DETECTION OF THE RECEPTOR FOR THE HUMAN UROKINASE-TYPE PLASMINOGEN-ACTIVATOR USING FLUORESCEINATED UPA

The urokinase-type plasminogen activator (uPA) is a serine protease th at plays a crucial role in blood coagulation and in tumor invasion and metastasis. uPA is a relatively large polypeptide and binds the uPA r eceptor (uPAR) with high affinity and specificity. Therefore, it was a good candidate for direct labeling with a fluorochrome for detection of the uPAR. We have produced a fluorescein (FITC)-labeled human uPA u sing a conjugation procedure that did not significantly alter its bind ing characteristics to the uPAR. Thirty nM FITC-uPA efficiently stains 2 x 10(5) uPAR-transfected mouse cells in suspension, as determined b y flow cytometric analysis. One mu g of FITC-uPA efficiently stains 2 x 10(5) uPAR transfectants grown on slides and analyzed by fluorescenc e optical microscopy. Human cell lines expressing the endogenous uPAR were stained with similar efficiency. Fixation in paraformaldehyde onl y slightly reduced the efficiency of staining of both transfectants an d cell lines. These characteristics allow the use of FITC-uPA in both static and dynamic morphological studies of uPAR-expressing cells.