Mapping part of the functional epitope for ligand binding on the receptor for urokinase-type plasminogen activator by site-directed mutagenesis

The urokinase-type plasminogen activator receptor (uPAR) is a glycolipid an chored multidomain member of the Ly-6/uPAR protein domain superfamily. Stud ies by site-directed photoaffinity labeling, chemical cross-linking, and li gand-induced protection against chemical modification have highlighted the possible involvement of uPAR domain I and particularly loop 3 thereof in li gand binding (Ploug, RI. (1998) Biochemistry 37, 16494-16505). Guided by th ese results we have now performed an alanine scanning analysis of this regi on in uPAR by site-directed mutagenesis and subsequently measured the effec ts thereof on the kinetics of uPA binding in real-time by surface plasmon r esonance. Only four positions in loop 3 of uPAR domain I exhibited signific ant changes in the contribution to the free energy of uPA binding (Delta De lta G greater than or equal to 1.3 kcal mol(-1)) upon single-site substitut ions to alanine (i.e. Arg(53), Leu(55), Tyr(57), and Leu(66)). The energeti c impact of these four alanine substitutions was not caused by gross struct ural perturbations, since all monoclonal antibodies tested having conformat ion-dependent epitopes on this domain exhibited unaltered binding kinetics, These sites together with a three-dimensional structure for uPAR may provi de an appropriate target for rational drug design aimed at developing new r eceptor binding antagonists with potential application in cancer therapy.