Chain uPA Research Articles

Effects of Extracellular DNA on Plasminogen Activation and Fibrinolysis

The increased levels of extracellular DNA found in a number of disorders involving dysregulation of the fibrinolytic system may affect interactions between fibrinolytic enzymes and inhibitors. Double-stranded (ds) DNA and oligonucleotides bind tissue-(tPA) and urokinase (uPA)-type plasminogen activators, plasmin, and plasminogen with submicromolar affinity. The binding of enzymes to DNA was detected by EMSA, steady-state, and stopped-flow fluorimetry. The interaction of dsDNA/oligonucleotides with tPA and uPA includes a fast bimolecular step, followed by two monomolecular steps, likely indicating slow conformational changes in the enzyme. DNA (0.1-5.0 μg/ml), but not RNA, potentiates the activation of Glu- and Lys-plasminogen by tPA and uPA by 480- and 70-fold and 10.7- and 17-fold, respectively, via a template mechanism similar to that known for fibrin. However, unlike fibrin, dsDNA/oligonucleotides moderately affect the reaction between plasmin and α(2)-antiplasmin and accelerate the inactivation of tPA and two chain uPA by plasminogen activator inhibitor-1 (PAI-1), which is potentiated by vitronectin. dsDNA (0.1-1.0 μg/ml) does not affect the rate of fibrinolysis by plasmin but increases by 4-5-fold the rate of fibrinolysis by Glu-plasminogen/plasminogen activator. The presence of α(2)-antiplasmin abolishes the potentiation of fibrinolysis by dsDNA. At higher concentrations (1.0-20 μg/ml), dsDNA competes for plasmin with fibrin and decreases the rate of fibrinolysis. dsDNA/oligonucleotides incorporated into a fibrin film also inhibit fibrinolysis. Thus, extracellular DNA at physiological concentrations may potentiate fibrinolysis by stimulating fibrin-independent plasminogen activation. Conversely, DNA could inhibit fibrinolysis by increasing the susceptibility of fibrinolytic enzymes to serpins.

Regulation of Plasminogen Activator Inhibitor-1 Expression by Tumor Suppressor Protein p53

H1299 lung carcinoma cells lacking p53 (p53-/-) express minimal amounts of plasminogen activator inhibitor-1 (PAI-1) protein as well as mRNA. p53(-/-) cells express highly unstable PAI-1 mRNA. Transfection of p53 in p53(-/-) cells enhanced PAI-1 expression and stabilized PAI-1 mRNA. On the contrary, inhibition of p53 expression by RNA silencing in non-malignant human lung epithelial (Beas2B) cells decreased basal as well as urokinase-type plasminogen activator-induced PAI-1 expression because of accelerated degradation of PAI-1 mRNA. Purified p53 protein specifically binds to the PAI-1 mRNA 3'-un-translated region (UTR), and endogenous PAI-1 mRNA forms an immune complex with p53. Treatment of purified p53 protein with anti-p53 antibody abolished p53 binding to the 3'-UTR of PAI-1 mRNA. The p53 binding region maps to a 70-nucleotide PAI-1 mRNA 3'-UTR sequence, and insertion of the p53-binding sequence into beta-globin mRNA destabilized the chimeric transcript. Deletion experiments indicate that the carboxyl-terminal region (amino acid residues 296-393) of p53 protein interacts with PAI-1 mRNA. These observations demonstrate a novel role for p53 as an mRNA-binding protein that regulates increased PAI-1 expression and stabilization of PAI-1 mRNA in human lung epithelial and carcinoma cells.

Elevated urokinase‐specific surface receptor expression is maintained through its interaction with urokinase plasminogen activator

Urokinase plasminogen activator (uPA) and its receptor (uPAR) are overexpressed in various neoplasms, and play a key role in tumor progression and metastasis. In this study, we examined uPA and uPAR expression in a variety of human breast cancer cell lines and found that lines with elevated uPA expression also exhibited high uPAR expression, suggesting the possibility that uPA and uPAR are regulated in concert. To test this possibility, we introduced antisense uPA RNA and antisense uPAR RNA in MDA-MB-231 and BT-549 lines that express high levels of uPA and uPAR. Antisense uPA RNA not only downregulated uPA expression, but also greatly reduced uPAR expression in both lines. However, antisense uPAR RNA-reduced uPAR expression with no apparent inhibitory effect on the levels of uPA. These results indicate that expression of uPAR requires uPA but not vice versa. With a panel of uPA and uPAR monoclonal antibodies (mAbs), we observed that the mAbs disrupting uPA and uPAR interaction, rather than mAb inhibiting uPA protease activity, reduced uPAR expression. Moreover, adding soluble single chain uPA (scuPA) to MDA-MB-231 or BT-549 cells expressing antisense uPA mRNA-restored uPAR expression. These findings suggest that uPA dictates uPAR expression and that uPA binding to uPAR transmits signals for uPAR expression. Finally, we provided evidence that Fyn, a Src family kinase, is involved in uPA-induced uPAR expression.

Outside-In Signaling upon Assembly on Endothelial Cells of the Proteins of the Plasma Kallikrein/Kinin System.

Investigations from our laboratory have shown that there is a multiprotein receptor system on endothelial cells (HUVEC) consisting of gC1qR, uPAR, and cytokeratin 1 for high molecular weight kininogen (HK) and factor XII (XII) (Blood 97:2342; 99:3585) that serves as a presentation receptor for prekallikrein (PK) activation. When PK binds to HK on HUVEC, it is activated to plasma kallikrein by the serine protease prolylcarboxypeptidase (JBC 277:17962; Blood 103:4554). HK is also known to have anti-proliferative and anti-angiogenic activity. We asked if there is outside-in signaling through this HUVEC receptor complex. Initial investigations determined if single chain urokinase (ScuPA) or XII stimulates Erk 1 and 2 (MAPK42 and 44) (MAPK) in HUVEC. Independently ScuPA (5–200 nM) or XII (15–200 nM) in the presence of 0.05 mM zinc ion stimulates HUVEC MAPK expression and it is blocked by 0.1 mM PD98059, 30 nM wortmann, or 0.05 mM LY294002. Two chain uPA or APMSF-treated ScuPA upregulates MAPK to the same extent as ScuPA. Similarly, XIIa or APMSF-treated XII upregulates MAPK like XII. Since HK, ScuPA, XII and vitronectin (VN) all bind to the same region on uPAR (JBC 279:16621), studies focused on the role of uPAR in these activities. Mab3B10 to the HK, ScuPA, XII, and VN binding site on uPAR's Domain 2 (D2) blocks ScuPA or XII upregulation of MAPK. Peptides LRG20, YLP20, PGS20, or FHN20 from uPAR's D2 (L166-N200) block ScuPA or XII upregulation of MAPK. Similarly, HKa (1 micromolar) and peptides from the HK Domain 5 (G469-H498) HUVEC binding region (GGH18, HKH20) (JBC 270:19256) block ScuPA- or XII-induced MAPK expression. Treatment of HUVEC with 5-20 mM methyl-beta-cyclodextran (MBCD) or fillipin (1 microgram/ml), agents known to disrupt lipid rafts, do not interfere with ScuPA- or XII-induced MAPK upregulation. These combined data indicate that zymogen ScuPA or XII, two proteins with growth factor regions, directly signal through uPAR to upregulate MAPK to possibly induce cellular proliferative activities. HK or its activated fragments bound to uPAR inhibits these potential growth promoting activities. These studies imply that HKa's anti-proliferative and anti-angiogenic activities are mediated through its interaction with uPAR. These investigations also indicate that, in addition to a presentation complex for PK activation, the HUVEC multiprotein receptor complex for HK, XII, and ScuPA has an auto-regulating outside-in signaling system in endothelials cells.

Induction of p53 by Urokinase in Lung Epithelial Cells

Urokinase plasminogen activator (uPA) is a serine protease that catalyzes the conversion of plasminogen to plasmin. The plasminogen/plasmin system includes the uPA, its receptor, and its inhibitor (plasminogen activator inhibitor-1). Interactions between these molecules regulate cellular proteolysis as well as adhesion, cellular proliferation, and migration, processes germane to the pathogenesis of lung injury and neoplasia. In previous studies, we found that uPA regulates cell surface fibrinolysis by regulating its own expression as well as that of the uPA receptor and plasminogen activator inhibitor-1. In this study, we found that uPA alters expression of the tumor suppressor protein p53 in Beas2B airway epithelial cells in both a time- and concentration-dependent manner. These effects do not require uPA catalytic activity because the amino-terminal fragment of uPA lacking catalytic activity was as potent as two chain active uPA. Single chain uPA also enhanced p53 expression to the same extent as intact two chain active uPA and the amino-terminal fragment. Pretreatment of cells with anti-beta1 integrin antibody blocked uPA-induced p53 expression. uPA-induced p53 expression occurs without increased p53 mRNA expression. However, uPA induced oncoprotein MDM2 in a concentration-dependent manner. uPA-induced p53 expression does not require activation of tyrosine kinases. Inactivation of protein-tyrosine phosphatase SHP-2 inhibits both basal and uPA-induced p53 expression. Plasmin did not alter uPA-mediated p53 expression. The induction of p53 expression by exposure of lung epithelial cells to uPA is a newly recognized pathway by which urokinase may influence the proliferation of lung epithelial cells. This pathway could regulate pathophysiologic alterations of p53 expression in the setting of lung inflammation or neoplasia.

Integrin αMβ2 Orchestrates and Accelerates Plasminogen Activation and Fibrinolysis by Neutrophils

Plasmin, the pivotal thrombolytic enzyme, is generated on the surface of many cell types, where urokinase receptor (uPAR)-bound urokinase (uPA) activates cell-bound plasminogen (Plg). It has been reported that neutrophils mediate endogenous thrombolysis involving a uPA-dependent mechanism, and we previously demonstrated that both uPAR and integrin alpha(M)beta(2) recognize uPA to control cell migration and adhesion. In the present study, we report that the alpha(M)beta(2) regulates neutrophil-dependent fibrinolysis. Phorbol 12-myristate 13-acetate (PMA)-stimulated but not resting neutrophils dissolved fibrin clots, and this activity was not only uPA- and Plg-dependent but also alpha(M)beta(2)-dependent. Purified alpha(M)beta(2) directly bound uPA (K(d) = 40 nm) and Plg (K(d) = 1 microm) in a dose-dependent and saturable manner. In Plg activation assays, addition of purified alpha(M)beta(2), but not a control protein, to a single chain uPA (sc-uPA)/Plg mixture, decreased the K(m) from 2 to 0.1 microm, thereby augmenting the overall reaction efficiency by 50-fold. The binding of sc-uPA to alpha(M)beta(2) was critical for the alpha(M)beta(2)-mediated enhancement of plasmin (Plm) generation, because this effect was lost when WT-sc-uPA was replaced with a kringle-less mutant (DeltaK-sc-uPA), which does not bind to alpha(M)beta(2). Plm inactivation by alpha(2)-antiplasmin was significantly delayed when Plm was preincubated with purified, soluble alpha(M)beta(2). When Plg was added to PMA-stimulated neutrophils, both uPA and Plg were co-immunoprecipitated with alpha(M)beta(2.) Thus, assembly of Plg and uPA on integrin alpha(M)beta(2) regulates Plm activity and, thereby, plays a crucial role in neutrophil-mediated thrombolysis.

Induction of Plasminogen Activator Inhibitor-1 by Urokinase in Lung Epithelial Cells

The plasminogen/plasmin system, urokinase-type plasminogen activator (uPA), its receptor (uPAR), and its inhibitor (PAI-1), influence extracellular proteolysis and cell migration in lung injury or neoplasia. In this study, we sought to determine whether tcuPA (two chain uPA) alters expression of its major inhibitor PAI-1 in lung epithelial cells. The expression of PAI-1 was evaluated at the protein and mRNA level by Western blot, immunoprecipitation, and Northern blot analyses. We found that tcuPA treatment enhanced PAI-1 protein and mRNA expression in Beas2B lung epithelial cells in a time- and concentration-dependent manner. The tcuPA-mediated induction of PAI-1 involves post-transcriptional control involving stabilization of PAI-1 mRNA. Inactivation of the catalytic activity of tcuPA had little effect on PAI-1 induction and the activity of the isolated amino-terminal fragment was comparable with full-length single- or two-chain uPA. In contrast, deletion of either the uPA receptor binding growth factor domain or kringle domain (kringle) from full-length single chain uPA markedly attenuated the induction of PAI-1. Induction of PAI-1 by exposure of lung epithelial cells to uPA is a newly recognized pathway by which PAI-1 could regulate local fibrinolysis and urokinase-dependent cellular responses in the setting of lung inflammation or neoplasia.

Release of Soluble Urokinase Receptor from Vascular Cells*

Urokinase-type plasminogen activator (uPA) and its cell surface-receptor (uPAR) regulate cellular functions linked to adhesion and migration and contribute to pericellular proteolysis in tissue remodelling processes. Soluble uPAR (suPAR) is present in the circulation, peritoneal and ascitic fluid and in the cystic fluid from ovarian cancer. We have investigated the origin and the vascular distribution of the soluble receptor, which accounts for 10-20% of the total receptor in vascular endothelial and smooth muscle cells. Phase separation analysis of the cell conditioned media with Triton X-114 indicated that suPAR associates with the aqueous phase, indicative of the absence of the glycolipid anchor. There was a polarized release of suPAR from cultured endothelial cells towards the basolateral direction, whereas the membrane-bound receptor was found preferentially on the apical surface. Both, uPAR and suPAR became upregulated 2-4 fold after activation of protein kinase C with phorbol ester, which required de-novo protein biosynthesis. Interleukin-1beta (IL-1beta), basic fibroblast growth factor (bFGF) or vascular endothelial growth factor increased suPAR release from endothelial cells, whereas platelet derived growth factor-BB, bFGF or IL-1beta stimulated suPAR release from vascular smooth muscle cells. Immune electron microscopy indicated that in atherosclerotic vessels (s)uPAR was observed on cell membranes as well as in the extracellular matrix. These findings indicate that (s)uPAR from vascular cells is upregulated by proangiogenic as well as proatherogenic growth factors and cytokines, is preferentially released towards the basolateral side of endothelial cells and accumulates in the vessel wall.

Urokinase mediates fibrinolysis in the pulmonary microvasculature

The role of urokinase-type plasminogen activator (uPA) and its receptor (uPAR) in fibrinolysis remains unsettled. The contribution of uPA may depend on the vascular location, the physical properties of the clot, and its impact on tissue function. To study the contribution of urokinase within the pulmonary microvasculature, a model of pulmonary microembolism in the mouse was developed. Iodine 125 (125I)–labeled fibrin microparticles injected intravenously through the tail vein lodged preferentially in the lung, distributing homogeneously throughout the lobes. Clearance of125I-microemboli in wild type mice was rapid and essentially complete by 5 hours. In contrast, uPA−/− and tissue-type plasminogen activator tPA−/− mice, but not uPAR−/− mice, showed a marked impairment in pulmonary fibrinolysis throughout the experimental period. The phenotype in the uPA−/− mouse was rescued completely by infusion of single chain uPA (scuPA). The increment in clot lysis was 4-fold greater in uPA−/− mice infused with the same concentration of scuPA complexed with soluble recombinant uPAR. These data indicate that uPA contributes to endogenous fibrinolysis in the pulmonary vasculature to the same extent as tPA in this model system. Binding of scuPA to its receptor promotes fibrinolytic activity in vivo as well as in vitro. The physical properties of fibrin clots, including size, age, and cellular composition, as well as heterogeneity in endothelial cell function, may modify the participation of uPA in endogenous fibrinolysis.

Urokinase mediates fibrinolysis in the pulmonary microvasculature

AbstractThe role of urokinase-type plasminogen activator (uPA) and its receptor (uPAR) in fibrinolysis remains unsettled. The contribution of uPA may depend on the vascular location, the physical properties of the clot, and its impact on tissue function. To study the contribution of urokinase within the pulmonary microvasculature, a model of pulmonary microembolism in the mouse was developed. Iodine 125 (125I)–labeled fibrin microparticles injected intravenously through the tail vein lodged preferentially in the lung, distributing homogeneously throughout the lobes. Clearance of125I-microemboli in wild type mice was rapid and essentially complete by 5 hours. In contrast, uPA−/− and tissue-type plasminogen activator tPA−/− mice, but not uPAR−/− mice, showed a marked impairment in pulmonary fibrinolysis throughout the experimental period. The phenotype in the uPA−/− mouse was rescued completely by infusion of single chain uPA (scuPA). The increment in clot lysis was 4-fold greater in uPA−/− mice infused with the same concentration of scuPA complexed with soluble recombinant uPAR. These data indicate that uPA contributes to endogenous fibrinolysis in the pulmonary vasculature to the same extent as tPA in this model system. Binding of scuPA to its receptor promotes fibrinolytic activity in vivo as well as in vitro. The physical properties of fibrin clots, including size, age, and cellular composition, as well as heterogeneity in endothelial cell function, may modify the participation of uPA in endogenous fibrinolysis.

Tumor dormancy induced by downregulation of urokinase receptor in human carcinoma involves integrin and MAPK signaling.

Mechanisms that regulate the transition of metastases from clinically undetectable and dormant to progressively growing are the least understood aspects of cancer biology. Here, we show that a large ( approximately 70%) reduction in the urokinase plasminogen activator receptor (uPAR) level in human carcinoma HEp3 cells, while not affecting their in vitro growth, induced a protracted state of tumor dormancy in vivo, with G(0)/G(1) arrest. We have now identified the mechanism responsible for the induction of dormancy. We found that uPA/uPAR proteins were physically associated with alpha5beta1, and that in cells with low uPAR the frequency of this association was significantly reduced, leading to a reduced avidity of alpha5beta1 and a lower adhesion of cells to the fibronectin (FN). Adhesion to FN resulted in a robust and persistent ERK1/2 activation and serum-independent growth stimulation of only uPAR-rich cells. Compared with uPAR-rich tumorigenic cells, the basal level of active extracellular regulated kinase (ERK) was four to sixfold reduced in uPAR-poor dormant cells and its stimulation by single chain uPA (scuPA) was weak and showed slow kinetics. The high basal level of active ERK in uPAR-rich cells could be strongly and rapidly stimulated by scuPA. Disruption of uPAR-alpha5beta1 complexes in uPAR-rich cells with antibodies or a peptide that disrupts uPAR-beta1 interactions, reduced the FN-dependent ERK1/2 activation. These results indicate that dormancy of low uPAR cells may be the consequence of insufficient uPA/uPAR/alpha5beta1 complexes, which cannot induce ERK1/2 activity above a threshold needed to sustain tumor growth in vivo. In support of this conclusion we found that treatment of uPAR-rich cells, which maintain high ERK activity in vivo, with reagents interfering with the uPAR/beta1 signal to ERK activation, mimic the in vivo dormancy induced by downregulation of uPAR.

Interaction of Single-Chain Urokinase with its Receptor Induces the Appearance and Disappearance of Binding Epitopes within the Resultant Complex for Other Cell Surface Proteins

Binding of urokinase-type plasminogen activator (uPA) to its glycosylphosphatidylinositol-anchored receptor (uPAR) initiates signal transduction, adhesion, and migration in certain cell types. To determine whether some of these activities may be mediated by associations between the uPA/uPAR complex and other cell surface proteins, we studied the binding of complexes composed of recombinant, soluble uPA receptor (suPAR) and single chain uPA (scuPA) to a cell line (LM-TK- fibroblasts) that does not express glycosylphosphatidylinositol (GPI)-anchored proteins to eliminate potential competition by endogenous uPA receptors. scuPA induced the binding of suPAR to LM-TK- cells. Binding of labeled suPAR/scuPA was inhibited by unlabeled complex, but not by scuPA or suPAR added separately, indicating cellular binding sites had been formed that are not present in either component. Binding of the complex was inhibited by low molecular weight uPA (LMW-uPA) indicating exposure of an epitope found normally in the isolated B chain of two chain uPA (tcuPA), but hidden in soluble scuPA. Binding of LMW-uPA was independent of its catalytic site and was associated with retention of its enzymatic activity. Additional cell binding epitopes were generated within suPAR itself by the aminoterminal fragment of scuPA, which itself does not bind to LM-TK- cells. When scuPA bound to suPAR, a binding site for alpha 2-macroglobulin receptor/LDL receptor-related protein (alpha 2 MR/LRP) was lost, while binding sites for cell-associated vitronectin and thrombospondin were induced. In accord with this, the internalization and degradation of cell-associated tcuPA and tcuPA-PAI-1 complexes proceeded less efficiently in the presence of suPAR. Further, little degradation of suPAR was detected, suggesting that cell-bound complex dissociated during the initial stages of endocytosis. Thus, the interaction of scuPA with its receptor causes multiple functional changes within the complex including the dis-appearance of an epitope in scuPA involved in its clearance from the cell surface and the generation of novel epitopes that promote its binding to proteins involved in cell adhesion and signal transduction.

Dependence of Human Vascular Cell Surface Proteolysis on Expression of the Urokinase Receptor.

To delineate the role of binding of urokinase type plasminogen activator (uPA) to its receptor (uPAR) in the local generation of plasmin by endothelium, we transfected spontaneously transformed immortalized human vascular endothelial cells that express high levels of uPA but low levels of uPAR with human uPAR complementary DNA. Compared with nontransfected cell, the stably transformed clonal cell line exhibited (a) a >10-fold increase in steady-state uPAR mRNA levels documented with Northern blot analysis (n = 3), (b) a 2.8-fold increase in cell surface expression of uPAR protein quantified by enzyme linked immunosorbent assay (n = 3), (c) a 2.9-fold increase in specific binding of radiolabeled single chain uPA (n = 4), and (d) markedly increased matrix adhesion. The participation of uPAR in cell surface proteolysis was apparent based on a 3.0-fold increase in cell associated plasmin activity (n = 3) and a 2.3-fold increase in lysis of noncrosslinked fibrin clots (n = 5). Thus, local generation of plasmin and consequent degradation of fibrin are likely to be promoted by cell surface localization of uPA by uPAR in cellular constituents of the vessel wall. Furthermore, genetic engineering of endothelium to enhance expression of uPAR may confer resistance to thrombosis or restenosis associated with endovascular stents.

Serine protease and metallo protease cascade systems involved in pericellular proteolysis

Cultures of transformed fibroblasts actively involved in extracellular matrix degradation have been examined for initial activation of serine and metallo protease cascade systems. Rous sarcoma virus transformed chick embryo fibroblasts (RSVCEF), in contrast to transformed mammalian cells, produce active, two chain urokinase-type plasminogen activator (tcu-PA). Active tcu-PA is found in serum-free, plasmin-free conditioned medium from RSVCEF cultures as determined by two independent methods, immunoprecipitation and differential DFP sensitivity. RSVCEF cultures synthesize and secrete inactive, single chain uPA (scu-PA) which is converted to tcu-PA in a time dependent manner by a catalytic mechanism that appears to involve a functioning uPA receptor on the surface of intact cells. The enzyme activity responsible for this conversion may represent the initiating catalytic event in the PA/plasminogen serine protease cascade system. A 70 kDa prometalloprotease capable of degrading denatured collagen following its activation also is significantly elevated in RSVCEF cultures over that of normal CEF. Trace amounts of the active 62 kDa form of the metalloprotease (gelatinase) is found in the transformed RSVCEF cultures indicating that these cultures produce a natural activator of the prometalloprotease. Plasmin and/or PA do not appear to be the activator of this enzyme as determined by indirect inhibition assays and direct assays employing purified enzymes. The possible central position of pro PA and the 70 kDa prometalloprotease in an interacting, complex protease cascade system involved in extracellular matrix degradation is discussed.

Serine phosphorylation of biosynthetic pro-urokinase from human tumor cells

Phosphorylation is a potent mechanism regulating the activity of many intracellular enzymes. We have discovered that the product of the human urokinase plasminogen activator gene, pro-uPA, is phosphorylated in serine in at least two human cell lines. Phosphorylation occurs within the cell during biosynthesis, and phosphorylated intracellular pro-uPA is secreted into the medium. Of the secreted pro-uPA molecules, 20–50% are phosphorylated in serine, thus representing a meaningful fraction of the total biosynthetic pro-uPA. Although the sites of phosphorylation have not yet been determined, at least two such sites must exist; in fact plasmin cleavage of phosphorylated single chain pro-uPA yields a two chain uPA in which both chains are phosphorylated. A specific function for pro-uPA phosphorylation has not yet been identified; however, it is tempting to speculate that, as in many other cases, phosphorylation may affect the activity of the enzyme, its response to inhibitors or the conversion of pro-uPA zymogen to active two-chain uPA. This would represent an additional way of regulating extracellular proteolysis, an important pathway involved in both intra- and extravascular phenomena like fibrinolysis, cell migration and invasiveness.

Receptor-mediated internalization and degradation of urokinase is caused by its specific inhibitor PAI-1.

The receptor for urokinase plasminogen activator (uPA) has been previously shown not to internalize its ligand, but rather to focalize its activity at the cell surface, allowing a regulated cell surface plasmin dependent proteolysis. The receptor in fact binds the proenzyme pro-uPA and allows its very efficient conversion to the active two chains form. Receptor bound active uPA can also interact with its specific type 1 inhibiror (PAI-1) which is therefore able to inhibit the cell surface plasmin formation. In this paper we show that the uPA-PAI-1 complex bound to the uPA receptor is internalized and degraded. U937 cells were incubated at 4 degrees C with labeled uPA-PAI-1 (and other ligands), the temperature then raised to 37 degrees C and the fate of the ligand followed for 3 h thereafter. The uPA-PAI-1 complex was internalized into the cells (i.e. could not be dissociated by acid treatment) and thereafter degraded (i.e. appeared in the supernatant in a non TCA-precipitable form). Other ligands (free uPA, ATF and DFP-treated uPA) were not internalized nor degraded. The degradation of the uPA-PAI-1 complex is preceded by internalization and is inhibited by chloroquine, an inhibitor of lysosomal protein degradation. These data suggest the existence of a cellular cycle of uPA. After synthesis pro-uPA is secreted, bound to the receptor and activated to two chain uPA. On the surface, uPA can activate surface bound plasminogen to produce surface bound plasmin. In the presence of PAI-1 uPA activity is inhibited and plasmin production interrupted, while the uPA-PAI-1 complex is internalized and degraded.