title: Identification of amino acid residues important for ligand binding to Fas.
abstract: The interaction of Fas (CD95), a member of the tumor necrosis factor receptor (TNFR) family, and its ligand (FasL) triggers programmed cell death (apoptosis) and is involved in the regulation of immune responses. Although the Fas-FasL interaction is conserved across species barriers, little is currently known about the molecular details of this interaction. Our aim was to identify residues in Fas that are important for ligand binding. With the aid of a Fas molecular model, candidate amino 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 FasL binding was examined by measuring the ability of these proteins to inhibit FasL-mediated apoptosis of Jurkat cells and bind FasL in ELISA and BIAcore assays. Mutation of two amino acids, R86 and R87 (D2), to serine totally abolished the ability of Fas to interact with its ligand, whereas mutants K84S, L90S, E93S (D2), or H126S (D3) showed reduced binding compared with wild-type Fas. Two mutants (K78S and H95S) bound FasL comparably to wild type. Therefore, the binding of FasL involves residues in two domains that correspond to positions critical for ligand binding in other family members (TNFR and CD40) but are conserved between murine and human Fas.
authors.affiliation: Bristol-Myers Squibb Pharmaceutical Research Institute, Seattle, Washington 98121, USA.
authors.forename: G C
authors.initials: GC
authors.lastname: Starling
authors.forename: J
authors.initials: J
authors.lastname: Bajorath
authors.forename: J
authors.initials: J
authors.lastname: Emswiler
authors.forename: J A
authors.initials: JA
authors.lastname: Ledbetter
authors.forename: A
authors.initials: A
authors.lastname: Aruffo
authors.forename: P A
authors.initials: PA
authors.lastname: Kiener
mesh_terms: D001665:Binding Sites; D002460:Cell Line; D053222:Fas Ligand Protein; D006801:Humans; D046911:Macromolecular Substances; D008562:Membrane Glycoproteins / Q000737:chemistry*; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D011994:Recombinant Proteins; D013329:Structure-Activity Relationship; D019014:fas Receptor / Q000737:chemistry*
chemical_list: C505661:FASLG protein, human; D053222:Fas Ligand Protein; D046911:Macromolecular Substances; D008562:Membrane Glycoproteins; D011994:Recombinant Proteins; D019014:fas Receptor