Two Adjacent Trimeric Fas Ligands Are Required for Fas Signaling and Formation of a Death-Inducing Signaling Complex

• DOI: 10.1128/mcb.23.4.1428-1440.2003
• Publication Date: 2003-01-29T22:44:28Z
• Authors: Nils Holler; Aubry Tardivel; Magdalena Kovacso...; Sylvie Hertig; Olivier Gaide; Fabio Martinon; Antoine Tinel; David Deperthes; Silvio Calderara; Therese Schulthess; Jürgen Engel; Pascal Schneider; Jürg Tschopp
• Keywords: 0301 basic medicine; B-Lymphocytes; Caspase 8; Death Domain Receptor Signaling Adaptor Proteins; Fas Ligand Protein; Cell Death; Fas-Associated Death Domain Protein; CD40 Ligand; Apoptosis; Caspase 9; 3. Good health; Adaptor Proteins, Signal Transducing; Adiponectin; Amino Acid Sequence; Animals; Antigens, CD95/metabolism; Apoptosis/physiology; B-Lymphocytes/metabolism; CD40 Ligand/genetics; CD40 Ligand/metabolism; Carrier Proteins/metabolism; Caspase 8; Caspase 9; Caspases/metabolism; Cell Death/physiology; Cells, Cultured; Collagen/metabolism; Death Domain Receptor Signaling Adaptor Proteins; Dimerization; Fas Ligand Protein; Fas-Associated Death Domain Protein; Humans; Immunoglobulin G/genetics; Immunoglobulin G/metabolism; Intercellular Signaling Peptides and Proteins; Membrane Glycoproteins/genetics; Membrane Glycoproteins/metabolism; Mice; Mice, Inbred C57BL; Molecular Sequence Data; Proteins/genetics; Proteins/metabolism; Receptors, Tumor Necrosis Factor/metabolism; Recombinant Fusion Proteins/genetics; Recombinant Fusion Proteins/metabolism; Signal Transduction; 03 medical and health sciences; Caspases; Immunoglobulin G; Animals; Humans; Adiponectin; Amino Acid Sequence; Collagen; Carrier Proteins; Dimerization; Cells, Cultured; Adaptor Proteins, Signal Transducing

The membrane-bound form of Fas ligand (FasL) signals apoptosis in target cells through engagement of the death receptor Fas, whereas the proteolytically processed, soluble form of FasL does not induce cell death. However, soluble FasL can be rendered active upon cross-linking. Since the minimal extent of oligomerization of FasL that exerts cytotoxicity is unknown, we engineered hexameric proteins containing two trimers of FasL within the same molecule. This was achieved by fusing FasL to the Fc portion of immunoglobulin G1 or to the collagen domain of ACRP30/adiponectin. Trimeric FasL and hexameric FasL both bound to Fas, but only the hexameric forms were highly cytotoxic and competent to signal apoptosis via formation of a death-inducing signaling complex. Three sequential early events in Fas-mediated apoptosis could be dissected, namely, receptor binding, receptor activation, and recruitment of intracellular signaling molecules, each of which occurred independently of the subsequent one. These results demonstrate that the limited oligomerization of FasL, and most likely of some other tumor necrosis factor family ligands such as CD40L, is required for triggering of the signaling pathways.