor in A431 cells

We have found the existence of specific receptors for the plasminogen activator, urokinase, in A431 human epidermoid carcinoma cells, cultures in plasminogen-free conditions. Two subsets of receptors have been recognized on the basis of 125I-labelled urokinase binding analysis: about 1 X 10(3) high-affinity (Kd = 5.0 X 10(-11) M) and 1 X 10(5) low-affinity (Kd = 9 X 10(-9) M) receptors per cell. The electron microscopic observation of a urokinase: ferritin conjugate has shown single and clustered receptors at the cell surface. Down-regulation of the receptors (T1/2 = 3.77 h) follows the binding of urokinase. The binding does not involve an intact catalytic site and is inhibited by a monoclonal antibody against the Mr 17500 proteolytic fragment of the A chain of urokinase.     

The plasminogen system and cell surfaces: evidence for plasminogen and urokinase receptors on the same cell type

The capacity of cells to interact with the plasminogen activator, urokinase, and the zymogen, plasminogen, was assessed using the promyeloid leukemic U937 cell line and the diploid fetal lung GM1380 fibroblast cell line. Urokinase bound to both cell lines in a time- dependent, specific, and saturable manner (Kd = 0.8-2.0 nM). An active catalytic site was not required for urokinase binding to the cells, and 55,000-mol-wt urokinase was selectively recognized. Plasminogen also bound to the two cell lines in a specific and saturable manner. This interaction occurred with a Kd of 0.8-0.9 microM and was of very high capacity (1.6-3.1 X 10(7) molecules bound/cell). The interaction of plasminogen with both cell types was partially sensitive to trypsinization of the cells and required an unoccupied high affinity lysine-binding site in the ligand. When plasminogen was added to the GM1380 cells, a line with high intrinsic plasminogen activator activity, the bound ligand was comprised of both plasminogen and plasmin. Urokinase, in catalytically active or inactive form, enhanced plasminogen binding to the two cell lines by 1.4-3.3-fold. Plasmin was the predominant form of the bound ligand when active urokinase was added, and preformed plasmin can also bind directly to the cells. Plasmin on the cell surface was also protected from its primary inhibitor, alpha 2-antiplasmin. These results indicate that the two cell lines possess specific binding sites for plasminogen and urokinase, and a family of widely distributed cellular receptors for these components may be considered. Endogenous or exogenous plasminogen activators can generate plasmin on cell surfaces, and such activation may provide a mechanism for arming cell surfaces with the broad proteolytic activity of this enzyme.     

The urokinase plasminogen activator receptor in the regulation of the actin cytoskeleton and cell motility

Cell migration is a complex process requiring tight control of several mechanisms including dynamic reorganization of the actin cytoskeleton and adhesion to the extracellular matrix. The GPI-anchored urokinase plasminogen activator receptor (uPAR) has an important role in the regulation of cell motility in many cell types. This is partly due to the localization of proteolytic activity on the cell surface by binding of the serine protease uPA. Results accumulated over the last decade suggest that uPAR is also involved in motility control through other mechanisms. These include induction of signal transduction events after ligation with uPA, binding to the extracellular matrix molecule vitronectin (VN), and association with integrins and other transmembrane partners. In this review these mechanisms will be discussed with a special emphasis on how the GPI-linked receptor transmits signals to the intracellular milieu and how uPAR participates in the regulation of actin cytoskeleton reorganization and cell adhesion during cell migration.     

Role of Mitogen-Activated Protein Kinases in Stimulation of Smooth Muscle Cell Migration by Urokinase

The urokinase-type plasminogen activator, or urokinase, stimulates proliferation, adhesion, and migration of different type cells both due to its proteolytic activity and by activation of intracellular transduction pathways after interaction with the external cell surface. It is suggested that activation of p42/p44^erk1,2 MAP-kinases in response to specific receptor binding to the urokinase N-terminal domain is the key event in initiation of cell migration. However, we have found that the central kringle-domain of urokinase has its own target on the cell surface, and that its binding causes a migration response of human smooth muscle cells (SMCs). In the present study, we have shown that the urokinase kringle-domain is required to activate the p38 MAP kinase cascade, and that its inhibition leads to suppression of the migration response of SMCs. On the contrary, stimulation of the p42/p44^erk1,2 MAP-kinase cascade is determined only by proteolytic activity of urokinase and does not depend on its binding to SMCs. Selective inhibition of the p42/p44^erk1,2 MAP-kinase cascade produced a depression of the SMC migration induced by catalytically active urokinase, but did not affect the migration induced by non-active urokinase. It is concluded that binding of the urokinase kringle-domain to a yet unidentified target at the SMC surface is required for activation of the p38 MAP-kinase cascade and of the cell migration. Urokinase was shown to stimulate phosphorylation and activation of regulatory light myosin chains that are required to increase the cytoskeleton dynamics and SMC motility. The participation of p42/p44^erk1,2 and p38 MAP-kinase cascade in the realization of this effect is discussed.     

Interaction of single-chain urokinase-type plasminogen activator with human endothelial cells

The interaction of urokinase-type plasminogen activators with receptors on the surface of endothelial cells may play an important role in the regulation of fibrinolysis and cell migration. Therefore, we investigated whether human umbilical vein endothelial cells (HUVEC) express receptors for single-chain urokinase (scu-PA) on the cell surface and examined the effect of such binding on plasminogen activator activity. Binding of 125I-labeled scu-PA to HUVEC, performed at 4 degrees C, was saturable, reversible, and specific (k+1 4 +/- 1 X 10(6) min-1 M-1, k-1 6.2 +/- 1.4 X 10(-3) min-1, Kd 2.8 +/- 0.1 nM; Bmax 2.2 +/- 0.1 X 10(5) sites/cell; mean +/- S.E.). Binding of radiolabeled scu-PA was inhibited by both natural and recombinant wild-type scu-PA, high molecular weight two-chain u-PA (tcu-PA), catalytic site-inactivated tcu-PA, an amino-terminal fragment of u-PA (amino acids 1-143), and a smaller peptide (amino acids 4-42) corresponding primarily to the epidermal growth factor-like domain. Binding was not inhibited by low molecular weight urokinase or by a recombinant scu-PA missing amino acids 9-45. Cell-bound scu-PA migrated at its native molecular mass on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In the presence of plasminogen, scu-PA bound to endothelial cells generated greater plasmin activity than did scu-PA in the absence of cells. In contrast, when tcu-PA was added directly to HUVEC, sodium dodecyl sulfate-stable complexes formed with cell or matrix-associated plasminogen activator inhibitors with a loss of plasminogen activator activity. These studies suggest that endothelial cells in culture express high affinity binding sites for the epidermal growth factor domain of scu-PA. Interaction of scu-PA with these receptors may permit plasminogen activator activity to be expressed at discrete sites on the endothelial cell membrane.     

Plasminogen activators and their inhibitors in the neuromuscular system: II. Serpins and serpin: protease complex receptors increase during in vitro myogenesis

In the course of studies on the regulation of plasminogen activator-mediated extracellular matrix degradation in muscle we found the presence of a factor, a cellular inhibitor of serine proteases having features similar to the serpin protease nexin I (PNI). This factor was present in the medium and at maximum concentration following fusion of skeletal muscle cells in culture. The ability of the PNI homologue in mouse muscle to inhibit ECM degradation by urokinase in myoblast medium was compared to that of human PNI purified from human fibroblasts. Stable (to SDS) 1:1 molar ratio complex formation between PNI and proteases, the proposed means by which these enzymes are regulated and removed, was also detected. Cell surface receptors for protease:PNI complexes, the specific binding sites for inactive complex internalization, were found on multinucleated myotubes, while little or no receptor activity was detected on myoblasts. These data suggest that developmental regulation of a) increased PNI proteolytic inhibitory activity expression and b) the appearance of protease:inhibitor complex receptors on muscle cell surfaces during myogenesis may constitute important regulatory features of muscle surface proteolytic activity. They complement previous studies of proteoglycan metabolism in muscle, which itself contains molecules capable of regulating the activity of myotube surface proteases.     

The urokinase receptor (uPAR) and the uPAR-associated protein (uPARAP/Endo180): membrane proteins engaged in matrix turnover during tissue remodeling

The breakdown of the barriers formed by extracellular matrix proteins is a pre-requisite for all processes of tissue remodeling. Matrix degradation reactions take part in specific physiological events in the healthy organism but also represent a crucial step in cancer invasion. These degradation processes involve a highly organized interplay between proteases and their cellular binding sites as well as specific substrates and internalization receptors. This review article is focused on two components, the urokinase plasminogen activator receptor (uPAR) and the uPAR-associated protein (uPARAP, also designated Endo180), that are considered crucially engaged in matrix degradation. uPAR and uPARAP have highly diverse functions, but on certain cell types they interact with each other in a process that is still incompletely understood. uPAR is a glycosyl-phosphatidylinositol-anchored glycoprotein on the surface of various cell types that serves to bind the urokinase plasminogen activator and localize the activation reactions in the proteolytic cascade system of plasminogen activation. uPARAP is an integral membrane protein with a pronounced role in the internalization of collagen for intracellular degradation. Both receptors have additional functions that are currently being unraveled. The present discussion of uPAR and uPARAP is centered on their protein structure and molecular and cellular function.     

A Flexible Multidomain Structure Drives the Function of the Urokinase-type Plasminogen Activator Receptor (uPAR)

The urokinase-type plasminogen activator receptor (uPAR) provides a rendezvous between proteolytic degradation of the extracellular matrix and integrin-mediated adhesion to vitronectin. These processes are, however, tightly linked because the high affinity binding of urokinase regulates the binding of uPAR to matrix-embedded vitronectin. Although crystal structures exist to define the corresponding static bi- and trimolecular receptor complexes, it is evident that the dynamic property of uPAR plays a decisive role in its function. In the present study, we combine small angle x-ray scattering, hydrogen-deuterium exchange, and surface plasmon resonance to develop a structural model describing the allosteric regulation of uPAR. We show that the flexibility of its N-terminal domain provides the key for understanding this allosteric mechanism. Importantly, our model has direct implications for understanding uPAR-assisted cell adhesion and migration as well as for translational research, including targeted intervention therapy and non-invasive tumor imaging in vivo.     

Urokinase plasminogen activator induces human smooth muscle cell migration and proliferation via distinct receptor-dependent and proteolysis-dependent mechanisms

In order to define the relative contribution of the proteolytic domain and the receptor-binding domain of urokinase plasminogen activator (uPA) toward its mitogenic properties we studied the effects of different uPA isoforms on migration and proliferation of human aortic smooth muscle cells (hSMC). The isoforms tested included native human glycosylated uPA, and two recombinant uPA forms, namely a recombinant uPA with wild type structure (r-uPA), and a uPA-mutant in which the first 24 N-terminal amino acid residues of the receptor binding domain were replaced by 13 foreign amino acid residues (r-uPAmut). Cell migration was evaluated using a micro-Boyden chamber assay, and cell proliferation assessed by measurement of [3H]-thymidine incorporation into DNA. Competition binding studies on hSMC using 125I-r-uPA as ligand demonstrated that r-uPA and r-uPAmut exhibited equivalent displacement profiles. However, migration of hSMC was promoted by r-uPA and not by r-uPAmut. r-uPA-induced migration occurred at concentrations (half-maximally effective concentration of 2 nM) approximating the Kd for uPA-uPAR binding (1 nM). r-uPA-induced migration was not affected by the plasmin inhibitor aprotinin. In contrast to their differential chemotactic properties, uPA, r-uPA and r-uPAmut, which possess similar proteolytic activities, all stimulated [3H]-thymidine incorporation in hSMC. Since the [3H]-thymidine incorporation response to each isoform occurred at concentrations (> 50 nM) much higher than necessary for uPAR saturation by ligand (1 nM), this mitogenic response may be independent of binding to uPAR. [3H]-thymidine incorporation responses to r-uPA and -uPAmut were sensitive to the plasmin inhibitor aprotinin, and uPA stimulated DNA synthesis was inhibited by plasminogen activator inhibitor. We conclude that hSMC migration in response to uPA depends upon on its binding to uPAR, whereas uPA-stimulated DNA synthesis in these cells requires proteolysis and plasmin generation.     

Helicobacter pylori interactions with plasminogen

Helicobacter pylori is the causative agent of chronic gastritis, peptic ulcer, and gastric malignancies. A number of virulence factors have been described including several adhesins, a cytotoxin, neutrophil-activating protein, and expression of binding of extracellular matrix proteins, like collagen type IV, laminin, and vitronectin. H. pylori strains commonly express binding of soluble plasminogen. Coccoid forms also express binding. Plasminogen binding was optimal at pH 7.0. The binding is mediated by two cell surface proteins of 42 and 57 kDa. Scatchard plot analysis showed a straight line with a K(d) of 7 x 10(-7) M. Lysine and E-aminocaproic acid inhibited binding. The binding domain on the plasminogen molecule is the fifth kringle, miniplasminogen. Plasminogen is converted to plasmin by tissue plasminogen activator. During H. pylori infection the activity of tissue plasminogen activator is decreased and that of urokinase increased. This is reversed after eradication therapy. The plasminogen binding and conversion to plasmin is the only proteolytic activity of H. pylori, and may enhance tissue penetration and be involved in carcinogenesis.     

In vivo invasion of modified chorioallantoic membrane by tumor cells: the role of cell surface-bound urokinase

The ability of the chick embryo chorioallantoic membrane (CAM) to withstand invasion by tumor cells can be intentionally compromised by altering its morphological integrity. Using a newly developed quantitative assay of invasion we showed that intact CAMs were completely resistant to invasion by tumor cells, wounded CAMs did not pose a barrier to penetration, and CAMs that were wounded and then allowed to reseal displayed partial susceptibility to invasion. The invasion of resealed CAMs required catalytically active plasminogen activator (PA) of the urokinase type (uPA); the invasive efficiency of tumor cells was reduced by 75% when tumor uPA activity or tumor uPA production was inhibited. The invasive ability of human tumor cells, which have surface uPA receptors but which do not produce the enzyme, could be augmented by saturating their receptors with exogenous uPA. The mere stimulation of either uPA or tissue plasminogen activator production, in absence of binding to cell receptors, did not result in an enhancement of invasiveness. These findings suggest that the increased invasive potential of tumor cells is correlated with cell surface-associated proteolytic activity stemming from the interaction between uPA and its surface receptor.     

Interaction between kringle and growth-factor-like domains in the urokinase molecule: Possible role in stimulation of chemotaxis

The results presented in this paper suggest the presence of an interaction between the kringle- and the growth-factor-like urokinase domains. This interaction regulates chemotactic properties of urokinase. We also show that interaction of urokinase with its "classical" receptor (uPAR) has a "permissive" effect on the interactions between the kringle domain and other targets on the cell surface. On the basis of our data we can suggest that uPAR serves as an "adaptor" for urokinase, and the binding of urokinase kringle domain to its receptor causes immediate activation of intracellular signaling and induction of cell migration.     

Random peptide bacteriophage display as a probe for urokinase receptor ligands

The urokinase receptor is a multi-functional protein that plays a central role in cell surface plasminogen activation, cell migration, and cell adhesion. We previously demonstrated that high affinity peptide ligands for the urokinase receptor, which are urokinase competitors, can be obtained from a 15mer peptide library (Goodson et al., 1994). In order to probe for additional urokinase receptor binding sites we affinity selected the same bacteriophage library on complexes of soluble urokinase receptor (suPAR) and the receptor binding domain of urokinase, residues 1-48 (uPA1-48). Bacteriophage were isolated which bound to suPAR and suPAR:uPA1-48 complexes with high yield. The peptide sequences encoded by these bacteriophage were distinct from those obtained previously on urokinase receptor expressing cells, and comprise two groups based upon effects on su-PAR:1-anilino-8-napthalene sulfonate (ANS) fluorescence, and vitronectin binding competition. Alanine scanning mutagensis of the soluble peptides was used to define minimal regions and key residues for suPAR binding by competition with the parent bacteriophage. A comparison of these results with sequences of domains of both vitronectin and integrin alpha-chains, which have been reported to be important for urokinase receptor binding, suggests that the homology with the peptide sequences selected is functionally significant.     

A urokinase receptor-associated protein with specific collagen binding properties

The plasminogen activation cascade system, directed by urokinase and the urokinase receptor, plays a key role in extracellular proteolysis during tissue remodeling. To identify molecular interaction partners of these trigger proteins on the cell, we combined covalent protein cross-linking with mass spectrometry based methods for peptide mapping and primary structure analysis of electrophoretically isolated protein conjugates. A specific tri-molecular complex was observed upon addition of pro-urokinase to human U937 cells. This complex included the urokinase receptor, pro-urokinase, and an unknown, high molecular weight urokinase receptor-associated protein. The tryptic peptide mixture derived from a cross-linked complex of pro-urokinase and the latter protein was analyzed by nanoelectrospray tandem mass spectrometric sequencing. This analysis identified the novel protein as the human homologue of a murine membrane-bound lectin with hitherto unknown function. The human cDNA was cloned and sequenced. The protein, designated uPARAP, is a member of the macrophage mannose receptor protein family and contains a putative collagen-binding (fibronectin type II) domain in addition to 8 C-type carbohydrate recognition domains. It proved capable of binding strongly to a single type of collagen, collagen V. This collagen binding reaction at the exact site of plasminogen activation on the cell may lead to adhesive functions as well as a contribution to cellular degradation of collagen matrices.     

Binding of the NG2 proteoglycan to kringle domains modulates the functional properties of angiostatin and plasmin(ogen)

Interactions of the developmentally regulated chondroitin sulfate proteoglycan NG2 with human plasminogen and kringle domain-containing plasminogen fragments have been analyzed by solid-phase immunoassays and by surface plasmon resonance. In immunoassays, the core protein of NG2 binds specifically and saturably to plasminogen, which consists of five kringle domains and a serine protease domain, and to angiostatin, which contains plasminogen kringle domains 1-3. Apparent dissociation constants for these interactions range from 12 to 75 nm. Additional evidence for NG2 interaction with kringle domains comes from its binding to plasminogen kringle domain 4 and to miniplasminogen (kringle domain 5 plus the protease domain) with apparent dissociation constants in the 18-71 nm range. Inhibition of plasminogen and angiostatin binding to NG2 by 6-aminohexanoic acid suggests that lysine binding sites are involved in kringle interaction with NG2. The interaction of NG2 with plasminogen and angiostatin has very interesting functional consequences. 1) Soluble NG2 significantly enhances the activation of plasminogen by urokinase type plasminogen activator. 2) The antagonistic effect of angiostatin on endothelial cell proliferation is inhibited by soluble NG2. Both of these effects of NG2 should make the proteoglycan a positive regulator of the cell migration and proliferation required for angiogenesis.     

Role of cell-surface lysines in plasminogen binding to cells: identification of alpha-enolase as a candidate plasminogen receptor.

Plasminogen binding to cell surfaces results in enhanced plasminogen activation, localization of the proteolytic activity of plasmin on cell surfaces, and protection of plasmin from alpha 2-antiplasmin. We sought to characterize candidate plasminogen binding sites on nucleated cells, using the U937 monocytoid cell as a model, specifically focusing on the role of cell-surface proteins with appropriately placed lysine residues as candidate plasminogen receptors. Lysine derivatives with free alpha-carboxyl groups and peptides with carboxy-terminal lysyl residues were effective inhibitors of plasminogen binding to the cells. One of the peptides, representing the carboxy-terminal 19 amino acids of alpha 2-antiplasmin, was approximately 5-fold more effective than others with carboxy-terminal lysines. Thus, in addition to a carboxy-terminal lysyl residue, other structural features of the cell-surface proteins may influence their affinity for plasminogen. Affinity chromatography has been used to isolate candidate plasminogen receptors from U937 cells. A major protein of Mr 54,000 was recovered and identified as alpha-enolase by immunochemical and functional criteria. alpha-Enolase was present on the cell surface and was capable of binding plasminogen in ligand blotting analyses. Plasminogen binding activity of a molecular weight similar to alpha-enolase also was present in a variety of other cell types. Carboxypeptidase B treatment of alpha-enolase abolished its ability to bind plasminogen, consistent with the presence of a C-terminal lysyl residue. Thus, cell-surface proteins with carboxy-terminal lysyl residues appear to function as plasminogen binding sites, and alpha-enolase has been identified as a prominent representative of this class of receptors.     

Recycling of the urokinase receptor upon internalization of the uPA:serpin complexes.

The GPI-anchored urokinase plasminogen activator receptor (uPAR) does not internalize free urokinase (uPA) but readily internalizes and degrades uPA:serpin complexes in a process that requires the alpha2-macroglobulin receptor/low density lipoprotein receptor-related protein (alpha2MR-LRP). This process is accompanied by the internalization of uPAR which renders it resistant to phosphatidylinositol-specific phospholipase C (PI-PLC). In this paper we show that during internalization of uPA:serpins at 37 degrees C, analysed by FACScan, immunofluorescence and immunoelectron microscopy, an initial decrease of cell surface uPAR was observed, followed by its reappearance at later times. This effect was not due to redistribution of previously intracellular receptors, nor to the surface expression of newly synthesized uPAR. Recycling was directly demonstrated in cell surface-biotinylated, uPA:PAI-1-exposed cells in which biotinylated uPAR was first internalized and subsequently recycled back to the surface upon incubation at 37 degrees C. In fact, uPAR was resistant to PI-PLC after the 4 degrees C binding of uPA:PAI-1 to biotinylated cells, but upon incubation at 37 degrees C PI-PLC-sensitive biotinylated uPAR reappeared at the cell surface. Binding of uPA:PAI-1 by uPAR, while essential to initiate the whole process, was, however, dispensable at later stages as both internalization and recycling of uPAR could be observed also after dissociation of the bound ligand from the cell surface.     

Production and purification of urokinase: a comprehensive review

An increased emphasis on prevention of fatalities due to thrombovascular disorders is broadening opportunities for several cardiovascular agents, especially plasminogen activators, for preventing strokes and heart attacks. Hence, urokinase, as one of the most potent plasminogen activators is attracting a great deal of attention. Developments in cell lines and bioprocess technology have made it possible to produce urokinase from in vitro cell culture. Attempts are now underway to enhance urokinase production from cell culture through media manipulation, bioreactor cultivation, and innovative purification techniques. Downstream processing also poses an intricate problem due to the complexity of cell culture extracts, susceptibility of urokinase to autocatalytic and proteolytic degradation and due to the presence of plasminogen activator inhibitors in the culture media. Hence, enhancing cellular productivity and downstream product recovery continue to be major challenges as discussed in this review. Furthermore, an approach for integrated upstream and downstream processing is needed to develop an economically viable technology. In the present review the emerging trends in urokinase production and purification have been discussed in detail.     

Regulation of urokinase/urokinase receptor interaction by heparin-like glycosaminoglycans

We show here that the interaction between the urokinase-type plasminogen activator and its receptor, which plays a critical role in cell invasion, is regulated by heparan sulfate present on the cell surface and in the extracellular matrix. Heparan sulfate oligomers showing a composition close to the dimeric repeats of heparin (glucosamine-NSO(3)(6-OSO(3))-iduronic acid(2-OSO(3))) n = 5 and n > 5, where iduronic acid may alternate with glucuronic acid, exhibit affinity for urokinase plasminogen activator and confer specificity on urokinase/urokinase receptor interaction. Cell surface clearance of heparan sulfate reduces the affinity of such interaction with a parallel decrease of specific urokinase binding in the presence of an unaltered expression of receptor. Transfection of human urokinase plasminogen activator receptor in normal Chinese hamster ovary fibroblasts and in Chinese hamster ovary cells defective for the synthesis of sulfated glycosaminoglycans results in specific urokinase/receptor interaction only in nondefective cells. Heparan sulfate/urokinase and receptor/urokinase interactions exhibit similar K(d) values. We concluded that heparan sulfate functions as an adaptor molecule that confers specificity on urokinase/receptor binding.     

Modulation of the urokinase receptor in human colon cell lines by N,N-dimethylformamide

The present study documents the effect of the planar, polar differentiation promoter N,N-dimethylformamide (DMF) on urokinase binding to colon carcinoma cells. Exposure of the colon carcinoma cell lines to the agent resulted in enhanced specific binding of radioactive urokinase to all cells tested. Insulin binding to the cells was, however, unaffected by DMF. A DMF exposure period of 45 h was required to observe maximum urokinase binding to two representative cell lines FET and RKO. Optimal stimulation of both cell lines occurred with 0.8% DMF. Scatchard analysis revealed the dissociation constants to be unchanged by the agent with the increased binding of radioactive plasminogen activator reflecting an up-regulation of binding sites. In this regard, the cell line RKO upon exposure to DMF, displayed approx. 700,000 receptors/cell, the highest value published, to date, for any cell line.