Separate domains of the human Fas ligand dictate self-association and receptor binding

The Fas receptor rapidly induces apoptosis when activated by ligand binding or by cross-linking with anti-Fas antibody, The Fas ligand (Fast), a member of the tumor necrosis factor family of ligands, is a IO-kilodalton type II transmembrane protein which is cleaved to produce soluble ligand. Although the Fas-FasL interaction plays a critical role in peripheral T cell homeostasis and cytotoxic T lymphocyte-mediated target cell killing, the requirements for human Fast receptor binding and oligomerization have not been defined. Here we report two distinct domains of the ligand which are responsible for self-association and binding to the Fas receptor. A COOH-terminal sequence of the Fast was found to be required for binding and biological activity, as verified by deletion mutagenesis, use of the NOK-1 blocking antibody and the humanized gld Fast mutation, N-Linked glycosylation of the Fast was not required for biological activity. However, the Fast expression level was dependent upon the three N-linked glycosylation sites. Moreover, the ability of the Fast to self-associate was not dependent upon transmembrane or cytoplasmic sequences, but was localized to a 47-amino acid region in its extracellular domain, These results indicate that the FasL-Fas receptor complex depends upon independent motifs located within the extracellular domain of the Fast.