The urokinase receptor

Fibrinoly.sis(l9!94) 8, Suppl. 1, 172-181 01994 Longman Group Ltd

The Urokinase Receptor

J.-D. Vassalli

The present review was written with the aim of providing a personal perspective on cellular aspects of extracellular proteolysis, with particular emphasis on the spatial control of urokinase-type plasminogen activator activity. In this context, the demonstration, some 10 years ago, that this enzyme can bind with high affinity to a cell surface site, the urokinase receptor, has helped to better understand the variety of mechanisms that cooperate to achieve the fine spatial regulation of extracellular proteolysis required to accommodate a degree of tissue plasticity while maintaining the overall stability of tissue architecture. Recent results on other possible roles of the urokinase receptor, in particular in signal transduction, will also be discussed in an attempt to reconcile the diverse functions that are mediated by urokinase and its cellular binding site.

EXTRACELLULAR

cations of their surroundings. Extracellular proteolysis is required for plasticity of tissue structure and function, it is indispensable for maintenance of fluidity of the extracellular milieu, in intercellular spaces as well as in the vascular compartment, in ductular structures and in body cavities. Inadequate extracellular proteolysis, either insufftcient or excessive, can be highly detrimental to tissue architecture and function. In view of this, it is probably not surprising that a multiplicity of mechanisms has evolved that control the timing, the intensity and the localization of this biochemically irreversible process. Dysregulations of extracellular proteolysis are primary or accessory events in the pathogenesis of many diseases, and research efforts aimed at a better understanding of the processes involved are likely to provide novel or improved therapeutic approaches. The progress in thrombolytic therapy is just one of the promising developments that have arisen from a more detailed understanding of extracellular proteases and of the molecular and cellular mechanisms responsible for controlling their activity.

PROTEOLYSIS

Extracellular proteolysis is one of the multiple ways whereby a cell can influence its environment. Enzymatic systems capable of degrading components of extracellular matrices or of activating precursors or latent forms of growth factors can play a critical part in the biology of cells within tissues. The progressive identification and characterization of such enzymatic systems has revealed the variety of biological phenomena that rely on extracellular proteolysis. From the earliest stages of embryogenesis, i.e. fertilization and postfertilization reactions, through the determination of developmental axes, the implantation of the mammalian embryo, the formation of blood vessels and the migration of cells during organogenesis as well as in the adult, to pathogenic and potentially lethal alterations of normal tissue function, extracellular proteases are major players in the dynamic interactions between cells and their environment. In fact it is not unlikely that, at one or multiple stages of their life cycle, all cells of the organism rely on extracellular proteolysis to catalyze modifi-

J.-D. Vassalli, Department of Morphology, Medical School, 121 I Geneva 4, Switzerland.

University

THE PLASMINOGEN SYSTEM

ACTIVATORS / PLASMIN

Among the proteolytic cascades active in the extracellular environment, the plasminogen activators (PA)/plasmin system’ is perhaps that which has received the most attention, Initially perceived as involved primarily in fibrinolysis, because of the extremely broad spectrum of physiological and pathological circumstances in which it is expressed, this system has progressively become widely accepted as being more generally implicated in the turnover of additional extracellular constituents besides fibrin. Plasmin can directly cleave many components of extracellular matrices, and it can also catalyze the degradation of plasmin-resistant substrates such as native collagens through the activation of zymogens of collagenolytic enzymes; the PA/plasmin system is thus capable of playing a predominant role in extracellular proteolysis throughout the organism. A possible limitation to this unifying view is that plasminogen, a

of Geneva

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Fibrinolysis

plasma protein secreted by hepatocytes, might not be present in certain extracellular compartments that are separated from blood plasma by anatomical boundaries impermeable to proteins. For instance, the central nervous system is isolated from plasma by the blood-brain barrier and seminiferous tubules are limited by the blood-testis barrier. Similarly, early embryos may not have plasminogen available before hepatic differentiation. This hypothetical limitation may however have to be reconsidered: plasminogen is present in the yolk of the chicken egg,* and there is growing evidence in favour of extrahepatic sites of plasminogen synthesis. This was initially demonstrated in the case of seminiferous tubules, shown to synthesize a protein indistinguishable from plasminogen3 and more recently the possibility of plasminogen synthesis in the central nervous system has been supported by the finding that microglial cells contain plasminogen mRNA and synthesize the protein, .4,5 furthermore, plasminogen mRNA can be identified in preparations of total murine brain RNA63 In addition to being very broadly, perhaps ubiquitously, expressed, and therefore of potential relevance in many different biological contexts, the PA/plasmin system can also serve as a paradigm to illustrate the complexity of extracellular proteolytic cascades. In all mammalian species studied, two PAS have been identified, tissue-type PA (tPA) and urokinase-type PA (uPA). They are the product of related genes belonging to the serine protease gene family. In the context of the present review, the most relevant difference between the two enzymes resides in structural determinants present in the non-catalytic regions of the proteins that are responsible for the differential targeting of the two enzymes to distinct regions of the extracellular environment; this will be described in more detail below. The two PAS share their major substrate, plasminogen, as well as specific inhibitors (PA inhibitors, PAIs) that are part of the serine protease inhibitors (serpin) family. One striking aspect of the PA/plasmin system is the extremely wide range of hormones, growth factors and environmental circumstances that can influence, in one or another cell type, the levels of expression of PA and PA1 genes. In most cases studied, regulation is at the level of transcription of the genes, although exceptions to this rule, i.e. instances of translational control of tPA mRNA (in mammalian oocytes’), and of regulated secretion of tPA (by endothelial cells8), indicate that multiple mechanisms can be employed to ensure temporally adequate expression of PA-catalyzed extracellular proteolysis. One intriguing question raised by the existence of two PA genes in mammals is whether the two enzymes play distinct roles. Some cell types produce only one PA, either tPA or uPA, whereas other cells produce both. No obvious and non-controversial pattern has emerged from comparisons of circumstances in which it is one or the other enzyme that is expressed. The most generally accepted view is that tPA is preferentially expressed in cases where fibrinolysis is required, while uPA production may be related to cell migration. However, in at

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least one case, that of ovarian granulosa cells, one enzyme (uPA) is produced in one animal species (the mouse) and the other enzyme (tPA) is produced in a closely related species (the rat).9 This suggests that the two enzymes may in fact play the same role, and that they can therefore ‘replace’ each other. In support of such an idea is the existence of only one PA (most comparable to mammalian uPA) in birds.” If the two PAS are exchangeable, why are there two at least in mammals, what evolutionary advantage would have derived from the duplication of an ancestral PA gene? One possibility is that the advantage does not reside in the protein-encoding region of the genes but rather in their regulatory sequences: two genes with different control elements probably represent a significant enrichment in the potential for transcriptional or post-transcriptional regulation, irrespective of whether the two gene products are functionally equivalent. In other words it may be in their non-coding regions that rests the key to the enigma of the two PAS. Alternatively, or in addition, the remarkable differences in binding to cell surfaces and to components of extracellular matrices between the two PAS (see below) suggest that the sites of PA-catalyzed proteolysis in the extracellular milieu may differ according to whether tPA or uPA is being produced.

THE UROKINASE-TYPE PA Like other members of the serine protease gene family, uPA is a two-chain enzyme. The amino-terminal A chain is characterized by two structural determinants, a ‘growth factor’ module and a kringle. The growth factor domain (GFD) was initially described based on a degree of resemblance of this part of the protein with EGF, in particular with respect to the position of cysteine residues. ’’ Recent structural studies have confirmed the proposed EGF-like folding of the amino-terminal portion of the A chain. ‘* Interestingly, this part of the molecule undergoes an unusual post-synthetic modification, the addition of a fucose residue.” The kringle domain resembles similar structures present in other members of the gene family. The major structural differences between tPA and uPA reside in their A chains: tPA has additional domains, a ‘finger’-like structure and a second kringle domain. The catalytic B chain of uPA endows the enzyme with the properties of an arginineesterase. A cleaved form of uPA, consisting of the intact B chain and a small carboxy-terminal region of the A chain, has a substrate specificity and specific catalytic activity comparable to that of the intact enzyme; thus the amino-terminal part of the A chain, including the growth-factor and kringle domains. is not involved in the enzymatic properties of uPA. As is the case for most serine proteases, and in contrast to tPA, the single chain form of uPA (pro-uPA) has only very low catalytic activity.14 A search for enzymes or circumstances capable of activating pro-uPA has uncovered a few candidates, the best of which is probably plasmin. As will be detailed below, it may be

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through the assembly on the cell surface of a multimolecular complex involving uPA, plasmin and their zymogens that the most catalytically-efficient system of plasmin generation is achieved. uPA was initially identified in urine, and it is indeed produced by epithelial cells lining discrete regions of the nephron. is However, the enzyme can be produced by many cell types, in essentially all tissues. Overall, expression of the uPA gene appears to occur preferentially in two different biological contexts: in certain types of epithelial cells and in cells engaged in migratory processes. In addition to kidney epithelial cells, cells from other organs comprising alveolar, tubular or ductular structures, for instance in the genital tract, the mammary gland, and other exocrine tissues, can produce uPA. The presence of the enzyme in urine suggests that it is secreted apically, at least by certain normal epithelial cells, and that its role, if any, may be exerted in the luminal compartment. However, in the absence of pulsechase experiments that would allow to follow the fate of newly synthesized enzyme molecules, it remains to be determined what proportion of the enzyme is secreted apically or baso-laterally. Another point that needs to be clarified is whether there are structural determinants in the (pro)uPA molecule responsible for its secretory targeting into urine. In addition to epithelial cells in the nephron, cells from the caudal part of the epididymis, the vas deferens and the seminal vesicle release uPA in the lumen of the male mouse genital tract. I6 It is at present unclear whether apical secretion of uPA is a constant for all epithelial tissues that produce the enzyme. Another important question to be solved in this respect is the possible dysregulation of apical targeting of uPA in the context of malignant progression of epithelial tumours; such a dysregulation could be involved in endowing epithelial cells with the capacity of degrading their basement membrane, a critical phase in the invasive process. The uncertainties summarized here make it difficult to come to clear conclusions with respect to the role of uPA in epithelial tissues; among the hypothesis that can be proposed for the enzyme in the lumen is a role for uPA-catalyzed proteolysis in the maintenance of tubular patency, similar to the role postulated for tPA in the vascular compartment. In the case of the male genital tract, uPA released by epithelial cells could be important for the biology of sperm cells. Cells that are engaged in migratory processes must overcome anatomical boundaries such as vascular and epithelial basement membranes. It therefore appears reasonable to consider that they must have the capacity of clearing a path through which they can proceed. Among specific examples of migratory processes involving uPA-producing cells, the implantation of the mammalian embryo is one of the most spectacular: invasive trophoblasts produce uPA, and the proteolytic activity thus generated may help the blastocyst penetrate the endometrium and carve out a space for the embryo to develop. Inflammatory cells, polymorphonuclear leukocytes and monocytes/macrophages, are also

highly migratory and invasive: from their sites of production in the bone marrow, through the circulation, to the tissular sites of inflammation, they have to cross the vascular endothelium and other barriers. Inflammatory agonists, including certain cytokines, trigger uPA production by these cells, whereas anti-inflammatory agents, such as glucocorticosteroids, decrease expression of the uPA gene. I7 Another type of cell endowed with striking migratory properties, both in the embryo and in the adult, is the vascular endothelial cell. Under the influence of angiogenie factors, in particular basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF), capillary endothelial cells are induced to breakdown their underlying basement membrane, to migrate and invade the extracellular matrix, and finally to reconstitute a continuous network of branching capillaries. Angiogenesis is a key phenomenon during embryonic development, and the same set of cellular events occurs cyclically in the adult female, during formation of the corpus luteum and development of the endometrium. Angiogenic agents cause endothelial cells to produce uPA, and angiogenic behaviour correlates with uPA-catalyzed proteolytic activity.‘8-22 Interestingly, while endothelial cells also produce tPA, perhaps constitutively, it is primarily uPA that is affected by angiogenic agents, supporting the notion that it is the latter enzyme that is preferentially associated with cell migration. In vitro studies of angiogenesis have also revealed one critical aspect of extracellular proteolysis in physiological circumstances: to be most effective in the context of cell migration, the proteolytic remodelling of the extracellular matrix during cell migration should involve the localized destruction of its constituents, in a way that avoids deleterious damage to the overall structure of the invaded tissue. If the underlying scaffold of the tissue is destroyed by proteases acting in an uncontrolled way, cell migration is not possible. An illustration of this principle is provided by endothelial cells induced to invade a three-dimensional matrix composed of fibrin: the PA-catalyzed proteolytic activity generated by the cells destroys the matrix, and no new capillary-like structures can form. By contrast, if proteolysis is kept under control by the inclusion of an inhibitor of plasmin in the medium used for culture, endothelial cells form tubes in the fibrin matrix.23 Work on endothelial cells, and also on many other cell types, has revealed that the production of PAIs can be triggered by the same modulators that induce the synthesis of PAS. Interestingly, the precise timing of enzyme and inhibitor production often does not coincide, so that at different times the net overall result of the protease-antiprotease balance can be proteolytic or antiproteolytic.24 The importance of such a fine tuning of the proteolytic balance is illustrated by the aberrant morphogenetic behavior of hemangioma-derived endothelial cells, that fail to produce a capillary-like network when placed in a three-dimensional matrix; correcting the excessive uPA-catalyzed proteolytic activity expressed by these tumour-derived cells results

Fibrinolysis

in a restoration of their capacity instead of large cystic structures.25

to form fine tubes

THE UPA RECEPTOR The studies summarized above, and many others, have revealed the importance of controlling extracellular proteolysis and the impressive spectrum of regulatory processes that are brought into play to modulate with precision the timing of uPA-catalyzed proteolysis. However, spatial control of this potentially damaging process would also appear to be critical, since diffusion of a secreted protein such as uPA may result in degradation of inappropriate substrates. Interestingly, both tPA and plasminogen can bind to insoluble extracellular macromolecules, such as fibrin, fibronectin or laminin,2h and this binding can limit their diffusion and/or target their activity to specific substrates. In the context of studies aimed at following the fate of uPA following its secretion by monocytes and related cell lines, and in particular its interaction with PAI-2, we observed the binding of exogenously-added ‘251-labeled enzyme to cells of the U937 line. Interestingly, while the ‘251-labeled uPA preparation used in this experiment contained both the full-size Mr 55 000 enzyme and its Mr 33000 cleavage product, only the intact protein appeared to bind to the cells. Thus, in one experiment, we had revealed a novel property of uPA, i.e. to bind to cell surfaces, and had a hint as to the region of the molecule involved in this binding, i.e. the amino-terminal fragment that is missing in the Mr 33 000 form of the protein. Further experiments allowed us to better characterize the kinetics of UPA binding: Scatchard analysis revealed a single class of binding sites, with an affinity of 4x10-‘eM, i.e. in the range of the plasma concentration of uPA, and competition studies with other proteins, including tPA, did not uncover alternative ligands. These results led us to propose the existence, on monocytes and related cells, of a plasma membrane binding site, a receptor, specific for this enzyme. Functional studies revealed that receptor-bound uPA was not rapidly endocytosed or degraded and that it maintained proteolytic activity, suggesting that the receptor may serve to localize plasminogen activation near the cell surface.” The identification of a cell surface receptor for secreted UPA introduced a new concept relevant to the control of extracellular proteolysis. While transcriptional modulations of protease and antiprotease gene expression probably play major parts in determining the timing and extent of extracellular proteolysis, the restricted diffusion afforded by binding of the enzyme to the cell surface could serve both to limit the pericellular zone affected and to concentrate and focus protease activity to the immediate environment of the cell, or perhaps to subdomains of the cell surface. This appeared as an optimal configuration to facilitate cell migration, in that the proteolytic activity generated could cause the localized destruction of the peri-cellular

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matrix, perhaps in front of the leading edge of migrating cells, without destroying the tissue scaffold. This concept also reinforced the notion that uPA, now perceived as an enzyme acting near the cell surface, might be the PA most directly involved in cell migration.

THE RECEPTOR-BINDING DOMAIN OF UPA The initial studies summarized above had revealed the importance of the amino-terminal region of the A chain of uPA in its binding to the receptor. This was confirmed by the demonstration that the isolated amino-terminal region could bind to the receptor, with an affinity comparable to that of intact uPA.** With the help of synthetic peptides, it was then possible to identify the GFD, and especially the amino acids between positions 12 and 32, as being directly implicated in the binding.29 Recent solution structure studies of the GFD of UPA have revealed key structural differences with homologous domains in growth factors such as EGF or TGFa, accounting for the specificity of uPA binding.12 The GFD domain of mouse UPA differs in several positions from its human counterpart. These differences account for a marked species-specificity of uPA binding: mouse uPA binds to mouse cells, but not to human cells, and conversely human UPA binds to human cells only.30This species specificity is an obstacle to studies of expression of the UPA receptor on murine cells, since human UPA cannot be used for this purpose: murine uPA is more difficult to obtain than the human enzyme and, furthermore, iodination of the murine protein appears to affect its binding, perhaps because of the presence of a tyrosine residue in its GFD. However, the species specificity has also been used to advantage, in in vivo experiments demonstrating the role of paracrine interactions between uPA and its receptor in tumour invasion.

CHARACTERIZATION RECEPTOR

OF THE uPA

Analysis of the UPA binding site by chemical cross-linking of radiolabelled ligand with disuccinimidyl suberate or with formaldehyde revealed a protein of Mr -45000.‘” Detergent partitioning using the Triton X114 heat-induced phase separation technique demonstrated the amphiphilic nature of the receptor: unoccupied receptor, endogenous uPA-receptor complexes, receptor-bound ‘251-uPA and cross-linked ‘251-uPAreceptor complexes all partitioned in the detergent phase, while the free ligand was aqueous.30 The crosslinking and detergent partitioning procedures have proven useful to reveal UPA receptors in a variety of cell types. The biochemical characterization and purification of the UPA receptor demonstrated that it is a glycoprotein of Mr 55 000-60000,“~ 32 and that its amphiphilic nature is due to a glycerophospholipid (GPI) anchor.3’ In this

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Receptor

context it is interesting to note that the receptor is deficient on peripheral blood leukocytes from patients with paroxysmal nocturnal hemoglobinuria,34 which have a defect in the mechanism of GPI-anchoring of membrane proteins. Cloning and sequencing of the human uPA receptor cDNA revealed the domain structure of the protein,35 and allowed the identification of its ligand binding region. 36*37Different forms of the uPA receptor and of its mRNA have been identified in different circumstances,38,39 including cleaved forms lacking the ligand binding domain40 that can be generated by urokinase and/or plasmin cleavage, and soluble forms lacking at least part of the GPI anchor.41 All these structural aspects are covered in other reviews in the same issue of this journal, and they will therefore not be further discussed here. The possibility that the uPA receptor may be a multisubunit cell surface protein has not been systematically explored. Cross-linking of cell surface-bound ‘251-uPA with disuccinimidyl suberate reveals additional bands than the Mr -100000 complex with the characterized another cross-linking agent, receptor; however, formaldehyde, does not yield these larger Mr species.” Immunoprecipitation of biosynthetically labelled cell proteins with an anti uPA receptor antibody co-purifies a Mr 38 000 polypeptide, that is phosphorylated on tyrosine residue(s) when the receptor is occupied by uPA.~* This suggests that the uPA receptor can associate with other cellular proteins, a finding of relevance to the modulation of its expression on the cell surface and/or to its possible role as a signal transducing receptor (see below).

EXPRESSION

OF THE uPA RECEPTOR

Initially discovered on human blood monocytes and related cell lines,27,28,43 the uPA receptor has been identified on a number of additional cell types. Amongst these are other circulating cells, including polymorphonuclear leukocytes, 44 lymphocytes and natural killer cells,45,46 vascular endothelia147,48 and smooth muscle cells,49 alveolar macrophages5’ myogenic cells5’ keratinocytes,52*53 placental trophoblasts.54 The biochemical characteristics of the receptor on these different cells appear very similar, with, however, differences in the pattern or extent of glycosylation. A more complete view of the cell types that can express the uPA receptor in vivo will be aided by the availability of reagents, nucleic acid probes and antibodies, allowing its study in experimental animals.- 38,55,56In the mouse, for instance, binding of uPA to spermatozoa during their transit through the male genital tract suggests that these cells express uPA receptors: in vitro studies have revealed that spermatozoa collected from the testis and the caput epididymis can bind exogenous murine, but not human, uPA, i.e. following a species-specificity of binding similar to that found for other cell types bearing characterized uPA receptors. I6 A molecular characterization of the uPA binding sites on male gametes is however

required to determine whether they correspond to the typical uPA receptor. Expression of uPA receptors by malignant cells would be in accord with the notion that cell surface localization of uPA activity is important for cell migration, and therefore for invasion and metastasis. Only few published studies have as yet addressed this issue, but the available reports indicate that malignant cells in at least a fraction of human tumours do express the uPA receptor. This was first shown for cancer cells at invasive foci of human colon adenocarcinomas,57,58 and more recently for breast carcinomas59-6’ and advanced melanomas.62 Considerable variability in the levels of expression were observed within individual tumours, and between tumours of different patients. More extensive studies will be required to determine if uPA receptor expression may have a prognostic value for certain human cancers.

REGULATION EXPRESSION

OF uPA RECEPTOR

Expression of the uPA receptor is a highly dynamic parameter of cell phenotype: receptor number, receptor affinity and receptor distribution can all be modulated on a given cell type. This is best illustrated for monocytes and related cells. Differentiation, either ‘spontaneous’ during in vitro culture or following exposure to phorbol myristate acetate, is accompanied by a progressive increase of up to 40-fold in receptor number.28,43 This increase can be accounted for by a parallel increase in receptor mRNA, and, at least in part, in receptor gene transcription. 63 Concomitantly, there is a progressive -lO-fold decrease in receptor affinity.64 The molecular basis for this change in affinity has not been completely elucidated, but it could correspond to a change in the receptor glycosylation pattern, which has been shown to influence affinity. 65 It is intriguing that the changes in receptor number and affinity appear to have opposing effects on the amount of uPA that can be bound to the cells; however, this would depend on the concentration of uPA in the cellular micro-environment, and at relatively high concentrations of uPA, which might be achieved around cells in tissues, the increase in the amount of cell-bound uPA would be substantial, despite the lower affinity of the receptor. Distribution of the uPA receptor on ‘resting’ monocytes, as revealed by cellular autoradiography of bound ‘251-uPA, appears relatively random. When these cells are placed in a chemotactic gradient, however, there is a rapid redistribution of the receptor to the leading edge of the migrating cells. 66 This striking polarization of receptor expression is in accord with the notion that the uPA receptor serves to focus plasminogen activation to the region of the plasma membrane were extracellular proteolysis is most likely required for migration. This is reminiscent of the localization of plasma membrane uPA at focal contacts between fibroblasts and their underlying substratum,67,68 where the enzyme might

Fibrinolysis

serve to loosen cell-matrix contacts for migration, or perhaps during the cell cycle. Modulation of UPA receptor expression has also been observed in other cell types. For instance, the Mo3 antigen, which has been shown to correspond to the UPA receptor, is an activation antigen of human T lymphocytes as well as of monocytes.46 Another cell type in which modulation of uPA receptor has been studied in some detail is the endothelial cell. When induced to migrate in response to angiogenic signals, in vivo and in vitro, these cells express increased levels of uPA.~’ This is mediated by cytokines, in particular bFGF, acting at least in part in an autocrine manner, and it involves a rapid and pronounced upregulation of both uPA and uPA receptor expression. 19.69 In these cells also, the increase in receptor number occurs in parallel with a decrease in receptor affinity. Thus, under conditions in which they are to degrade their underlying basement membrane and to invade the surrounding extracellular matrix, cells increase their surface uPA activity by making more of the enzyme and expressing more plasma membrane uPA receptors; this supports the notion that the uPA/uPA receptor interaction could be an important parameter of cell migration.

ROLE OF THE UPA RECEPTOR: uPA-CATALYZED PROTEOLYSIS

FOCUSING

Throughout this review, the idea that the uPA receptor could serve to focus plasminogen activation and plasmin activity has been extensively discussed, and it will not be dwelled upon further. However, some additional aspects relevant to this hypothesis should be mentioned here. The zymogen pro-uPA binds to the receptor with an affinity comparable to that of the active enzyme;“,” binding of pro-uPA does not appear to affect its conformation in a way that would render it catalytically active, but neither does it prevent its proteolytic activation. Thus, binding of pro-uPA endows the cell surface with a reservoir of proteolytic activity that can be rapidly recruited. Receptor-bound uPA is only marginally less susceptible to PAIs than is the free enzyme, and it is therefore not protected from these inhibitors.‘* Complexes between uPA and PAIs bind to the receptor. However, by contrast to the free enzyme that can remain on the cell surface for many hours, uPA/PAI complexes are rapidly cleared by endocytosis and then degraded.“.‘” Endocytosis appears to involve the interaction of the complexes with a different receptor, a,macroglobulin receptor/LRP.74 Plasminogen and plasmin can bind to cell surfaces through as yet incompletely defined interactions, involving the lysine-binding sites on the kringles.75 The simultaneous presence of pro-uPA, uPA, plasminogen and plasmin on the cell surface results in the

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assembly of a highly efficient system of plasmin generation.76-78 In particular, the activation of pro-uPA to uPA is markedly accelerated in this configuration. . While soluble plasmin is extremely rapidly inactivated by its specific serpin-class inhibitor a,-antiplasmin, association with plasma membranes protects the enzyme from this macromolecular inhibitor.75 Taken together, these observations provide strong support for the notion that the PA/plasmin system is designed in part to function in close association with the cell surface. In this context, the uPA receptor would obviously play a determining role. One particularly appealing aspect of this system, in which cell surface proteolytic activity involves binding of proteases to cell surface determinants, is that it allows paracrine-type interactions: the (pro)enzymes can be synthesized and secreted by one given set of cells, but can also serve other cells in the tissue to acquire the capacity to catalyze extracellular proteolysis in their own micro-environment. In this context, it is interesting to recall the situation described in the case of spermatozoa, that bind uPA produced by epithelial cells of the male genital tract,” as well as the observations reported for certain tumours, in which the uPA receptor may be present on the tumour cells themselves while uPA appears to be synthesized by cells in the stroma. Given that the uPA receptor may be involved in the spatial control of extracellular proteolysis, what are the candidate substrates for the cell surface PA/plasmin system? Components of extracellular matrices can be directly degraded by plasmin, and certain zymogens of the matrix metalloprotease family are activated by plasmin; hence a direct or indirect catabolic effect on the extracellular matrix appears highly likely. Another type of effect that is of great potential interest relates to the biology of growth factors. The latent forms of TGF-Bs, and probably of certain other members of the TGF-IJ family, are activated by plasmin; in a cellular system, the presence of uPA receptors was found to increase greatly the plasmin-mediated activation of latent TGFl3.79.8oHepatocyte growth factor/scatter factor, a mitogen, motogen and morphogen for a variety of cell types, is a structural homologue of plasminogen but without enzymatic activity or potential; the single-chain precursor is biologically inactive, and its extracellular cleavage by uPA is required for maturation to the active form.8’ Basic-FGF associates with heparan sulphate proteoglycans (HSPG) in the extracellular matrix, and PA-mediated proteolysis releases biologically active bFGF - HSPG complexes from the matrix.s2 Thus the PA/plasmin system, acting on masked or precursor forms of growth factors, could play a part in the cytokine-mediated control of tissue function. The uPA receptor, by focusing the activity of this system, could favour the localized activation or uncovering of growth factors, thereby restricting their activity to a limited population of cells, or perhaps even to sub-domains of individual cells. A vast body of correlative and experimental evidence

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suggests that uPA expression is important for cellular invasion in vivo. However, direct evidence in favour of a role for receptor-associated uPA in this process is much more limited. Analysis of the in vivo behavior of transfected tumour cell lines has shown that co-expression of uPA and receptor is required to endow cells with maximal invasivity or metastatic potentia1.83s4 In one experimental system, saturation of the uPA receptor by mutant catalytically-inactive uPA dramatically inhibited metastasis.85 The latter model is particularly interesting, since in principle neither total uPA levels nor receptor occupancy differed between the highly metastatic cell line and its mutant uPA-expressing derivative: the only difference resided in the inability of catalytically-competent (pro)uPA to associate with the receptor, suggesting that uPA activity localized by binding to the receptor was the critical determinant indispensable for metastatisation.

ROLE OF THE uPA RECEPTOR: TRANSDUCTION

SIGNAL

A steadily increasing flow of reports describing effects of the uPA receptor or of uPA/receptor interactions that may not be directly or exclusively related to a role in focusing extracellular proteolysis suggests that alternative or additional functions should be envisioned for the receptor. In particular, there is growing evidence for an activation of signal transduction pathways by uPA receptor occupancy. Thus, the uPA-induced tyrosine phosphorylation of a receptor-associated Mr 38 000 protein suggests that uPA can act as a growth factor on monocyte-like cells by activating a protein tyrosine kinase.42 On a different type of cells, uPA and its isolated amino terminal region stimulated the formation of diacylglycerol in a manner resembling that which is activated by insulin. s6 In yet another type of cells, both enzymatically active and inactivated uPA induced a marked and transient increase in c-f&r mRNA abundance, that could be prevented by preincubation of the cells in the presence of inhibitors of protein tyrosine kinases.*’ At least three different types of effects of uPA on cultured cells have been reported to be independent of uPA’s enzymatic activity and have therefore been attributed to a possible signalling capacity of the uPA receptor. They include a mitogenic response of certain cell lines,88 an autocrine interaction involved in differentiation and adhesion of myeloid cell lines89,90 and a requirement for the uPA receptor in endothelial cell and monocyte migration.79,9’ Some of the features of uPA and of the receptor deserve to be mentioned in the context of a possible role in signalling. ??

presence of fucose did not appear to influence binding, it was critical for the growth factor activity of uPA or its isolated amino terminal region on one cell line9*. This is the first evidence for a specific effect of fucosylation, an unusual posttranslational modification. The GPI-mode of anchoring of the uPA receptor suggests that the characterized receptor protein cannot, by itself, generate a signal on the cytosolic side of the plasma membrane. However, there is ample precedent for GPl-anchored cell surface molecules transducing signals, in particular through association with protein of a uPAtyrosine kinases. 93 The identification induced tyrosine phosphorylation42 is thus compatible with known pathways of signalling by GPI-anchored receptors. Receptor-bound pro-uPA and uPA remain on the cell surface for many hours, unless the enzyme is inactiof vated by a PAI. .66 there is no down-regulation receptor expression following its occupancy by the ligand. This needs to be taken into account when signalling through the uPA receptor is invoked. The polarization of uPA receptors on cells migrating in a chemotactic gradient66 may be viewed as compatible with a localized signalling, affecting discrete regions of the cell; if this signalling was to influence the cytoskeletal organization, for instance, the receptor could play a part in controlling cell movement.

A fucose residue has been demonstrated to be directly linked to threonine 18 of uPA, i.e. within the region of the protein involved in receptor binding.i3 While the

In summary, although evidence is accumulating that the uPA receptor is capable of signal transduction, no clear pattern of pathways involved or of effects triggered has as yet emerged. There is little doubt that these issues will attract increasing attention in the near future.

ROLE OF THE UPA RECEPTOR: TO RECONCILE APPARENTLY HYPOTHESES

AN ATTEMPT CONFLICTING

The last two sections have summarized the hypotheses that have been put forward with respect to the possible role(s) of the uPA receptor. Is a role in focusing cell surface proteolytic activity compatible with a putative signalling activity affecting intracellular reactions? Although it is possible that ultimately only one, or perhaps even neither one, of these postulated functions will turn out to be correct, they may be reconciled in at least two ways, which are not mutually exclusive. ??

The role of the PA/plasmin system in growth factor activation has been summarized above, and so has the influence of the uPA receptor in this process. Effects obtained upon the exposure of cells to inactive uPA or to uPA fragments lacking the catalytic region of the enzyme 79*88-9ocould be due not to receptor signalling but to receptor masking, with a resultant decrease in surface uPA activity and perhaps an increase in soluble uPA activity: such changes could affect the yield of active forms of certain growth factors, resulting in

Fibrinolysis

??

cellular responses to alterations in growth factor levels. In this view, all effects related to the UPA receptor could in fact be primarily caused by changes in extracellular proteolysis. A less extreme hypothesis is that some effects reported to be due to UPA receptor signalling are mediated through other growth factors. Both the focusing of UPA activity to the leading edge of migrating cells and certain of the effects that are proposed to be elicited by UPA receptor signalling, in particular those related to cellular adhesion and to chemotaxis,89-91 are relevant to one biological function: cell migration. UPA and its receptor could help cells migrate in tissues, by proteolytically clearing a path in the extracellular matrix and simultaneously by controlling, perhaps through processes of protein tyrosine phosphorylation, the cytoskeletal machinery. The localization of uPA, and therefore probably of the receptor, at focal contacts of cells with their substratum, in close proximity to intracellular sites rich in vinculin, is in accord with this possibility.67*68In this view, the uPA/uPA receptor interaction would be multifunctional, but with one ultimate goal, to contribute to directional cell migration. One appealing aspect of such a system would be particularly manifest in conditions of paracriny: binding of UPA to the receptor would simultaneously signal cytoskeletal rearrangement and endow the cell with a proteolytic potential that it may require in order to migrate. An extension of the concept of cell migration to more discrete rearrangements of the interactions between cells and their immediate extracellular environment, such as occur during the cell cycle, for instance at cytokinesis, may be appropriate in the context of this discussion.

CONCLUSIONS The impressive body of knowledge that has been accumulated in less than 10 years on the UPA receptor, in particular on its structure, will help resolve some of the questions raised in this review. Also, the recent availability of mice lacking a functional UPA gene94, prepared through the gene ‘knock out’ technology, will doubtless contribute major insights. Similarly, attempts at preparing mice lacking the UPA receptor gene will define the circumstances in which receptor expression is relevant to physiology or pathology. Understanding the UPA receptor may help manage certain human diseases. The role of UPAin tumour invasion and metastasis will perhaps be more accurately perceived when the contribution of the receptor is taken into account. It is also important to recall that the UPA receptor, initially found on monocytes, is expressed on a number of cell lineages involved in immune and inflammatory reactions. Receptor expression, or the interaction between the receptor and its ligand, could become targets for antitumour or anti-inflammatory therapy.

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ACKNOWLEDGEMENTS I am most grateful to all my colleagues from the University of Geneva Medical School with whom I have collaborated in studying the urokinase receptor. This field of investigation was initiated with D. Baccino and D. Belin, and it has benefited from the contributions of A. Estreicher, MS. Pepper, P. Ragno, J. Huarte, A. Wohlwend, J. Miihlhauser, A.-P. Sappino, A. Gos, R. Stocklin, J.-L. Carpentier, R. Montesano and L. Orci. The work from our laboratory has been supported by grants from the Swiss Fonds national de la recherche scientifique. the Sir Jules Thorn Charitable Overseas Trust and the Commission fed&ale des maladies rhumatismales.

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