IDENTIFICATION OF AMINO-ACID-RESIDUES IMPORTANT FOR LIGAND-BINDING TOFAS

The interaction of Fas (CD95), a member of the tumor necrosis factor r eceptor (TNFR) family, and its ligand (FasL) triggers programmed cell death (apoptosis) and is involved in the regulation of immune response s. Although the Fas-FasL interaction is conserved across species barri ers, little is currently known about the molecular details of this int eraction. Our aim was to identify residues in Fas that are important f or ligand binding. With the aid of a Fas molecular model, candidate am ino acid residues were selected in the Fas extracellular domain 2 (D2) and D3 and subjected to serine-scanning mutagenesis to produce mutant Fas molecules in the form of Ig fusion proteins. The effects of these mutations on Fast binding was examined by measuring the ability of th ese proteins to inhibit Fast-mediated apoptosis of Jurkat cells and bi nd Fast in ELISA and BIAcore(TM) assays. Mutation of two amino acids, R86 and R87 (D2), to serine totally abolished the ability of Fas to in teract with its ligand, whereas mutants K84S, L90S, E93S (D2), or H126 S (D3) showed reduced binding compared with wild-type Fas. Two mutants (K78S and H95S) bound FasL comparably to wild type. Therefore, the bi nding of FasL involves residues in two domains that correspond to posi tions critical for ligand binding in other family members (TNFR and CD 40) but are conserved between murine and human Fas.