SYSTEMATIC MUTATIONAL ANALYSIS OF THE RECEPTOR-BINDING REGION OF THE HUMAN UROKINASE-TYPE PLASMINOGEN-ACTIVATOR

The amino-terminal fragment of human uPA (ATF; amino acids 1-135), whi ch contains the binding site for the uPA receptor (uPAR, CD87) was exp ressed in the yeast Saccharomyces cerevisiae. Recombinant yeast ATE mo dified and extended by an amino-terminal in-frame insertion of a His, tract, was purified from total protein extracts by nickel chelate affi nity chromatography and shown to be functionally active since it effic iently competes with uPA for binding to cell-surface-associated uPAR. The ATF expression plasmid served as a template for the construction o f a series of site-directed mutants in order to define those amino aci ds that are important for binding to uPAR. All mutant ATF proteins but one (deletion of Ser26) were expressed in a stable form (about 20-30 ng/mg total protein) and the binding capacity of each mutant was teste d by a uPA-ligand binding assay employing recombinant uPAR immobilized to a microtiter plate. Each of the 11 amino acids of loop B of the bi nding region of uPA (amino acids 20-30) were individually substituted with alanine. Lys23, Tyr24, Phe25, Ile28, and Trp30 were important det erminants for uPAR binding. A systematic alanine scan was also perform ed with chemically synthesized linear peptides spanning amino acids 14 -32 of ATE Comparable results to those with the yeast ATF mutants were obtained. In a different set of experiments, those amino acids of the uPAR-binding region of uPA that are only conserved between man and ba boon but not in other species were altered: whereas substitution of Th r18 by alanine or Asn32 by serine had hardly any effect, replacement o f Asn22 by tyrosine and Trp30 by arginine (both positions are strictly conserved in other mammals) led to ATF variants incapable of interact ing with human uPAR. Deletion of either Val20, Ser21, Lys23, His29 or Val20 plus Ser21, respectively, also generated non-reactive ATF mutant s. Finally, Lys23 in ATF was substituted with certain amino acids: whe reas the replacement of Lys23 by alanine, histidine or glutamine gener ated ATF variants with moderate uPAR-binding activity, the introductio n of a negatively charged amino acid (exchange of Lys23 by glutamic ac id) completely abolished uPAR-binding activity. The results presented for the ATF mutants and uPA-derived peptides may provide clues necessa ry to establish the nature of the physical interaction of uPA with its receptor and may help to develop uPA-derived peptide analogues as pot ential therapeutic agents to block tumor cell-associated uPA/uPAR inte raction.