A COMPETITIVE CHROMOGENIC ASSAY TO STUDY THE FUNCTIONAL INTERACTION OF UROKINASE-TYPE PLASMINOGEN-ACTIVATOR WITH ITS RECEPTOR

Urokinase-type plasminogen activator (uPA) converts plasminogen to pla smin which degrades various extracellular matrix components. uPA is fo cused to the cell surface via binding to a specific receptor (uPAR, al so termed CD87). uPAR-bound uPA mediates pericellular proteolysis in a variety of biological processes, e.g. cell migration, tissue remodeli ng and tumor invasion. We have developed a competitive microtiter plat e-based chromogenic assay which allows the analysis of uPA/uPAR intera ction. The plates are coated with recombinant uPAR expressed in Chines e hamster ovary (CHO) cells. Proteolytically active uPA (HMW-uPA) is a dded to the microtiter plate-attached uPAR. The amount of receptor-bou nd uPA is then determined indirectly via addition of plasminogen, whic h is activated to plasmin, followed by cleavage of a plasmin-specific chromogenic substrate. Substances interfering with binding of HMW-uPA to uPAR diminish the generation of plasmin, as indicated by a reductio n of cleaved chromogenic substrate. This assay was used to analyze the inhibitory capacity of a variety of proteins and peptides, respective ly, on the uPA/uPAR interaction: i) uPAR and uPAR-variants expressed i n CHO cells, yeast or E. coli, ii) the aminoterminal fragment (ATF) of human uPA or yeast recombinant pro-uPA, iii) synthetic peptides deriv ed from the sequence of the uPAR-binding region of uPA, and iv) antibo dies directed against uPAR. This assay may be helpful in identifying u PA and uPAR analogues or antagonists which efficiently block uPA/uPAR interaction.