The Journal of Experimental Medicine
VeriKine-HS Human IFN-Beta
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© The Rockefeller University Press, 0022-1007/1997/4/1487/ $5.00
The Journal of Experimental Medicine, Volume 185, Number 8, April 21, 1997 1487-1492

Brief Definitive Reports

Identification of Amino Acid Residues Important for Ligand Binding to Fas

Gary C. Starling, Jürgen Bajorath, John Emswiler, Jeffrey A. Ledbetter, Alejandro Aruffo, and Peter A. Kiener

From Bristol-Myers Squibb Pharmaceutical Research Institute, Seattle, Washington 98121

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 BIAcoreTM 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.

Address correspondence to Gary C. Starling, Bristol-Myers Squibb Pharmaceutical Research Institute, 3005 First Avenue, Seattle, WA 98121.


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