Mapping Part of the Functional Epitope for Ligand Binding on the Receptor for Urokinase-type Plasminogen Activator by Site-directed Mutagenesis

Henrik Gårdsvoll, Keld Danø, Michael Ploug
1999 Journal of Biological Chemistry  
The urokinase-type plasminogen activator receptor (uPAR) is a glycolipid anchored multidomain member of the Ly-6/uPAR protein domain superfamily. Studies by site-directed photoaffinity labeling, chemical cross-linking, and ligand-induced protection against chemical modification have highlighted the possible involvement of uPAR domain I and particularly loop 3 thereof in ligand binding (Ploug, M. (1998) Biochemistry 37, 16494 -16505). Guided by these results we have now performed an alanine
more » ... ing analysis of this region in uPAR by site-directed mutagenesis and subsequently measured the effects thereof on the kinetics of uPA binding in real-time by surface plasmon resonance. Only four positions in loop 3 of uPAR domain I exhibited significant changes in the contribution to the free energy of uPA binding (⌬⌬G > 1.3 kcal mol ؊1 ) upon single-site substitutions to alanine (i.e. Arg 53 , Leu 55 , Tyr 57 , and Leu 66 ). The energetic impact of these four alanine substitutions was not caused by gross structural perturbations, since all monoclonal antibodies tested having conformationdependent epitopes on this domain exhibited unaltered binding kinetics. These sites together with a three-dimensional structure for uPAR may provide an appropriate target for rational drug design aimed at developing new receptor binding antagonists with potential application in cancer therapy.
doi:10.1074/jbc.274.53.37995 pmid:10608868 fatcat:klvigs2vybffzfnvq5cjtlhnne