Vincent Ellis

Structure—function relationships in the receptor for urokinase-type plasminogen activator Comparison to other members of the Ly-6 family and snake venom α-neurotoxins

Plasminogen activation is regulated by the interaction between urokinase‐type plasminogen activator (uPA) and its specific glycolipid‐anchored cell surface receptor (uPAR). uPAR is composed of three homologous domains and is the only multi‐domain member of the Ly‐6 family of glycolipid‐anchored membrane proteins. Recent evidence has highlighted similarities between the individual domains of uPAR and the large family of secreted, single domain snake venom α‐neurotoxins, suggesting that uPAR may adopt the same gross folding pattern as these structurally well characterized proteins. Structural aspects of the binding between α‐neurotoxins and the acetylcholine receptor may have a major influence on future studies of the interaction between uPA and uPAR.

Cell-induced potentiation of the plasminogen activation system is abolished by a monoclonal antibody that recognizes the NH2-terminal domain of the urokinase receptor

We have raised four monoclonal antibodies recognizing different epitopes within the human cell‐surface receptor for urokinase‐type plasminogen activator (u‐PA). One of these antibodies completely abolishes the potentiation of plasmin generation observed upon incubation of the zymogens pro‐u‐PA and plasminogen with U937 cells. This antibody, which is also the only one to completely inhibit the binding of DFP‐inactivated [125I]‐u‐PA to U937 cells, is directed against the u‐PA binding NH2‐terminal domain of u‐PAR, a well‐defined fragment formed by limited chymotrypsin digestion of purified u‐PAR, demonstrating the functional independence of the u‐PA binding domain as well as the critical role of u‐PAR in the assembly of the cell‐surface plasminogen activation system.

The Urokinase Receptor: Involvement in Cell Surface Proteolysis and Cancer Invasion

Proteolytic enzymes are thought to play a primary role in the dissolution of the extracellular matrix that is necessary b r the passage of migrating cells through tissue barriers in a diversity of biological processes.lJ The migration and invasion of cells in physiological situations, such as trophoblast implantation, embryo morphogenesis, angiogenesis, inflammation, wound healing and other processes involving tissue destruction and remodelling, is of limited extent and duration and therehre must be subject to strict regulation to initiate, localize, and terminate the proteolytic activity. The same proteolytic enzyme systems are also thought to contribute to the invasive properties and metastatic potential of malignant tumor cells. However, the concept that this occurs basically as a consequence of the over-expression of the pmteolytic enzymes may be too simplistic. The migration of all cells requires, in addition to matrix dissolution, cell attachment to the matrix which could not occur in the presence of unlimited matrix degradation. Therefbre there is also a necessity b r these proteolytic processes to be highly regulated in tumor cells. This is particularly apparent when the complex multistep nature of the metastatic process is considered. For blood-spread metastases this involves the sequential detachment of cells from the primary tumor mass, the dissolution of basement membranes and interstitial connective tissue, the migration of the tumor cells first into and then out of the vascular system through the endothelial lining, bebre re-estabhhment at a distant site. In this respect the tumor metastasis may be regarded as one of the most complex cell migratory processes, and consequently there must be strict temporal and spatial control of the proteolytic processes leading to it. Several proteolytic enzyme systems may contribute to the overall process of extracellular matrix degradation, including plasminogen activators,l matrix metalloproteases (cg., collagenases),3 and cathepsin D,4 and it is likely that these systems can act either independently or in a concerted manner. However the generation of the broad specificity protease plasmin by plasrninogen activators is possibly central to this whole

Urokinase-type plasminogen activation in three human breast cancer cell lines correlates with their in vitro invasiveness

In order to invade and spread cancer cells must degrade extracellular matrix proteins. This degradation is catalysed by the concerted action of several enzymes, including the serine protease plasmin. Several experimental studies have shown that inhibition of plasmin formation reduces cancer cell invasion and metastasis, indicating a critical role of this proteolytic pathway in these processes. In order to further study the role of plasmin in cancer progression, we have characterized urokinase-type plasminogen activator (uPA) mediated plasmin formation in three human breast cancer cell lines. Using monoclonal antibodies against uPA and its receptor uPAR, we have investigated the contribution of uPA and uPAR to invasive capacity in an in vitro invasion assay. MDA-MB-231 BAG cells were found to express high protein levels of uPA, uPAR and PAI-1. MDA-MB-435 BAG cells produced low amounts of uPA, PAW and moderate amounts of uPAR, whereas MCF-7 BAG cells showed low levels of uPA, uPAR and PAM protein. In a plasmin generation assay MDA-MB-231 BAG cells were highly active in mediating plasmin formation, which could be abolished by adding either an anticatalytic monoclonal antibody to uPA (clone 5) or an anti-uPAR monoclonal antibody (clone R3), which blocks binding of uPA to uPAR. The two other cell lines lacked the capacity to mediate plasmin formation. In the Matrigel invasion assay the cells showed activity in this order: MCF-7 BAG < MDA-MB-435 BAG < MDA-MB-231 BAG. Testing MDA-MB-231 BAG cells in the Matrigel invasion assay revealed that invasion could be inhibited in a dose-dependent manner either by the clone 5 uPA antibody or by the clone R3 uPAR antibody, suggesting that the cell surface uPA system is actively involved in this invasive process. It is concluded that these three cell lines constitute a valuable model system for in vitro studies of the role of cell surface uPA in cancer cell invasion and has application in the search for novel compounds which inhibit mechanisms involved in uPA-mediated plasmin generation on cancer cells.