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 (FasL), a member of the tumor necrosis factor family of ligands, is a 40-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 FasL 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 FasL 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 FasL mutation. N-Linked glycosylation of the FasL was not required for biological activity. However, the FasL expression level was dependent upon the three N-linked glycosylation sites. Moreover, the ability of the FasL 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 FasL.
Orlinick
J R
JR
Department of Cell Biology and Anatomy, Cornell University Medical College, New York, New York 10021, USA.
Elkon
K B
KB
Chao
M V
MV
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D001704:Biopolymers; D019556:COS Cells; D002460:Cell Line; D018076:DNA, Complementary; D053222:Fas Ligand Protein; D006031:Glycosylation; D006801:Humans; D008562:Membrane Glycoproteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D017384:Sequence Deletion; D014162:Transfection
D000911:Antibodies, Monoclonal; D001704:Biopolymers; D018076:DNA, Complementary; C505661:FASLG protein, human; D053222:Fas Ligand Protein; D008562:Membrane Glycoproteins