The urokinase receptor

,Invited

Review

Fibrinolysis & Proteolysis (1998) 12 (4), 191-204 © Harcourt Brace & Co. Ltd 1998

The urokinase r e c e p t o r N. Behrendt, R. W. Stephens The Finsen Laboratory, Rigshospitalet, Strandboulevarden 49, Bldg 7.2, DK-2100 Copenhagen 0, Denmark

INTRODUCTION

The discovery of the urokinase receptor in 1985 ~-3 opened a new dimension in the study of extracellular proteolysis and matrix degradation. The proteolytic cascade system of plasminogen activation had long been recognized as performing a central role in these processes. 4,5 It was soon demonstrated that the novel receptor, uPAR, could localize the urokinase plasminogen activator (u-P/k) on a wide range of cell types, ~ and further to specific compartments on the surface of the cells in question, typically at sites involved in focal cellsubstratum and cell-cell contacts? It was realized that at these sites the bound ligand remained enzymatically active] and kinetic studies showed that the plasminogen activation cascade system became accelerated when the reactants were cell-bound, a,9 These observations were highly relevant for the hypothesis that the u-PA system does have an involvement in processes of pericellular tissue degradation such as those occurring in cancer invasion. As detailed below, subsequent work led to the elucidation of several aspects of the processes in which uPAR is involved and even the identification of novel roles of the receptor system, in addition to those directly related to proteolysis. The human uPAR gene ~° is situated on chromosome 19. ~ The receptor is highly expressed on the surface of activated macrophages and also occurs on various other leukocytes, being identical with the macrophage differentiation antigen M03, ~2 and also designated CD8Z ~3,14It is also found expressed, at relatively low levels, in certain normal tissues (as studied in the mouse15). Higher levels are found in various types of cancer. In cancer, the expression of uPAR seems to be part of a directed system of synthesis in which certain components of the proteolytic system are produced by cancer cells and others are produced by stromal cells, leading to assembly of the Received: 27 March 1998 Accepted: 24 April 1998 Correspondence to: Niels Behrendt, Tel. + 45 35 45 57 08, Fax: + 45 35 38 54 50, E-mail. [email protected]

complete system at the invasive front. The expression pattern of uPAR on normal and malignant cells has been reviewed with special emphasis to this tumor-stroma interaction 16 and will not be treated in detail here. The number of uPAR molecules per cell varies markedly, with values from a few thousands to several hundred thousands being reported. 6,~7Ligand binding is reversible with a Kd in the range of 0.1-1 nM and occurs with active u-PA as well as the zymogen pro-u-PA. 1,z17-2~ The interaction allows the proteolytic activation of the bound zymogen ~8 at the very site where plasminogen activation also occurs (see below). More recently, it has become clear that uPAR also binds to vitronectin in a manner that allows this ligand and u-PA to be bound simultaneously. 22 We will focus on the structure of the receptor protein, the structure-function relationship of its binding reactions, the enzymatic consequences for u-PA and pro-u-PA of binding to uPAR, and the presumed role of uPAR in cancer invasion and metastasis. In addition to the interactions covered here, certain other functions of uPAR have been identified which are treated in separate reviews in this volume. The reader is referred to these articles for discussion of such aspects as the function of uPAR in signal transduction, the role in ligand internalization and the functional interplay between uPAR and integrins. PROTEIN STRUCTURE

The human 23 and murine 24 uPARs have been purified and characterized and uPAR-encoding cDNAs from human, 25 murine, 26 rat 2z28and bovine 29,3° cells have been cloned and sequenced. The human cDNA encodes a 335 amino acid residue polypeptide, 25 including an NH zterminal signal peptide of 22 residues and a COOHterminal segment that is removed during processing for glycolipid membrane anchorage (see below). The mature receptor protein probably comprises 283 residues. 3',32 The uPAR protein (Fig. 1) is composed of three extracellular domains 21,33 with a low but discernible mutual sequence homology. 33,34The consensus sequence pattern 191

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CHO

CHO

CHO

I

CHO

3 Fig. 1 The molecular structure of the uPAR molecule. (A) Domain composition and posttranslational modifications, uPAR is formed by a single polypeptide chain which folds into three extracellular domains (D1, D2, D3). The protein has no intracellular part but is anchored in the plasma membrane by a COOH-terminal GPI-moiety (zigzag line). The scissors symbolize the protease-sensitivity of the linker region between D1 and D2; a cleavage in this region inactivates the binding potential towards uPA as well as vitronectin. The number of SSbridges in each domain is indicated. The disulfide connectivity pattern assumed to be general for the uPAR/Ly-6 domain family members is 1-5, 2-3, 4-6, 7-8, 9-10 (referring to the order of the cysteines in the sequence), except that D1 of uPAR has only the first four of these disulfides.39 Four N-linked carbohydrate side chains are present (CHO) and this carbohydrate constitutes a substantial part of the apparent molecular weight of the protein. (B) Over-all folding pattern of a member of the uPAR/Ly-6 domain family. The polypeptide backbone structure of CD59 (single extracellular domain) is shown as derived from NMR data. The NH2- and COOH- termini are indicated (N and C, respectively). The three loops are numbered according to sequence. The secondary structures include mostly beta structure (lighter shading), comprising one two-stranded (left) and one three-stranded sheet (centre and upper/right). A helical segment is present in loop 3 (dark shading, lower right). In a hypothetical, aligned structure of D1 of uPAR, Tyr~7which is a critical residue in the binding of uPA would be situated in this helical region. 85The three-dimensional model of CD59 is based on data deposited by C. M. Fletcher, R. A. Harrison, P. J. Lachmann and D. Neuhaus in the Brookhaven National Laboratory Protein Data Bank; structure code 1CDQ. The Rasmol program, version 2.6, was used for representation of the polypeptide backbone structure.

characterizing the domains of uPAR is refound in a group of proteins currently referred to as the uPAR/Ly-6 protein family. In addition to uPAR and the murine Ly-6 differentiation antigens, this family comprises the complement component CD59 (membrane inhibitor of reactive lysis; MIRL), the murine thymocyte / B-cell antigen ThB (with the homologous Ag E48 on h u m a n keratinocytes) and HVS-15 (a gene product of herpesvirus saimiri). 33-38 uPAR, however, is the only family m e m b e r identified so far which has more than one domain,* The disulfide pairing pattern of the first domain of uPAR 39 and that of CD594° have been solved, and the patterns identified clearly confirm the relatedness of these proteins (Fig. 1A). The three-dimensional structure of uPAR is not known but the NMR-based structure of the single domain of

*A recent report describes a cDNA encoding a bone protein, RoBo-1, which appearsto be composedof two domainsof the present domain type (Noel L S, ChampionB R, HolleyC L, et al, RoBo-l, a novelmemberof the urokinaseplasminogenactivatorreceptor/CD59/Ly-6/snaketoxin Familyselectivelyexpressedin rat bone and growth plate cartilage.J Biol Chem 1998; 273: 3878-3883). Fibrinolysis & Proteolysis (1998) 12(4), 191-204

CD59 has been solved, 41,42 and is assumed to resemble that of the individual domains of uPAR. The CD59 domain is composed of three loops that emanate from a disulfide cross-linked core. It contains mainly beta structure except for a short helical segment in the third loop (Fig. 1B). A group of snake neurotoxins, the (z-neurotoxins, share the disulfide pairing pattern of the uPAR/Ly-6 proteins and the three-dimensional structure of CD59 actually has a clear similarity to the solved ~-neurotoxii-i structures, suggesting that the extensive structural knowledge on the latter group of proteins can aid the understanding of the uPAR/Ly-6group. 43 uPAR is anchored in the plasma membrane through a glycosyl-phosphatidylinosityl (GPI) moiety. 31 The protein thus has no intracellular domain and no membranespanning peptide segment, a property central to any understanding of the membrane distribution and to the discussion of functions such as cellular signal transduction (see below). The GPI anchor is coupled covalently during a post-translational processing event in which the COOHterminal part of the nascent polypeptide is removed and the GPI structure becomes attached to a novel © Harcourt Brace & Co. Ltd 1998

The urokinase receptor

COOH-terminus which, in the case of human uPAR, is most likely GlY283as judged from mutagenesis studiesY The primary structure of the human uPAR contains five potential N-glycosylation sitesy of which four have been found to be utilized.44Indeed uPAR is heavily glycosylated, giving rise to a pronounced molecular heterogeneity, and leading to a substantial shift in electrophoretic mobility upon enzymatic deglycosylation.23 A number of molecular studies, including some of those cited herein, have been done using a geneticallyengineered, water-soluble uPAR variant (amino acid residues 1-277) which is devoid of the GPI moiety.45 This protein has the same ligand-binding properties as wild-type uPAR,46 and in the following it is considered functionally equivalent at the molecular level to the GPI-containing uPAR. STRUCTURAL BASIS FOR THE REACTION B E T W E E N u-PA A N D u P A R

In the u-PA molecule, the receptor binding site resides in the NHE-terminal growth factor-like domain (GFD). 47 The low molecular weight form of u-PA (LMW-u-PA which retains proteolytic activity but which is devoid of GFD and the kringle domain) thus does not bind to uPAR? ,2 Within the GFD, the omega-loop (residues 22-28) 4s,49 contains residues critical for binding, as shown by chemical protection analysis (involvement of Tyr2435) and sitedirected mutagenesis studies. 5° The delineation of uPAR's binding determinants towards u-PA is more complicated. The first domain of uPAR (D1) is critical for binding and has a slight binding capability of its own,33 but quantitative analyses have shown that its ligand affinity is more than 2000-fold lower than that of the intact three-domain uPAR.46Within D 1, Tyrs7, situated in the presumed third loop, has been identified as part of the molecular complex interface. 35 The N-bound glycan of D 1, situated on Asn52, also seems to have some importance for binding, since a genetically engineered uPAR variant without this glycan has a somewhat lower ligand affinity than the wild-type protein? 1 The very low ligand affinity of D1 in isolation as compared to the complete three-domain protein would suggest that the other domains of the uPAR molecule also contribute to the binding to u-PA. Indeed, studies by chemical 'zero-length' cross-linking point to the existence of ligand contact areas both within and outside D1. 2~ A uPAR-binding peptide which competes with u-PA for binding also interacts with determinants in both D1 and D(2+3), as shown by photoaffinity labelling.52 In addition, however, non-covalent interactions between the domains may be important in governing an active conformarion of D1 itself. 46 Removal of the third domain (D3) also leads to a pronounced loss of ligand affinity, resulting © Harcourt Brace & Co. Ltd 1998

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from a strongly increased dissociation rate for the complex formed by u-PA with the remaining part (D(l+2)) of uPAR.21 C L E A V E D A N D S O L U B L E F O R M S OF u P A R

The 'linker' region between D 1 and D2 is highly susceptible to proteolytic cleavage and in this part of the human uPAR, potential cleavage positions exist for both trypsinand chymotrypsin- like protease activities, and for leukocyte elastaseY ,43 Interestingly, the proteases capable of cleaving uPAR in this linker region include u-PA itself (despite the very narrow specificity of this enzyme), and plasmin? 3 A similar cleavage susceptibility does not exist in the region between D2 and D3. 2~ A cleaved form of uPAR (comprising D(2+3)) has actually been demonstrated on the surface of cultured human U937 cells, 5~ as well as in detergent extracts of transplanted human tumors grown on nude nlice.54 Blocking studies with monoclonal antibodies against u-PA have indicated that component(s) of the u-PA/plasmin system are indeed engaged in uPAR cleavage during cell culture9 Furthermore, in another study where the cleavage of uPAR was accomplished by addition of purified u-PA to the cultures, it was shown that the binding of u-PA to uPAR on the cell surface facilitates the cleavage, even though the bound u-PA probably does not cleave the same receptor molecule to which it is boundY The mechanism responsible for this phenomenon is unknown. As is evident from the discussion above, neither of the known cleavage products of uPAR (i.e. D(2+3) or D1) can bind to u-PA with appreciable affinity, and the cleavage may thus be considered an inactivation mechanism. It cannot be excluded, however, that one or both of the products may have functions other than u-PA binding, and in this connection, a role of the cleaved receptor in cellular signal transduction has been suggested? 6 Other mechanisms are evidently capable of releasing an apparently full-length uPAR (rather than just D 1) from the cell surface? 7-59It has not been determined, however, whether this release occurs following proteolytic cleavage close to the COOH-terminus, or by the action of phospholipases that might cleave the GPI anchor. A summary of data available on the occurrence of soluble uPAR(s) in biological samples follows in the section on uPAR as a prognostic indicator, below. T H E F U N C T I O N OF u P A R IN T H E P L A S M I N O G E N ACTIVATION SYSTEM

In the primary structure of u-PA, the receptor-binding domain (GFD) is remote from the domain harboring the proteolytic activity and in the folded u-PA molecule, Fibrinolysis & Proteolysis (1998) 12(4), 191-204

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NMR studies suggest that there is a large motional independence between the individual domains. 6° In accordance with this independence, binding of u-PA to purified uPAR has no effect on the amidolytic activity of the protease and only a modest attenuating effect on its activity towards plasminogen. 6~-63 This latter effect is probably caused by a weak steric interference with the accessibility of the enzyme to the large substrate molecule. Likewise, the plasmin-catalyzed activation of pro-uPA bound to purified receptor is slightly slower than the activation of unbound pro-u-PA 62,63 On the cell surface, however, the u-PA-uPAR complex is exposed to a complicated local molecular environment. Therefore, in this situation, the functional outcome of the binding of u-PA to uPAR does not merely reflect the properties of the isolated complex but rather the integrated function of the u-PA-uPAR complex with additional components. Several studies have shown that the reciprocal activation system of pro-u-PA and plasminogen64,65 becomes accelerated by certain cells, such as monocyte-like U937 cells, in a uPAR-dependent manner, s,66 It is of central importance that this accelerative phenomenon requires not only the binding of pro-u-PA to uPAR but also the binding of plasminogen to other, unidentified, components on the cell surface.8 The acceleration seems to be due to an effect on both reactions of the reciprocal system, i.e. the plasmincatalyzed activation of pro-u-PA s and the u-PA-catalyzed activation of plasminogen 6~ (Fig. 2B, C). It is likely that, even though the uPAR-complexed ligand functions with a slightly lowered efficiency in both reactions (as found in the purified system), the organization of the components on the cell surface more than compensates for this attenuation by means of concentration and orientation effects which favor the reactions. 6z This view is supported by a kinetic study with U937 cells on the reaction between active u-PA and plasminogen. 6a These analyses revealed that the accelerative effect of the cells is due to a strong decrease in the apparent Km of u-PA for the substrate plasminogen, reflecting the binding of the substrate on the cell surface, whereas at the same time the k~at of the reaction is actually slightly lowered, probably reflecting the above-mentioned partial steric hindrance of the plasminogen activation reaction. The function of pro-u-PA in this system has been disputed. It is commonly acknowledged that pro-u-PA is a true zymogen with an extremely low intrinsic proteolytic activity,64 and at this as well as other laboratories, this situation was found to be unchanged after binding of pro-u-PA to purified uPAR. 62,6~,65 This result is in accordance with the above-mentioned structural domain independence. On the contrary, others have reported a pronounced acquisition of activity by pro-u-PA as a result of uPAR binding. 6s In the coupled activation Fibrinolysis & Proteolysis (1998) 12(4), 191-204

system used in the latter experiments, however, a putative proteolytic activation of pro-u-PA by trace protease contaminants is difficult to exclude (discussion,62 published comments69;°). Using a similar system, other investigators have found that receptor binding does not lead to acquisition of activity per se, but that the uPAR-pro-u-PA complex can be rendered active by certain peptides acting as cofactors. 7~ In both studies, however, the exclusion of carrier proteins from the model incubations represents a problem in the interpretation, due to the inherent conformational instability of the participating proteins in very dilute solution (discussion62). In the one case it was in fact demonstrated that the inclusion of a carrier protein such as BSA abolished the observed effect,71 thus challenging the relevance of the phenomenon under physiological conditions. Whereas it is the view of the present authors that a uPAR-mediated, direct molecular modulation of the activity status of pro-u-PA is unlikely, it is important to note that the functional ability of pro-u-PA might still change on the cell surface without the need for an increase in the proteolytic activity. Thus, an orientation effect as discussed above might render the very low intrinsic activity of pro-u-PA relevant in those cases where the cell-bound substrate, plasminogen, becomes optimally situated in close proximity to the uPAR-bound proenzyme (Fig. 2A). Inhibitors of u-PA binding to uPAR (receptor antagonists, see below) are capable of dramatically reducing the ability of the cell-surface system to produce plasmin activityY This results in part from the lack of the accelerative effect described above, but in addition the role of protease inhibitors has to be considered (Fig. 2D). Cell-bound plasmin is efficiently protected against the abundant plasmin inhibitors, cs-antiplasmin6~ and %-macroglobulin, 72 thus strongly favoring the localization of the activation cascade to the cell surface. This is probably a prime factor in the directing of these proteolytic events to cell surfaces in the presence of the protease inhibitors of serumfl but in the absence of u-PA binding to uPAR, the cell surface reciprocal activation system no longer functions. A similar protection does not occur though, in the case of the fast-acting inhibitors of u-PA itself, uPAR-bound u-PA is still susceptible to regulation by reaction with the specific inhibitors, PAI-19 and PAI-2,z~ even though the receptor interaction leads to a somewhat lowered rate of reaction. TM I N T E R A C T I O N OF uPAR W I T H V I T R O N E C T I N

Vitronectin, an important adhesion protein of plasma and the extracellular matrix, is capable of binding to uPAR on the cell surface as well as to purified, soluble uPAR. 22 At least under certain conditions, uPAR appears to be the © Harcourt Brace & Co. Ltd 1998

The urokinase receptor

A

B

C

195

D

Fig. 2 Some important players in the proteolytic cascade system on the cell surface, uPAR (hatched) binds both the single-chain pro-u-PA and the active two-chain u-PA. Plasminogen (Pig) and plasmin (Pli) bind to unidentified components on the cell surface, allowing reactions with the uPAR-bound u-PA/pro-u-PA. The u-PA-uPAR complex is highly stable (tl/2: several hours, l°a whereas plasminogen and plasmin exchange quite rapidly between the cell-bound and the solution state. (A) The intrinsic proteolytic activity of pro-u-PA is very low and this property is not changed upon binding to uPAR. However, specific orientation effects may favor the interaction with cell-bound plasminogen, leading to an activation reaction that could serve as a potential initiation mechanism for the cell-bound cascade. (B) Proteolytic activation of uPAR-bound pro-u-PA is catalyzed by surface-bound plasmin and this reaction is favored relative to the situation in solution, probably due to orientation and concentration effects. (C) The resulting two-chain u-PA, still bound to uPAR, efficiently activates neighboring plasminogen in a reaction favored by the same factors as above. The combination of B and C leads to a strong acceleration of the feedback activation mechanism. (D) Whereas uPAR-bound u-PA is being inhibited by PAl-1 and PAl-2 almost as efficiently as free u-PA, a similar situation does not exist for plasmin. After plasminogen activation on the cell surface, the resulting cell-bound plasmin is protected against its major inhibitor, %-antiplasmin (o~2aPli),thus focusing the proteolytic potential at the site of activation.

major vitronectin-binding protein on some cell types (such as cuhured endothelial cells75), thus pointing to an adhesive role of this receptor in addition to its function in proteolysis. Binding to uPAR takes place both with native, monomeric vitronectin and with the multimeric (urea-denatured) protein, z~,z6 The uPAR interaction is directed to the NHE-terminal somatomedin B domain in vitronectin. 7zA region consisting of residues 364-380 close to the COOH-terminus may be involved in addition, as suggested by competition studies with synthetic peptides. TM The cleaved form of vitronectin (residues 1-379) which is present in plasma along with the full-length form (1-459) retains the uPAR binding capability. 76 Importantly, the well-established interaction between vitronectin and PAI-1 also involves the somatomedin B domain zg,s° and indeed, PAI-1 and uPAR compete for vitronectin binding, zS-zz The vitroneCtin binding motif in the uPAR molecule is probably as complex as is the u-PA binding region. Several monoclonal antibodies against D 1 of uPAR have been shown to inhibit vitronectin binding 7~,76 whereas other antibodies, directed against the remaining part (D(2+3)) of uPAR, were non-inhibitory. One antibody of the latter series, however, showed a partial blockage of binding in a different experimental system. 22 Competition studies with purified domains of uPAR point to the involvement of more than one domain in the vitronectin binding reaction, indicating that the intact uPAR is required for efficient binding, 76 just like the situation described above for the uPAR-u-PA interaction. © Harcourt Brace & Co. Ltd 1998

Importantly, even though the interactions of uPAR with u-PA and vitronectin have similar structural requirements, the receptor structure does permit the simultaneous binding of both of these ligands. Actually, it has been found in several experimental systems that pre-saturation of uPAR with u-PA even leads to augmentation of vitronectin binding. 22,75,76The molecular mechanism lying behind this phenomenon is not clear. Apparently, u-PA binding does not change the Kd of uPAR for vitronectin. Consequently, it has been suggested that uPAR can exist in more than one conformation and that the vitronectin-binding conformation is favored by complex formation with u-PAY It is" clear from the description above that a complicated interplay exists between uPAR, u-PA, vitronectin and PAI-1. This has led to the proposal that, under certain conditions, PAI-1 has a decisive role in governing the balance between uPAR-mediated cellular matrix adhesion and release. According to this model, 77 the interaction of uPAR with u-PA will promote cellular attachment as a consequence of increased adhesion to vitronectin. Under conditions where the cell is exposed to a surplus of PAI-1, however, the inhibitor will then not only bind to u-PA (leading to internalization of uPAR, see below) but also actively displace uPAR from vitronectin, resulting in renewed detachment. This model assumes a dominance of uPAR-vitronectin-mediated adhesion over the well-known interactions of cell membrane integrins with vitronectin, a situation that may vary markedly between cells of different lineage. Fibrinolysis & Proteolysis (1998) 12(4), 191-204

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ADDITIONAL FUNCTIONS uPAR-associated components on the cell

The binding of u-PA to uPAR may elicit a number of different secondary events, depending on conditions and on the cell type in question. In addition to the effects on the plasminogen activation cascade, responses as diverse as ligand internalization, signal transduction and changes in cellular adhesion properties have been observed as a result of the initial binding reaction (for details, see review articles elsewhere in this volume: Ehart et al and May et al). Common to these events, however, is the need for communication with other cell surface constituents, a need that is dictated by the fact that the GPI-anchored uPAR has no intracellular domain. Therefore, the identification of membrane proteins that interact with uPAR on the cell surface is a critical issue in the elucidation of the phenomena mentioned above. A brief treatment of these association reactions is provided here. When u-PA binds one of the plasminogen activator inhibitors, PAI-1 or PAI-2, the resulting complex becomes internalized by certain cells that possess uPAR. 8~,82 The internalization of u-PA:PAI-1 has been shown to result from a concerted action of uPAR and the low density lipoprotein receptor related protein (LRP, also known as the a2-macroglobulin receptor), s3-85 Other, LRP-related receptors may substitute for LRP in this process, s6,sz Ultimately, uPAR is recycled to the cell surface. 88 The cell surface components that mediate uPARdependent signal transduction have not been identified with certainty, even though the need for an 'adapter' molecule is clearly indicated? 6 Attempts have been made to approach this problem by co-capping and co-immunoprecipitation studies. On human leukocytes, uPAR was found to co-cap 89 and co-precipitate 9° with Mac-1 (also known as complement receptor type 3 (CR3); CD1 lb/CD18), a B2-integrin that takes part in proteinprotein as well as protein-cafoohydrate interactions. The occurrence of contacts between uPAR and Mac-1 on the cell surface is supported by several later studies using methods such as resonance energy transfer (RET) microscopy.9~,92The interaction is inhibited by N-acetylD-glucosamine, suggesting that the lectin function of Mac-1 is important for this phenomenon. 89 Functional analyses have suggested that the coupling between Mac-1 and uPAR is indeed involved in uPAR-dependent signalling. 93 On various other cell types, uPAR appears to interact with other (BI and ~3) integ rins?4'9~ Importantly, some of the uPAR-integrin interactions seem to play a role in modulating the binding properties of the integrins in question towards their ligands in the extracellular matrix, 94,96 by means of mechanisms that may involve direct molecular modulation as well as transmembrane Fibrinolysis & Proteolysis (1998) 12(4), 191-204

signalling. Conversely, the assembly of the uPAR-integrin complexes on the cell surface in some cases is dependent on the binding of the integrins to their respective ligands. 91,95 This observation may be important for an understanding of the directing of uPAR, on certain cell types, to areas of focal cell-substratum contact, as recognized at an early pointY r Other indirect interactions of uPAR with the cytoskeleton, suggested by phenomena such as the polarization of uPAR to the leading edge of migrating monocytes, s2 may also be mediated in part through integrins but during the polarization of human neutrophils, uPAR was actually found to dissociate from Mac-1 in an organized manner. 92 The question of molecular contacts between uPAR and other membrane proteins is clearly cell-specific and is related to the occurrence of particular microcompartments in the cell surface structure. In some, but not in all, cases GPI-anchored proteins appear to have a relatively high lateral mobility in the plasma membrane (discussiongs). On the other hand, on certain cell types various GPI-proteins are considered to be part of defined surface structures s u c h as caveolae? 9 In some cases, a co-localization of uPAR with caveolin (a marker protein characteristic of caveolae) has indeed been reported 1°° but other investigators have questioned the occurrence of uPAR in this compartment. 1°~ Whereas most of the interactions described above have been inferred either from indirect indications of molecular proximity or from functional interdependence, there are few examples where complexes of uPAR and other membrane proteins have been demonstrated directly at the molecular level On monocyte-like U937 cells, though, uPAR-bound pro-u-PA forms a complex with a high-molecular-weight membrane protein, and this complex can be fixed covalently by the enzyme tissue transglutaminase without prior disruption of the cell (Fig. 3)2°2t P H Y S I O L O G I C A L F U N C T I O N S OF uPAR: S T U D I E S ON u P A R - D E F I C I E N T M I C E

uPAR-deficient mice have been constructed by targeted gene inactivation, and so it has become evident that uPAR deficiency does not compromise fertility, development or hemostasis. 1°3,1°4 However, analysis of the phenotype when combined with tPA deficiency does support a physiological function for uPAR in surveillance of fibrin, particularly in the liver. 1°5 Recently a study with

tAn additional protein found recently to interact with uPAK on the cell surface is the cation-independent, mannose 6-phosphate/insulin-like growth factor-II receptor (Nykjaer A, Christensen E I, Vorum H, et al, Mannose 6-phosphate/insulin-like growth factor-II receptor to lysosomes via a novel binding interaction. J Cell Biol 1998; 141: 815-828.)

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The urokinase receptor

1

3

M t x 10 ~3 200 -

Table 1 Blocking the function of the urokinase receptor Method

Reference

ATF ATF peptides

Blasi, 1986138 Appella, 198747 Kobayashi, 199412° B0rgle, 1997139 Stephens, 19899 Kirchheimer, 1989149 Renne, 199166 Mohanam, 1993TM Luther, 1997142 Behrendt, 1993199 Ellis, 1993149 Crowley, 1993113 Wilhelm, 1994144 Kariko, 199414s Kook, 1994121 Quattrone, 1995146 Ossowski, 1996147 Yu, 1997123 Go, 1997148 Goodson, 19941~1

DFP-uPA 67~*

Anti-receptor antibodies

43Suramin 30-

Transfection with mutant uPA Recombinant soluble uPAR Ribozyme Antisense

2{) 14

Fig. 3 Ligand labeling of uPAR and a uPAR-associated protein on the cell surface. U937 cells were incubated with radiolabeled pro-u-PA followed by either chemical cross-linking with DSS (ligand labeling of uPAR; lane 1, solid arrow) or cross-linking with tissuetransglutaminase (ligand labeling of a uPAR associated protein; lane 3, open arrow). The samples were analyzed by SDS-PAGE under reducing conditions followed by autoradiography. None of these cross-linking products are formed if the cells are preincubated with a uPAR-blocking antibody (result not shown). Lane 2 shows the result obtained in the absence of cross-linking reagents where only non-covalently bound pro-uPA is evident. The electrophoretic mobility of M marker proteins is indicated; note that the apparent M r of the cross-linked conjugates includes the M r of pro-uPA itself. The experiment was performed as previously described.102

smooth muscle cells from uPAR-deficient mice has suggested that, at least in cell culture, uPAR-dependent plasminogen activation is also involved in activation of latent transforming growth factor beta. ~°6 ANTAGONISTS

OF u-PA BINDING

TO uPAR

The central role of uPAR in proteolytic, as well as adhesive, functions combined with results from localization studies and experimental cancer model systems, suggests a new intervention strategy for cancer treatment, based on the use of uPAR antagonists. Interference with u-PA-uPAR binding may have less toxic side-effects than direct inhibition of the catalytic activity of uP.& or plasmin, since for example in the mouse kidney there are high levels of u-VA 1°7which appears to have a physiological function 1°8 despite only very low levels of uPAR expression.~5 As yet, however, only few non-protein reagents with the required antagonistic properties have been described. In the course of molecular and functional studies on uPAR in vitro, it was found that the polysulfonated dinaphthyl compound, suramin, is capable of inhibiting the u-PA-uPAR interaction. ~°9 However, suramin is not a specific antagonist, and it has been known for several © Harcourt Brace & Co. Ltd 1998

197

Phage-displayed peptides Fusion Proteins/Conjugates ATF:PAI-2 fusion u-PA:saporin toxin conjugate ATF:HSA fusion u-PA:urinary trypsin inhibitor conjugate ATF:IgG fusion

Ballance, 1992112 Cavallaro, 1993 TM LU, 1994, 1996149,15° Kobayashi, 1995113 Crowley, 1993119 Min, 1996122 Kost, 1997TM

years that it can lead to toxic effects in vivo. ''° Another binding inhibitor was discovered by the phage display technique, leading to the construction of a 15-mer peptide which inhibits binding in vitro with an ICs0 as low as 10 nM. TM As an altemative to the use of synthetic binding inhibitors, there are several other possible strategies which may be applied as research tools in an effort to deprive tumor cells or tumor-recruited stromal cells of the function of uPAR, as summarized in Table 1. These include competitive ligands based on the GFD of u-PA and antibodies, but also various approaches to downregulate uPAR expression, such as the use of antisense and ribozyme methods. More recently, fusion proteins or chemical conjugates have been constructed that contain a uPAR ligand sequence together with a partner protein that sterically hinders accessibility of neighboring cell-surface receptors and prolongs the half-life of the competitor in the blood circulation. The range of possible fusion or conjugate protein partners for a uPAR binding sequence is of course limitless, and already includes inhibitors of u-PA and plasmin, 1~2,''~ toxins 114 and antibodies. Thus, as well as enabling interference with the cell-surface proteolytic system of tumor cells, the range of partner proteins for antagonists can be extended to facilitate the specific targeting of uPAR-expressing ceils. Among several Fibrinolysis & Proteolysis (1998) 12(4), 191-204

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akemative possibilities whose application could be considered, this could employ enzymes which process pro-drugs (e.g. using antibody-directed enzyme pro-drug therapy (ADEPT)/~5 antibodies directing cell-mediated cytotoxic reactions, ~ or even packaged delivery systems (e.g. virosomes)/~7 so as to exploit the many different methods of attack on cells which display uPAR on their surface). If cycling of uPAR through the cell membrane proves to be a dominant process in malignant cells, one may look to the model of the transferrin receptor where many applications for cancer therapy are already under study? TM EXPERIMENTAL TUMOR THERAPY DIRECTED A G A I N S T uPAR F U N C T I O N

The extension of in vitro cell-surface plasminogen activation studies with uPAR antagonists to in vivo model systems has supported a role for the u-PA-uPAR interaction in tumor growth and dissemination. Thus, in a model system with human prostate carcinoma cells grown in nude mice, an antagonist of u-PA-uPAR binding showed pronounced anti-metastatic effects, "9 and an anti-metastatic effect of a binding antagonist was also observed with murine Lewis lung carcinoma cells grown in syngeneic mice? 2° Antisense suppression of uPAR in squamous carcinoma cells resuked in inhibition of the local cellular invasion into a chicken chorioallantoic membrane, and an increase in tumor latency in the same system/21 Significant reduction of spontaneous metastases could also be demonstrated by inoculation of nude mice with human cancer cells expressing a mutated inactive form of u-PA.~9 Recently, another study showed that treatment with a uPAR antagonist inhibited angiogenesis and tumor growth in a syngeneic mouse tumor model? 22A further consequence of uPAR deprivation has also now been revealed that deserves further investigation: namely that antisense suppression of uPAR in tumor ceils can induce a protracted state of dormancy, as found using the same system with chicken chorioallantoic membrane as above? 23 It should be emphasized, however, that in vivo studies of uPAR blockade are not only still few in number, but also present some difficulties in attributing the observed effects solely to inhibition of uPAR function. Furthermore, a problem in the interpretation arises in those cases where, e.g. human cancer cells are studied in nude mice, since the u-PA-uPAR interaction is species-specific. 47 As mentioned in the Introduction, a tumor/stroma interaction appears to be central in the assembly of the proteolytic system in spontaneous cancers. It may be hoped that future studies will address these problems by the use of potent low-molecularweight antagonists that have high specificity for the Fibrinolysis & Proteolysis (1998) 12(4), 191-204

uPAR target, and which are effective in syngeneic (mouse) systems. uPAR AS A P R O G N O S T I C I N D I C A T O R IN CANCER PATIENTS

uPAR normally occurs at low levels of expression in a limited number of tissue locations in the mature adult, but expression is in many cases enhanced in cancer. Thus, in breast cancer, uPAR is expressed on some tumor cells and is widespread on the tumor-associated macrophages, but is undetectable in normal breast tissue? z4-~26In colorectal cancer uPAR mRNA expression and immunoreactivity are found in both tumor cells and tumor infiltrating macrophages, while no signals are detected outside the tumor tissue/27 The application of ELISA techniques to the assay of uPAR levels in extracts of resected tumor tissue from colorectal cancer, 128 lung cancer ~29 and breast cancer ~3° has demonstrated that high levels of uPAR protein in tumor tissue are related to poor patient survival. GrondahI-Hansen et al have reported on the prognostic value of ELISA-determined uPAR in detergent and non-detergent extracts prepared from the same 505 primary breast tumors. ~3° Univariate analysis showed that uPAR levels above the median value in the set of non-detergent extracts were significantly associated with a shorter overall survival. In a clinically-relevant subgroup of 201 node-positive postmenopausal patients, uPAR in non-detergent extracts had a particularly strong prognostic impact, and in this group uPAR appeared to be an independent, and the single most important, biochemical marker of both relapse-free and overall survival. By the use of a different ELISA, Duggan et al also showed the prognostic impact of uPAR measured in non-detergent extracts of 141 primary tumors. TM It is intriguing that the uPAR determined in non-detergent extracts provides a stronger prognostic parameter than measurement of uPAR in detergent extracts. The uPAR in non-detergent extracts most likely represents a soluble form released from the surface of tumor cells and/or stromal cells by extracellular enzymatic cleavage of uPAR? 7,132If this fraction consisted of free D 1 this might be expected from the fact that active u-PA, plasmin and various other proteases can cleave uPAR between the first and second domains (see above)? 5 However, it is notable that, while Western blots of detergent extracts of human breast tumor tissue suggest the presence of both intact uPAR and D(2 + 3) (HoyerHansen, unpublished), the only form of soluble uPAR so far found in blood plasma from healthy donors and cancer patients appears to be the full-length, threedomain protein. The occurrence of intact soluble uPAR in healthy human plasma and elevated levels in patients © Harcourt Brace & Co. Ltd 1998

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with paroxysmal noctumal hemoglobinuria was earlier conclusively demonstrated by immunoaffinity purification and cross-linking to ~2~l-labelledamino-terminal fragment (ATF) of human u-PA. 59,133 In these studies the soluble receptor purified from plasma was able to efficiently bind its ligand, and therefore must be considered to consist of all three domains (see above). Moreover, full-length uPAR was also indicated by the molecular weight of the labelled cross-linked ATF/uPAR complex? 9,~33The release of this form by tumor cells is indicated by the full-length soluble uPAR found in culture supernatant from human HT-1080 fibrosarcoma cells, ss and the successful use of a column of immobilized u-PA in the affinity-purification of soluble uPAR from plasma and ascites fluid of ovarian c a n c e r patients.S7 Thus, the occurrence of three-domain soluble uPAR in all the cases above is consistent with release of the ectodomains by either phospholipases cleaving the glycolipid anchor, or proteases, e.g. metalloproteases, that could possibly cleave the protein near the COOH-terminus. However, the tissue origin of the uPAR found in blood from healthy donors, the actual enzyme activity(s) responsible for the release of uPAR from the surface of either normal or tumor cells in vivo, and the functional significance of this release are all questions that are unresolved. It is clear that, if the soluble uPAR formed in tumor tissue can enter the blood of cancer patients, giving rise to increased peripheral blood levels of uPAR, this could provide highly useful information with regard to patient assessment. Appreciable amounts of soluble uPAR have been found previously in plasma and ascites fluid from ovarian cancer patients. 5z Our recent studies using selective epitope ELISA of human plasma uPAR have provided additional evidence that full-Iength threedomain uPAR is indeed the major form of uPAR present in peripheral blood from healthy donors as well as cancer patients. TM We have also reported that in patients with non-small-cell lung cancer 135 and in patients with advanced breast and colorectal cancer TM the plasma concentration of soluble uPAR is significantly increased as compared to healthy individuals. To support these studies of cancer patient plasma, it is clearly necessary to closely investigate the levels and the potentially different forms of soluble uPAR in blood from healthy donors as well as cancer patients. Measurement of these low levels found in plasma calls for improved ELISA techniques, and for this purpose we have recently developed an ELISA method which achieves an increased level of sensitivity and specificity?34 This assay has been tested in several different ways in purified systems and using internal controls of recombinant soluble uPAR added into a plasma pool. Using this methodology we are currently investigating the prognostic value of soluble uPAR © Harcourt Brace & Co. Ltd 1998

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measurements in preoperatively collected blood samples from patients with colorectal, breast, renal and ovarian cancers. Another useful approach to the prognostic assessment of uPAR in cancer patients is exemplified by the work of Heiss and colleagues. In a study of gastric carcinoma, 136it was shown that, while dissemination of tumor ceils to bone marrow is prevalent among these cancer patients, the patients who had uPAR-positive tumor cells in marrow were those who had the poorest survival. If it can be firmly established that soluble uPAR increases in plasma in patients with the major types of cancer then this opens up the prospect of enlarged prognostic studies, avoiding the restrictions imposed by the use of resected tumor tissue. Furthermore, it makes possible longitudinal studies of soluble uPAR during patient follow-up, and therefore assessment of its value in detection of tumor recurrence. The early indications are that in ovarian ~7 and colorectal cancers (Stephens et al, submitted) plasma uPAR is a predictor of prognosis and may be highly valuable in identifying high-risk patients, thus enabling focused use of adjuvant therapy. CONCLUSION

The urokinase receptor is a multifunctional protein shown in vitro to function in cell-surface proteolysis, cellular adhesion to extracellular matrix proteins and cellular signal transduction. The availability of uPAR knock-out mice will help the assessment of these roles in vivo, where at least a function in fibrinolysis is already apparent. At the molecular level, the function of uPAR is best described in relation to proteolysis, but also with respect to other roles of the receptor some patterns are beginning to emerge. In this regard, a key issue is the identification and molecular characterization of uPAR's interaction partners on the cell surface. The last few years have led to further progress in the understanding of the binding reaction between uPAR, u-PA and vitronectin. A central goal in this connection is the elucidation of the three-dimensional structure of uPAR. In addition to aiding studies on the structure-function relationship of the receptor, this would facilitate the further development of receptor binding antagonists. The available data suggesting a role of uPAR in cancer combined with the indications that uPAR deficiency does not lead to severe physiological consequences (as found in mice) suggest that uPAR could be blocked as part of a putative cancer therapy without severe side-effects. If further experimental studies on cancer animal models prove to consolidate the early findings with uPAR antagonists, this may make uPAR an important therapeutic target. In any event, there are already strong indications from investigations in cancer patients that uPAR will prove valuable Fibrinolysis & Proteolysis (1998) 12(4), 191-204

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as a p r o g n o s t i c m a r k e r . S t u d i e s o n uPAR m e a s u r e d in t u m o r e x t r a c t s are n o w b e i n g e x t e n d e d to i n c l u d e plasma measurements, which may enlarge the knowl e d g e a n d u s e f u l n e s s of t h i s p a r a m e t e r c o n s i d e r a b l y . T h e s e s t u d i e s m a y h o l d g o o d p r o s p e c t s for a n i m p r o v e m e n t in p a t i e n t a s s e s s m e n t , p a r t i c u l a r l y if t h e p o t e n t i a l for i d e n t i f i c a t i o n of h i g h - r i s k p a t i e n t s c a n b e realized.

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33. Behrendt N, Ploug M, Patthy L, Houen G, Blasi F, Dana K. The ligand-binding domain of the cell surface receptor for urokinase-type plasminogen activator. J Biol Chem 1991; 266: 7842-7847. 34. Palfree R G. The urokinase-type plasminogen activator receptor is a member of the Ly-6 superfamily [letter]. Immunol Today 1991; 12: 170. 35. Ploug M, Rahbek Nielsen H, Ellis V, Roepstorff P, Dana K. Chemical modification of the urokinase-type plasminogen activator and its receptor using tetranitromethane. Evidence for the involvement of specific tyrosine residues in both molecules during receptor-ligand interaction. Biochemistry 1995; 34: 12524-12534. 36. Mbrecht J C, Nicholas J, Cameron J R, Newman C, Fleckenstein B, Honess R W. Herpesvirus saimiri has a gene specifying a homologue of the cellular membrane glycoprotein CD59. Virology 1992; 190: 527-530. 37. Gumley T P, McKenzie I E Kozak C A, Sandrin M S. Isolation and characterization of cDNA clones for the mouse thymocyte B cell antigen (ThB). J Immunol 1992; 149: 2615-2618. 38. Brakenhoff R H, Gerretsen M, Knippels E M, et al. The h u m a n E48 antigen, highly homologous to the murine Ly-6 antigen ThB, is a GPI-anchored molecule apparently involved in keratinocyte cell-cell adhesion. J Cell Biol 1995; 129: 1677-1689. 39. Ploug M, Kjalke M, Ronne E, Weidle U, Hoyer-Hansen G, Dana K. Localization of the disulfide bonds in the NH~-terminal domain of the cellular receptor for h u m a n urokinase-type plasminogen activator. A domain structure belonging to a novel superfamily of glycolipid-anchored membrane proteins. J Biol Chem 1993; 268: 17539-17546. 40. Sugita Y, Nakano Y, Oda E, Noda K, Miura N H, Tomita M. Determination of carboxyl-terminal residue and disulfide bonds of MACIF (CD59), a glycosyl-phosphatidylinositolanchored membrane protein. J Biochem Tokyo 1993; 114: 473-477. 41.. Kieffer B, Driscoll P C, Campbell I D, Willis A C, van der Merwe P A, Davis S J. Three-dimensional structure of the extracellular region of the complement regulatory protein CD59, a new cell-surface protein domain related to snake venom neurotoxins. Biochemistry 1994; 33:4471-4482. 42. Flectcher C M, Harrison R A, Lachmann P J, Neuhaus D. Structure of a soluble, glycosylated form of the h u m a n complement regulatory protein CD59. Structure 1994; 2: 185-199. 43. Ploug M, Ellis V. Structure-function relationships in the receptor for urokinase-type plasminogen activator. Comparison to other members of the Ly-6 family and snake venom alpha-neurotoxins. FEBS Lett 1994; 349: 163-168. 44. Ploug M, Rahbek-Nielsen H, Nielsen P F, Roepstorff P, Dana K. Glycosylation profile of a recombinant urokinase-type plasminogen activator receptor expressed in Chinese hamster ovary cells. J Biol Chem 1998; 273: 13933-13943. 45. Ronne E, Behrendt N, Ploug M, et al. Quantitation of the receptor for urokinase plasminogen activator by enzymelinked immunosorbent assay. J Immunol Methods 1994; 167: 91-101. 46. Ploug M, Ellis V, Dana K. Ligand interaction between urokinase-type plasminogen activator and its receptor probed with 8-anilino-l-naphthalenesulfonate. Evidence for a hydrophobic binding site exposed only on the intact receptor. Biochemistry 1994; 33: 8991-8997. 47. Appella E, Robinson E A, Ullrich S J, et al. The receptor-binding sequence of urokinase. A biological function for the growthfactor module of proteases. J Biol Chem 1987; 262: 4437-4440.

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