Endo180)

lism in human monocytes and macrophages. Adv Exp Med Biol 467:541–52.

sity lipoprotein present in vivo? Curr Opin Lipidol 9:337–344.

van Reyk DM, Jessup W, Dean RT: 1999. Prooxidant and antioxidant activities of macrophages in metal-mediated LDL oxidation: the importance of metal sequestration. Arterioscler Thromb Vasc Biol 19:1119–1124.

Yoshida H, Ishikawa T, Hosoai H, et al.: 1999. Inhibitory effect of tea flavonoids on the ability of cells to oxidize low density lipoprotein. Biochem Pharmacol 58:1695–1703.

Yla-Herttuala S: 1998. Is oxidized low-den-

PII S1050-1738(01)00075-5

TCM

The Urokinase Receptor Associated Protein (uPARAP/Endo180): A Novel Internalization Receptor Connected to the Plasminogen Activation System Lars H. Engelholm, Boye S. Nielsen, Keld Danø, and Niels Behrendt* The urokinase-mediated plasminogen activation system plays a central role in the extracellular proteolytic degradation reactions in cancer invasion. In this review article we discuss a number of recent findings identifying a new cellular receptor protein, uPARAP, that interacts with components of this proteolytic system. uPARAP is a high molecular weight type-1 membrane protein, belonging to the macrophage mannose receptor protein family. On the surface of certain cells, uPARAP forms a ternary complex with the pro-form of the urokinase-type plasminogen activator (uPA) and its primary receptor (uPAR). While the biological consequences of this reaction have not yet been verified experimentally, a likely event is ligand internalization because uPARAP is a constitutively recycling internalization receptor. uPARAP also binds at least one component, collagen type V, in the extracellular matrix meshwork, pointing to a potential role in proteolytic substrate presentation. Additional ligands have been proposed, including collagenase-3 and glycoproteins capable of interacting with one of the multiple carbohydrate recognition-type domains of uPARAP. In various adult tissues uPARAP is present on fibroblasts, macrophages and a subset of endothelial cells. In fetal tissues the protein has also been demonstrated in certain bone forming regions. Hypotheses on the physiological function of uPARAP include regulatory roles in extracellular proteolysis. This type of function would be likely to direct the local turnover of proteases and their substrate degradation products and thus may add to the complicated interplay between several cell types in governing restricted tissue degradation. (Trends Cardiovasc Med 2001;11:7–13). © 2001, Elsevier Science Inc. Lars H. Engelholm, Boye S. Nielsen, Keld Danø, and Niels Behrendt are from The Finsen Laboratory, Rigshospitalet, Copenhagen Ø, Denmark. * Address correspondence to: Niels Behrendt, The Finsen Laboratory, Rigshospitalet, Strandboulevarden 49, Bldg. 7.2, DK-2100 Copenhagen Ø, Denmark. Tel.: 145 3545 5708; fax: (145) 3538 5450; e-mail: [email protected]. © 2001, Elsevier Science Inc. All rights reserved. 1050-1738/01/$-see front matter

TCM Vol. 11, No. 1, 2001

The regulatory mechanisms that govern specific proteolytic degradation reactions in the extracellular environment are decisive for the invasive processes of cancer as well as for a number of normal physiological events where tissue remodeling takes place (Danø et al. 1999). All of these phenomena depend on the cellular penetration of physical barriers composed of extracellular matrix proteins. These barriers are broken by means of proteolytic degradation and the enzymes responsible exert their function in the form of proteolytic cascade systems. The understanding of these strictly regulated processes at the molecular level requires the elucidation of numerous interactions between zymogens, active enzymes, inhibitors and receptors. At the physiological level, interplay between several cell types is involved in these processes. Thus, even though the organized proteolytic systems are needed for the cancer cells, these cells do not actively synthesize all of the proteins involved. Rather, a tumor–stroma interaction occurs where the stromal cells immediately surrounding and infiltrating the tumor are induced, by unknown mechanisms, to generate some of the components necessary (Coussens et al. 2000, Johnsen et al. 1998). The plasminogen activation (PA) system is a proteolytic cascade that is being studied intensively in this connection as well as in the context of cardiovascular homeostasis (Andreasen et al. 1997, Carmeliet and Collen 1995). In this system, the abundant pro-enzyme, plasminogen, becomes activated by one of two known, specific processing enzymes: the urokinase-type plasminogen activator (uPA) or the tissue-type plasminogen activator (tPA). This process leads to the formation of active plasmin, an aggressive serine protease with a trypsin-like activity, capable of degrading fibrin as well as several substrate proteins in the extracellular matrix (Danø et al. 1985). A number of important functions of this proteolytic system have been demonstrated at the physiological level by gene inactivation studies in mice. Plasminogen deficiency has thus been shown to lead to accumulation of fibrin deposits in the liver and various other organs (Bugge et al. 1995), to a delay in skin wound healing (Rømer et al. 1996), to an impaired cardiac wound healing after myocardial infarction (Creemers et al. 2000) and, in

7

mice with an additional disorder in lipid metabolism, also to an increased tendency for atherosclerosis (Xiao et al. 1997). In transgenic mice prone to develop mammary tumors due to expression of the Polyoma middle T antigen, plasminogen deficiency was found to lead to a decreased tumor metastasis to the lungs (Bugge et al. 1998). A central component in the PA system is the specific uPA receptor, uPAR, which binds uPA as well as its pro-enzyme, pro-uPA, thus localizing the proteolytic potential to specific regions on the surface of particular cells (Cubellis et al. 1986, Pöllänen et al. 1988, Vassalli et al. 1985). uPAR serves to promote plasminogen activation in a complicated interplay with plasminogen-binding components on the cell, probably involving a template mechanism for zymogen activation as well as a protection of surface-bound plasmin activity against serum inhibitors (Ellis and Danø 1991, Ellis et al. 1991). In cancer, the uPA/uPAR system clearly illustrates the above-mentioned tumor– stromal interplay, one example being colon adenocarcinoma where uPAR is expressed by a subset of the cancer cells and the tumor-infiltrating macrophages, whereas uPA is delivered by the surrounding fibroblast-like stromal cells (Pyke et al. 1991). It is through this concerted effort that the proteolytic system gets assembled at the invasive front. In studies with transplanted tumors in mice, antagonists against the uPA–uPAR interaction have been found to reduce tumor growth (Min et al. 1996, Tressler et al. 1999) and metastasis (Crowley et al. 1993). Gene inactivation studies suggest that the importance of uPAR in the functions of the PA system varies among the different roles that this system fulfills. Thus, vascular wound healing and arterial neointima formation appear to proceed independently of uPAR deficiency (Carmeliet et al. 1998) even though both processes are impaired in uPA-deficient mice (Carmeliet et al. 1997). On the other hand, a physiological role of uPAR in supporting fibrinolysis has indeed been documented in live mice. Fibrin deposits thus accumulate in the liver of mice with a combined deficiency in uPAR and tPA whereas no such deposits are observed in mice deficient in tPA alone (Bugge et al. 1996). uPAR is a glycoprotein of Mr z55,000 (Behrendt et al. 1990, Nielsen et al.

8

1988). cDNA cloning, alignment studies and protein chemical characterization have shown that the protein consists of three extracellular domains, all belonging to the “uPAR/Ly-6” domain family (Behrendt et al. 1991, Ploug and Ellis 1994, Roldan et al. 1990). The receptor has no transmembrane or intracellular part but is anchored in the plasma membrane by a COOH-terminal glycosyl-phosphatidylinositol (GPI) moiety (Ploug et al. 1991). Functional studies in vitro suggest that uPAR is a multifunctional receptor, communicating with other cell surface proteins at several levels. In addition to the above-mentioned interplay with plasminogen-binding components, uPAR has thus been implicated in roles as diverse as ligand internalization (Nykjaer et al. 1992), modulation of integrin specificity (Wei et al. 1996), cytoskeletal interactions allowing specific localization at focal contacts (Pöllänen et al. 1988) and cellular signal transduction (Resnati et al. 1996) [see Behrendt and Stephens (1998) for a discussion and additional references]. It is noteworthy that the lack of an intracellular domain in uPAR dictates the need for a transmembrane “adapter” protein in several functions (Resnati et al. 1996). Whereas most or all of the functions of uPAR are thus assumed to depend on interactions with other cellular proteins, the information on the latter components is only scarce. In this review article we discuss the properties of a recently characterized receptor protein that may fulfill some of the roles mentioned above. The new component, designated the uPAR-associated protein (uPARAP), interacts with pro-uPA and uPAR to form a ternary complex on the cell surface. Recently, two independent lines of study led to the cloning and characterization of human uPARAP. First, a search for cellular interaction partners of the uPA–uPAR system resulted in the isolation of the uPARAP–pro-uPA complex, providing the amino acid sequence information necessary for cDNA cloning (Behrendt et al. 2000). Second, a study aimed at the characterization of an otherwise unidentified internalization receptor, reactive with a specific antibody and designated Endo180, led to the identification of an antibody reactive clone in a bacterial expression library, allowing the cloning of the Endo180 cDNA (Sheikh et al. 2000). The sequences of

the two full-length products turned out to be identical and also made it clear that uPARAP/Endo180 is the human counterpart of a previously cloned murine cDNA encoding a lectin-like membrane protein (Wu et al. 1996). • Protein Characteristics The uPARAP cDNA encodes a protein of 1479 amino acids, including a presumptive 37 amino acid signal sequence at the NH2 terminus (Behrendt et al. 2000). The product is a type-1 membrane protein (Figure 1) with a typical transmembrane sequence and a small cytoplasmic domain at the COOH terminus. The extracellular part is composed of an NH2terminal, cysteine rich domain peculiar to this protein family (see below), followed by a fibronectin type-II like domain (Fn-II) and a series of eight consecutive

Figure 1. The domain composition of uPARAP. The domain composition was deduced from sequence alignment with the macrophage mannose receptor (Taylor et al. 1990, Wu et al. 1996, Behrendt et al. 2000). Cys-rich: the NH2-terminal cysteine rich domain that belongs to a domain family specific for the macrophage mannose receptor protein family; FN-II: fibronectin type-II domain; CRD: C-type carbohydrate recognition domain (note that these domains are structurally defined); TM: transmembrane segment; Cyto: cytoplasmic domain. An NH2-terminal signal sequence, presumably 37 residues, precedes the cysteine rich domain before expression on the cell surface.

TCM Vol. 11, No. 1, 2001

domains belonging to the C-type carbohydrate recognition domain (CRD) family. This domain structure is characteristic for a rather recently established protein family, the macrophage mannose receptor protein family (Taylor 1997). Including uPARAP, so far four proteins are known to belong to this family. The other three members are the macrophage mannose receptor (Taylor et al. 1990), the receptor for secretory phospholipase A2 (Ishizaki et al. 1994, Lambeau et al. 1994) and a membrane protein designated DEC-205/MR6 that has been suggested to be engaged in antigen uptake and processing in dendritic cells of the immune system (Jiang et al. 1995, McKay et al. 1998). Except for DEC-205/MR6 that has 10 CRDs instead of eight, all of the family members share the domain structure described above. Further evidence underlining the relation of uPARAP to this protein family stems from the structure of the human uPARAP gene (located on chromosome 17), which has an exon/intron structure very close to that of the macrophage mannose receptor gene (Sheikh et al. 2000). All of the protein family members seem to function as internalization receptors (Taylor 1997) and in accordance with this notion, studies on the Endo180 antigen have

shown that it is a membrane protein capable of internalizing antibodies directed against it, long before the cloning of the cDNA (Isacke et al. 1990). uPARAP is a glycoprotein, containing N-linked carbohydrate side chains of the complex type (Sheikh et al. 2000). The carbohydrate accounts for less than 10% of the apparent molecular weight in SDSPAGE analysis, but the details of the glycosylation are not known. An additional post-translational modification is the phosphorylation of at least one serine residue, probably located in the cytoplasmic domain (Sheikh et al. 2000) (see below). • Binding Reactions of uPARAP Studies on the cellular interplay with components of the plasminogen activation system initially demonstrated uPARAP as a membrane protein capable of interacting with pro-uPA in a uPAR-dependent reaction (Behrendt et al. 1993). More recently, it has become clear that a trimolecular complex involving all of these participants is indeed assembled on the cell surface (Behrendt et al. 2000); see Figure 2. In the formation of this complex it is likely that uPAR accounts for the initial binding of pro-uPA because no appreciable binding of pro-

Figure 2. Representation of uPARAP and its interaction partners. The binding of pro-uPA to uPAR is the prerequisite for formation of the ternary complex with uPARAP (left). Note that the GPI-anchored uPAR is unable to communicate with the interior of the cell, whereas uPARAP possesses an intracellular domain. Collagen V competes with pro-uPA for the interaction with uPARAP but does not dissociate the uPAR-pro-uPA part of the complex (right). Other ligands may include the matrix metalloprotease, collagenase-3 (MMP-13). A carbohydratebinding function of uPARAP is likely on theoretical grounds, but the experimental evidence is so far restricted to artificial glycoproteins.

TCM Vol. 11, No. 1, 2001

uPA to uPARAP is observed in the absence of uPAR (L. H. Engelholm and N. Behrendt, unpublished results). Furthermore, if the binding of pro-uPA to uPAR is blocked by means of a monoclonal anti-uPAR antibody, the interaction between pro-uPA and uPARAP is abolished (Behrendt et al. 1993). The formation of the trimolecular complex thus appears to be dependent on the (permanent or transient) juxtapositioning of uPAR and uPARAP on the cell surface, and this architecture may vary among different cell types that express the two proteins. It follows that the degree of complex formation is not necessarily a reflection of the absolute amounts of uPAR and uPARAP present; for a further discussion see Behrendt et al. (2000). The interaction between pro-uPA and uPARAP can be fixed and visualized by an enzymatic cross-linking procedure (Behrendt et al. 1993); see Figure 3. Competitive ligands for uPARAP can therefore be identified by their ability to block the reaction with labeled pro-uPA. This type of experiment revealed that a specific type of collagen, collagen V, binds strongly to uPARAP (Behrendt et al. 2000) (Figure 2). Collagen V is part of the fibrils of the lamina reticularis, lining the lamina densa in the basement membrane (Adachi et al. 1997). uPARAP thus interacts with both an important trigger enzyme in a proteolytic cascade system and a potential substrate for proteolytic degradation. Interestingly, in contrast to most other collagens, collagen V has been reported to be sensitive to degradation by trypsin-like enzymes (Niyibizi and Eyre 1989). Comparative studies with other types of collagen have so far not revealed any new binding candidates, but it is still an open issue whether additional collagen ligands exist for uPARAP. The collagen-binding function is most likely enabled by the Fn-II domain of uPARAP because this domain type has been shown to be involved in the collagen-binding activities of several other proteins (Banyai et al. 1990). It is noteworthy that another member of the present protein family, the phospholipase A2 receptor, also binds collagens in a reaction dependent on the Fn-II domain, as shown by domain deletion analysis (Ancian et al. 1995). In relation to collagen substrates, a highly interesting, but so far preliminary, finding is the implication of the rat uPARAP analogue in the cellular bind-

9

Figure 3. Electrophoretic demonstration of uPARAP and its binding partners. Human promyeloid histiocytic U937 cells were incubated with radiolabeled pro-uPA (Mr z50,000) after preincubation with unlabeled potential competitor proteins. The binding of pro-uPA to uPARAP leads to the formation of a radiolabeled product of Mr z200–250,000. This product is visualized by enzymatic covalent cross-linking followed by SDS polyacrylamide gel electrophoresis and autoradiography of the gel (Behrendt et al. 2000). UPARAP binding proteins such as collagen V (lane 4) or a specific rabbit antibody against uPARAP (lane 2; see Figure 4 for details) substantially reduces pro-uPA complex formation. No reduction is found with collagen type IV (lane 5) or with an irrelevant IgG (lane 3). The interplay between uPARAP and uPAR is shown by incubation of the cells with a blocking antibody against uPAR (lane 6). This treatment completely prevents the primary cellular binding of pro-uPA (non-cross-linked product) as well as the formation of the high molecular weight uPARAP complex. The electrophoretic mobilities of Mr marker proteins are indicated to the left.

ing of collagenase-3/MMP-13. It has been reported that the direct binding of collagenase-3 to rat osteosarcoma cells is mediated by a protein with a sequence known at the time only in terms of the corresponding mouse cDNA, whereas another receptor, the low-density lipoprotein receptor related protein (LRP), is essential for the subsequent internalization (Barmina et al. 1999). The former protein sequence was identified as that published by Wu et al. (1996), which can now be assigned to uPARAP. This observation opens the possibility that uPARAP is engaged in the regulation of two or

10

even several proteolytic systems. The actual interaction with collagenase-3, a member of the matrix metalloprotease family, could be of considerable significance since this enzyme is an aggressive extracellular protease with activity against native collagens and is expressed in several types of human tumors (Pendas et al. 2000). Finally, the multiple CRDs in the structure of uPARAP would suggest a lectin function (Figure 1). However, although all of the members of this protein family can probably bind certain glycoproteins, it is disputed whether this activity is part of their biological function in all cases. For example, whereas the macrophage mannose receptor definitely binds the carbohydrate side chains of various glycoproteins in a Ca21-dependent reaction (Taylor et al. 1992, Taylor and Drickamer 1993), another family member, the phospholipase A2 receptor, binds the phospholipase ligand in a protein–protein interaction (Lambeau et al. 1994). In the case of uPARAP, an examination of protein sequence motifs suggests that the first and the second CRD may support a Ca21-dependent lectin function (Sheikh et al. 2000, Taylor 1997). At the experimental level, binding has so far only been demonstrated with artificial glycoconjugates such as chemically glycosylated albumin derivatives or glycosylated agarose (Behrendt et al. 2000, Sheikh et al. 2000). Further experiments with natural glycoproteins are clearly required. It should be noted that recent studies on the cysteine-rich domain of the macrophage mannose receptor suggest that this domain type can also fulfill a carbohydrate-binding function (MartinezPomares et al. 1999). As discussed above, uPARAP is capable of ligand internalization like the other members of this protein family. In a series of elegant studies it was furthermore demonstrated that uPARAP is constitutively internalized independently of receptor occupancy and that after internalization it is recycled to the cell surface (Isacke et al. 1990). The molecular details of these processes are not known but it has been suggested that the serine phosphorylation mentioned above, which was found to be strongly enhanced following activation of protein kinase C, has a role in the modulation of the intracellular trafficking of the receptor (Sheikh et al. 2000). Being constitutively recy-

cled, it is possible that uPARAP would not discriminate between its ligands in this process and therefore the internalization may comprise most or all of the ligands mentioned above. It should be stressed, however, that the internalization experiments with uPARAP cited here were all done with the use of specific monoclonal antibodies; the ligands for uPARAP discussed in this review have so far not been studied in this context. • Tissue Distribution of uPARAP Even though knowledge concerning the tissue specificity of uPARAP expression is still fragmentary, it is clear that the protein has a rather restricted cellular distribution. Immunohistochemical studies have shown that uPARAP is present on fibroblasts in several tissues (Isacke et al. 1990). In the skin, the epidermis was negative, whereas uPARAP was demonstrated on dermal macrophages (Sheikh et al. 2000). The total population of peripheral blood leukocytes came out negative for uPARAP mRNA in a Northern blot analysis (Wu et al. 1996), but uPARAP expression could be induced by differentiation of monocytes in vitro and macrophage-like cells were also found positive in placenta sections (Sheikh et al. 2000). The expression level in the brain appears to be low (Wu et al. 1996), but the GenBank database nevertheless contains an entry representing the human uPARAP cDNA sequence, referring to a screening program of brain-derived proteins (Ishikawa et al. 1998). The adult liver is devoid of any uPARAP mRNA signal but, interestingly, a short uPARAP transcript that apparently arose from alternative splicing has been observed in the fetal liver, which in addition contained the fulllength transcript (Wu et al. 1996). It is not known whether the shorter uPARAP transcript leads to expression of a protein product. A central issue is the uPARAP expression status of the endothelium. In situ hybridization studies in the mouse have shown a pronounced uPARAP mRNA signal in various highly vascularized organs (Wu et al. 1996). Whereas this pattern might suggest a predominant endothelial expression, recent studies by immunohistochemistry indicate that, in human term placenta sections, the protein is only expressed in a subset of the endothelial cells (Sheikh et al. 2000). If a TCM Vol. 11, No. 1, 2001

regulated expression of uPARAP in endothelial subcompartments indeed takes place, the details of this process may prove very important. In a recent study on endothelial gene products preferentially expressed in tumors, the uPARAP sequence was identified among the 25 mostly elevated transcripts in the vessels of malignant compared with normal colorectal tissue (St. Croix et al. 2000). Another interesting and rather restricted expression of uPARAP was observed at the mRNA level in certain boneforming regions in mouse embryos (Wu et al. 1996), which may suggest a function of the protein in the dynamics of these processes. At the subcellular level, co-localization studies and examination by confocal microscopy have demonstrated that uPARAP has a punctate distribution on the cell surface, being localized in clathrin coated pits (Isacke et al. 1990, Sheikh et al. 2000). Furthermore, a comparison of permeabilized and non-permeabilized cells

has revealed that a major fraction of the uPARAP molecules are localized inside the cell in vesicles that are probably endosomes (Sheikh et al. 2000). These observations are in accordance with the proposed function of uPARAP as an internalization receptor. An immunohistochemical staining experiment demonstrating uPARAP in mesenchymal cells in a human first trimester placenta specimen is shown in Figure 4. The trophoblasts are uPARAP negative, as are also the endothelial cells in the example shown. • Conclusions and Hypotheses It is clear from the preceding sections that the knowledge concerning this new receptor is just starting to accumulate. However, the ability of uPARAP to interact with the pro-uPA–uPAR complex, the assignment of putative additional ligands and the general potential for internalization make it reasonable to formulate a

Figure 4. Demonstration of uPARAP in the human placenta. A specific rabbit antibody against uPARAP was raised by immunization with a synthetic peptide (residue 810–830) from the human uPARAP sequence. The antibody was used for immunohistochemical staining of a first trimester human placenta by a standard immunoperoxidase technique (Nielsen et al. 1997). The positive reaction is restricted to mesenchymal cells (m) in the villous stroma, whereas the trophoblastic cells (t) and in this field also the vessels (v) are negative. uPARAP positive vessels can be observed elsewhere in the section (not shown). Bar: 30 mm. Negative controls, including non-immune rabbit IgG and IgG from the immunized rabbit after depletion of the peptide-reactive fraction showed no staining. Insert: A 3-fold increased magnification reveals the particulate distribution of staining in the uPARAP positive cells. (B.S. Nielsen and C. Pierleoni, unpublished data).

TCM Vol. 11, No. 1, 2001

number of hypotheses for the biological function. As mentioned above, the constitutively recycling property of uPARAP opens the possibility that any bound ligand will be internalized and directed to endosomal degradation (Isacke et al. 1990). Interestingly, with regard to the uPA system such a function of uPARAP may resemble that of a well-established internalization receptor, LRP, that is active in the internalization of complexes formed between uPA and plasminogen activator inhibitor 1. On several cell types this function of LRP depends on the initial binding of uPA to uPAR (Nykjaer et al. 1992), just like the situation described above for the formation of the pro-uPA complex with uPARAP. It is obvious that the localized clearance and turnover of proteolytic components will have a major impact on the proteolytic balance in the tissue in question, and such a situation might contribute in an important manner to the multiple reactions of the complicated tumor–stroma interaction mentioned in the first section of this article. This underlines the importance of cellular localization studies of uPARAP in tissues undergoing active remodeling. Another role of uPARAP may be related to the binding of collagen V and, putatively, additional ligands which are substrates for extracellular proteolysis, such as other collagens or uPARAP-reactive glycoproteins. uPAR is a preferred site for the initiation of the proteolytic cascade system (Ellis et al. 1989, Ellis and Danø 1991) and the binding of substrate proteins to the neighboring uPARAP might serve as a substrate presentation mechanism. Such a function would not necessarily be restricted to plasmin sensitive substrates, since plasmin in turn is able to activate other classes of proteases with different specificity, notably including pro-collagenase-1, pro-stromelysin-1, progelatinase B (Murphy et al. 1991, 1999) and to some extent also pro-collagenase3 (Knäuper et al. 1996). The last-mentioned activation mechanism may receive renewed attention in the light of the proposed interaction between collagenase-3 and uPARAP. In addition, or as an alternative to these mechanisms, the binding of proteolytic degradation products to uPARAP may provide a route for the clearance of the substrate proteins in question, while the binding to intact matrix proteins might serve adhesive functions.

11

Finally, as mentioned in the introductory section, several functions are mediated by uPAR through communication with unidentified cell surface proteins. A role of uPARAP in these phenomena is not necessarily limited to ligand binding and internalization processes. An interesting issue is thus the possibility of uPARAP playing a role in uPARdependent signal transduction; recent evidence points to the involvement of other internalization receptors in signal transduction events, [i.e., a signaling role of the low-density lipoprotein receptor family of proteins (Rice and Curran 1999)]. Even though any function of uPARAP in this context is so far speculative, it is reasonable to emphasize that the transmembrane property and the existence of a phosphorylation sensitive cytoplasmic domain of uPARAP are features that could contribute to diverse means of cellular communication.

References

Behrendt N, Rønne E, Danø K: 1993. A novel, specific pro-urokinase complex on monocyte-like cells, detected by transglutaminase-catalyzed cross-linking. FEBS Lett 336:394–396. Behrendt N, Rønne E, Ploug M, et al.: 1990. The human receptor for urokinase plasminogen activator. NH2-terminal amino acid sequence and glycosylation variants. J Biol Chem 265:6453–6460. Behrendt N, Stephens RW: 1998. The urokinase receptor. Fibrinolysis Proteolysis 12:191–204. Bugge TH, Flick MJ, Danton MJ, et al.: 1996. Urokinase-type plasminogen activator is effective in fibrin clearance in the absence of its receptor or tissue-type plasminogen activator. Proc Natl Acad Sci USA 93:5899– 5904. Bugge TH, Flick MJ, Daugherty CC, Degen JL: 1995. Plasminogen deficiency causes severe thrombosis but is compatible with development and reproduction. Genes Dev 9:794–807. Bugge TH, Lund LR, Kombrinck KK, et al.: 1998. Reduced metastasis of Polyoma virus middle T antigen-induced mammary cancer in plasminogen-deficient mice. Oncogene 16:3097–3104.

Adachi E, Hopkinson I, Hayashi T: 1997. Basement-membrane stromal relationships: interactions between collagen fibrils and the lamina densa. Int Rev Cytol 173: 73–156.

Carmeliet P, Collen D: 1995. Gene targeting and gene transfer studies of the plasminogen/plasmin system: implications in thrombosis, hemostasis, neointima formation, and atherosclerosis. FASEB J 9:934–938.

Ancian P, Lambeau G, Lazdunski M: 1995. Multifunctional activity of the extracellular domain of the M-type (180 kDa) membrane receptor for secretory phospholipases A2. Biochemistry 34:13,146–13,151.

Carmeliet P, Moons L, Dewerchin M, et al.: 1998. Receptor-independent role of urokinase-type plasminogen activator in pericellular plasmin and matrix metalloproteinase proteolysis during vascular wound healing in mice. J Cell Biol 140:233–245.

Andreasen PA, Kjøller L, Christensen L, Duffy MJ: 1997. The urokinase-type plasminogen activator system in cancer metastasis: a review. Int J Cancer 72:1–22. Banyai L, Trexler M, Koncz S, Gyenes M, Sipos G, Patthy L: 1990. The collagenbinding site of type-II units of bovine seminal fluid protein PDC-109 and fibronectin. Eur J Biochem 193:801–806. Barmina OY, Walling HW, Fiacco GJ, et al.: 1999. Collagenase-3 binds to a specific receptor and requires the low density lipoprotein receptor-related protein for internalization. J Biol Chem 274:30,087–30,093. Behrendt N, Jensen ON, Engelholm LH, Mørtz E, Mann M, Danø K: 2000. A urokinase receptor-associated protein with specific collagen binding properties. J Biol Chem 275:1993–2002. Behrendt N, Ploug M, Patthy L, Houen G, Blasi F, Danø K: 1991. The ligand-binding domain of the cell surface receptor for urokinase-type plasminogen activator. J Biol Chem 266:7842–7847.

12

Carmeliet P, Moons L, Herbert JM, et al.: 1997. Urokinase but not tissue plasminogen activator mediates arterial neointima formation in mice. Circ Res 81:829–839. Coussens LM, Tinkle CL, Hanahan D, Werb Z: 2000. MMP-9 supplied by bone marrowderived cells contributes to skin carcinogenesis. Cell 103:481–490. Creemers E, Cleutjens J, Smits J, et al.: 2000. Disruption of the plasminogen gene in mice abolishes wound healing after myocardial infarction. Am J Pathol 156:1865– 1873. Crowley CW, Cohen RL, Lucas BK, Liu G, Shuman MA, Levinson AD: 1993. Prevention of metastasis by inhibition of the urokinase receptor. Proc Natl Acad Sci USA 90:5021–5025. Cubellis MV, Nolli ML, Cassani G, Blasi F: 1986. Binding of single-chain prourokinase to the urokinase receptor of human U937 cells. J Biol Chem 261:15,819–15,822. Danø K, Andreasen PA, Grøndahl-Hansen J, Kristensen P, Nielsen LS, Skriver L: 1985.

Plasminogen activators, tissue degradation, and cancer. Adv Cancer Res 44:139–266. Danø K, Rømer J, Nielsen BS, et al.: 1999. Cancer invasion and tissue remodeling— cooperation of protease systems and cell types. APMIS 107:120–127. Ellis V, Behrendt N, Danø K: 1991. Plasminogen activation by receptor-bound urokinase. A kinetic study with both cell-associated and isolated receptor. J Biol Chem 266:12,752–12,758. Ellis V, Danø K: 1991. Plasminogen activation by receptor-bound urokinase. Semin Thromb Hemost 17:194–200. Ellis V, Scully MF, Kakkar VV: 1989. Plasminogen activation initiated by single-chain urokinase-type plasminogen activator. Potentiation by U937 monocytes. J Biol Chem 264:2185–2188. Isacke CM, van der Geer P, Hunter T, Trowbridge IS: 1990. p180, a novel recycling transmembrane glycoprotein with restricted cell type expression. Mol Cell Biol 10:2606– 2618. Ishikawa K, Nagase T, Suyama M, et al.: 1998. Prediction of the coding sequences of unidentified human genes. X. The complete sequences of 100 new cDNA clones from brain which can code for large proteins in vitro. DNA Res 5:169–176. Ishizaki J, Hanasaki K, Higashino K, et al.: 1994. Molecular cloning of pancreatic group I phospholipase A2 receptor. J Biol Chem 269:5897–5904. Jiang W, Swiggard WJ, Heufler C, et al.: 1995. The receptor DEC-205 expressed by dendritic cells and thymic epithelial cells is involved in antigen processing. Nature 375:151–155. Johnsen M, Lund LR, Rømer J, Almholt K, Danø K: 1998. Cancer invasion and tissue remodeling: common themes in proteolytic matrix degradation. Curr Opin Cell Biol 10:667–671. Knäuper V, Will H, Lopez-Otin C, et al.: 1996. Cellular mechanisms for human procollagenase-3 (MMP-13) activation. Evidence that MT1-MMP (MMP-14) and gelatinase a (MMP-2) are able to generate active enzyme. J Biol Chem 271:17,124–17,131. Lambeau G, Ancian P, Barhanin J, Lazdunski M: 1994. Cloning and expression of a membrane receptor for secretory phospholipases A2. J Biol Chem 269:1575–1578. Martinez-Pomares L, Crocker PR, Da Silva R, et al.: 1999. Cell-specific glycoforms of sialoadhesin and CD45 are counterreceptors for the cysteine-rich domain of the mannose receptor. J Biol Chem 274: 35,211–35,218. McKay PF, Imami N, Johns M, et al.: 1998. The gp200-MR6 molecule which is functionally associated with the IL-4 receptor modulates

TCM Vol. 11, No. 1, 2001

B cell phenotype and is a novel member of the human macrophage mannose receptor family. Eur J Immunol 28:4071–4083. Min HY, Doyle LV, Vitt CR, et al.: 1996. Urokinase receptor antagonists inhibit angiogenesis and primary tumor growth in syngeneic mice. Cancer Res 56:2428–2433. Murphy G, Docherty AJ, Hembry RM, Reynolds JJ: 1991. Metalloproteinases and tissue damage. Br J Rheumatol 30(Suppl 1):25–31. Murphy G, Stanton H, Cowell S, et al.: 1999. Mechanisms for pro matrix metalloproteinase activation. APMIS 107:38–44. Nielsen BS, Sehested M, Kjeldsen L, Borregaard N, Rygaard J, Danø K: 1997. Expression of matrix metalloprotease-9 in vascular pericytes in human breast cancer. Lab Invest 77:345–355. Nielsen LS, Kellerman GM, Behrendt N, Picone R, Danø K, Blasi F: 1988. A 55,000– 60,000 Mr receptor protein for urokinasetype plasminogen activator. Identification in human tumor cell lines and partial purification. J Biol Chem 263:2358–2363. Niyibizi C, Eyre DR: 1989. Bone type V collagen: chain composition and location of a trypsin cleavage site. Connect Tissue Res 20:247–250. Nykjaer A, Petersen CM, Møller B, et al.: 1992. Purified alpha 2-macroglobulin receptor/ LDL receptor-related protein binds urokinase.plasminogen activator inhibitor type-1 complex. Evidence that the alpha 2-macroglobulin receptor mediates cellular degradation of urokinase receptor-bound complexes. J Biol Chem 267:14,543–14,546. Pendas AM, Uria JA, Jimenez MG, Balbin M, Freije JP, Lopez-Otin C: 2000. An overview of collagenase-3 expression in malignant tumors and analysis of its potential value as a target in antitumor therapies. Clin Chim Acta 291:137–155. Ploug M, Ellis V: 1994. Structure-function relationships in the receptor for urokinasetype plasminogen activator. Comparison to other members of the Ly-6 family and snake venom alpha-neurotoxins. FEBS Lett 349:163–168. Ploug M, Rønne E, Behrendt N, Jensen AL, Blasi F, Danø K: 1991. Cellular receptor for urokinase plasminogen activator. Carboxyl-terminal processing and membrane anchoring by glycosyl-phosphatidylinositol. J Biol Chem 266:1926–1933.

colon adenocarcinomas. Am J Pathol 138:1059–1067. Resnati M, Guttinger M, Valcamonica S, Sidenius N, Blasi F, Fazioli F: 1996. Proteolytic cleavage of the urokinase receptor substitutes for the agonist-induced chemotactic effect. EMBO J 15:1572–1582. Rice DS, Curran T: 1999. Mutant mice with scrambled brains: understanding the signaling pathways that control cell positioning in the CNS. Genes Dev 13:2758–2773. Roldan AL, Cubellis MV, Masucci MT, et al.: 1990. Cloning and expression of the receptor for human urokinase plasminogen activator, a central molecule in cell surface, plasmin dependent proteolysis. EMBO J 9:467–474. Rømer J, Bugge TH, Pyke C, et al.: 1996. Impaired wound healing in mice with a disrupted plasminogen gene. Nat Med 2:287– 292. Sheikh H, Yarwood H, Ashworth A, Isacke CM: 2000. Endo180, an endocytic recycling glycoprotein related to the macrophage mannose receptor is expressed on fibroblasts, endothelial cells and macrophages and functions as a lectin receptor. J Cell Sci 113:1021–1032. St Croix B, Rago C, Velculescu V, et al.: 2000. Genes expressed in human tumor endothelium. Science 289:1197–1202. Taylor ME: 1997. Evolution of a family of receptors containing multiple C-type carbohydrate-recognition domains. Glycobiology 7:R5–R8. Taylor ME, Bezouska K, Drickamer K: 1992. Contribution to ligand binding by multiple

TCM Vol. 11, No. 1, 2001

Taylor ME, Conary JT, Lennartz MR, Stahl PD, Drickamer K: 1990. Primary structure of the mannose receptor contains multiple motifs resembling carbohydrate-recognition domains. J Biol Chem 265:12,156– 12,162. Taylor ME, Drickamer K: 1993. Structural requirements for high affinity binding of complex ligands by the macrophage mannose receptor. J Biol Chem 268:399–404. Tressler RJ, Pitot PA, Stratton JR, et al.: 1999. Urokinase receptor antagonists: discovery and application to in vivo models of tumor growth. APMIS 107:168–173. Vassalli JD, Baccino D, Belin D: 1985. A cellular binding site for the Mr 55,000 form of the human plasminogen activator, urokinase. J Cell Biol 100:86–92. Wei Y, Lukashev M, Simon DI, et al.: 1996. Regulation of integrin function by the urokinase receptor. Science 273:1551–1555. Wu K, Yuan J, Lasky LA: 1996. Characterization of a novel member of the macrophage mannose receptor type C lectin family. J Biol Chem 271:21,323–21,330. Xiao Q, Danton MJ, Witte DP, et al.: 1997. Plasminogen deficiency accelerates vessel wall disease in mice predisposed to atherosclerosis. Proc Natl Acad Sci USA 94:10,335–10,340.

PII S1050-1738(01)00076-7

TCM

MEETING REPORTS Are you attending a conference that would be of interest to other cardiologists? If you are interested in preparing a report of conference highlights for TCM please contact: Kenneth R. Chien, MD, PhD Editor-in-Chief Trends in Cardiovascular Medicine Editorial Office, 655 Avenue of the Americas New York, NY 10010

Pöllänen J, Hedman K, Nielsen LS, Danø K, Vaheri A: 1988. Ultrastructural localization of plasma membrane-associated urokinase-type plasminogen activator at focal contacts. J Cell Biol 106:87–95. Pyke C, Kristensen P, Ralfkiaer E, et al.: 1991. Urokinase-type plasminogen activator is expressed in stromal cells and its receptor in cancer cells at invasive foci in human

carbohydrate-recognition domains in the macrophage mannose receptor. J Biol Chem 267:1719–1726.

Phone:

212-633-3918

Fax:

212-633-3977

13