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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.

Urokinase (uPA) has the striking ability to cleave its receptor, uPAR, thereby inactivating the binding potential of this molecule. Here we demonstrate that the glycosylphosphatidylinositol (GPI) anchor of uPAR, which is attached to the third domain, is an important determinant in governing this reaction, even though the actual cleavage occurs between the first and second domains. Purified full-length GPI-anchored uPAR (GPI-uPAR) proved much more susceptible to uPA-mediated cleavage than recombinant truncated soluble uPAR (suPAR), which lacks the glycolipid anchor. This was not a general difference in proteolytic susceptibility since GPI-uPAR and suPAR were cleaved with equal efficiency by plasmin. Since the amino acid sequences of GPI-uPAR and suPAR are identical except for the C-terminal truncation, the different cleavage patterns suggest that the two uPAR variants differ in the conformation or the flexibility of the linker region between domains 1 and 2. This was supported by the fact that an antibody to the peptide AVTYSRSRYLE, amino acids 84-94 in the linker region, recognizes GPI-uPAR but not suPAR. This difference in the linker region is thus caused by a difference in a remote hydrophobic region. In accordance with this model, when the hydrophobic lipid moiety was removed from the glycolipid anchor by phospholipase C, low concentrations of uPA could no longer cleave the modified GPI-uPAR and the reactivity to the peptide antibody was greatly decreased. Naturally occurring suPAR, purified from plasma, was found to have a similar resistance to uPA cleavage as phospholipase C-treated GPI-uPAR and recombinant suPAR.

The urokinase plasminogen activator receptor-associated protein/Endo180 (uPARAP/Endo180) is a newly discovered member of the macrophage mannose receptor family that was reported to interact with ligand-bound urokinase plasminogen activator receptor (uPAR), matrix metalloprotease-13 (MMP-13), and collagen V on the cell surface. We have determined the sites of expression of this novel receptor during murine postimplantation development. uPARAP/Endo180 was expressed in all tissues undergoing primary ossification, including the developing bones of the viscerocranium and calvarium that ossify intramembranously, and developing long bones undergoing endochondral ossification. uPARAP/Endo180 mRNA was expressed by both immature osteoblasts and by mature osteocalcin-producing osteoblasts-osteocytes, and was coexpressed with MMP-13. Interestingly, osteoblasts also expressed uPAR. Besides bone-forming tissues, uPARAP/Endo180 expression was detected only in a mesenchymal condensation of the midbrain and in the developing lungs. The data suggest a function of this novel protease receptor in bone development, possibly mediated through its interactions with uPAR, MMP-13, or collagen V.

Certain monoclonal antibodies are capable of inhibiting the biological binding reactions of their target proteins. At the molecular level, this type of effect may be brought about by completely different mechanisms, such as competition for common binding determinants, steric hindrance or interference with conformational properties of the receptor critical for ligand binding. This distinction is central when employing the antibodies as tools in the elucidation of the structure-function relationship of the protein in question. We have studied the effect of monoclonal antibodies against the urokinase plasminogen activator receptor (uPAR), a protein located on the surface of various types of malignant and normal cells which is involved in the direction of proteolytic degradation reactions in the extracellular matrix. We show that surface plasmon resonance/biomolecular interaction analysis (BIA) can be employed as a highly useful tool to characterize the inhibitory mechanism of specific antagonist antibodies. Two inhibitory antibodies against uPAR, mAb R3 and mAb R5, were shown to exhibit competitive and non-competitive inhibition, respectively, of ligand binding to the receptor. The former antibody efficiently blocked the receptor against subsequent ligand binding but was unable to promote the dissociation of a preformed receptor-ligand complex. The latter antibody was capable of binding the preformed complex, forming a transient trimolecular assembly, and promoting the dissociation of the uPA/uPAR complex. The continuous recording of binding and dissociation, obtained in BIA, is central in characterizing these phenomena. The identification of a non-competitive inhibitory mechanism against this receptor reveals the presence of a determinant which influences the binding properties of a remote site in the molecular structure and which could be an important target for a putative synthetic antagonist.

To investigate the role of plasmin(ogen) in mammary tumor development and progression, plasminogen-deficient mice were crossed with transgenic mice expressing Polyoma middle T antigen under the control of the mouse mammary tumor virus long terminal repeat. Virgin females carrying the Polyoma middle T antigen uniformly developed multiple, bilateral mammary tumors, regardless of the presence or absence of circulating plasminogen. Both the age at which these tumors became palpable and subsequent tumor growth were indistinguishable between plasminogen-deficient mice and plasminogen-expressing littermates. However, plasminogen was found to greatly modify the metastatic potential in this model system; lung metastasis in plasminogen-deficient mice was significantly reduced as compared to littermate controls with respect to frequency of occurrence, total number of metastases, and total metastatic tumor burden. Plasminogen activators, as well as other key factors that govern the conversion of plasminogen to plasmin, were expressed within the mammary tumors, suggesting that the plasminogen/plasmin system may promote metastasis by contributing to tumor-associated extracellular proteolysis. The data provide direct evidence that plasmin(ogen) is a tumor progression factor in PymT-induced mammary cancer, and support the hypothesis that hemostatic factors play an important role in tumor biology.