uPA (urokinase type plasminogen activator) is a thrombolytic protease which has been implicated in certain non-thrombolytic physiological processes, most notable the invasive growth or migration of metastatic cells and of macrophages. The role of uPA in cell migration appears to involve binding of this protease to specific cell surface receptors. I hope to engineer uPA derived molecules which are proteolytically inactive and which bend with enhance avidity to the uPA receptor. These molecules may serve as competitive inhibitors of native uPA receptor binding and in this way be useful therapeutic agents by inhibiting physiological processes requiring receptor bound uPA. Towards achieving this end, I plan, in phase I of this project, to develop a uPA receptor binding assay. This assay will involve yeast secretion of uPA molecules onto nitrocellulose filters and probing of these filters with 1251-labeled, purified receptor molecules. The assay will be designed to be used in phase II of this project for rapid screening of yeast colonies secreting uPA molecules which have altered receptor binding. I also plan, in phase I to construct a library in E.coli of vectors with mutations within the cDNA encoding the receptor binding region of uPA. In phase II, I plan to screen this library and to follow strategies leading to the engineering of uPA molecules with enhanced receptor binding and to test the ability of these molecules to competitively inhibit binding by wild-type molecules. In collaboration with others, I plan to test the ability of these molecules as inhibitors of invasive cell migration.
