Fas- and Tumor Necrosis Factor-mediated Apoptosis Uses the Same Binding Surface of FADD to Trigger Signal Transduction

Abstract

FADD is known to function as a common signaling conduit in Fas-and tumor necrosis factor (TNF)-mediated apoptosis. The convergent death signals from the Fas receptor and TNF receptor 1 are transferred to FADD by death domain interactions triggering the same cellular event, caspase-8 activation. In this work, we investigated whether the same binding surface of FADD is used for both signaling pathways by using FADD death domain mutants. Mutations in helices ␣2 and ␣3 of the FADD death domain, the interacting surface with the Fas death domain, affected TNF-mediated apoptosis to various extents. This indicated that TNF-mediated apoptosis uses the same binding surface of the FADD death domain as Fas-mediated apoptosis. The binding specificity is not the same, however. Some mutations affected the binding affinity of the Fas death domain for the FADD death domain, but did not influence TNFmediated apoptosis and vice versa. Interestingly, all mutants tested that affected TNF-mediated apoptosis have structural perturbations, implying that the structural integrity, involving helices ␣2 and ␣3 in particular, is critical in TNF-mediated apoptosis. Our results suggest that different signaling molecules use a similar structural interaction to trigger the same cellular event, such as caspase-8 recruitment, which could be typical in convergent signal transduction.The tumor necrosis factor (TNF) receptor family consists of type I transmembrane proteins characterized by cysteine-rich repeats in their extracellular ligand-binding domains (1). A subset of the family, comprising CD95/Fas, TNFR1, p75 nerve growth factor receptor, DR3 (death receptor 3), and the two TRAIL (TNF-related apoptosis-inducing ligand) receptors (DR4/TRAIL receptor 1 and DR5/TRAIL receptor 2), shares a conserved motif in the cytoplasmic regions known as the death domain, which is essential for transducing the apoptotic signal (2-4). Upon receptor ligation, the death domain acts as the docking site for homotypic interaction with death domain-containing cytoplasmic proteins such as FADD (Fas-associated death domain protein) (5, 6) and TRADD (TNFR1-associated death domain protein) (7). FADD binds directly to Fas and is also recruited to TNFR1, DR3, and possibly other related receptors via TRADD (3,8). The death effector domain at the N-terminal end of FADD then interacts with a related motif in the prodomain of caspase-8 (5, 9) or caspase-10b (10). Activation of these upstream cysteine proteases is thought to trigger a proteolytic cascade, which apparently constitutes the execution of apoptosis. Under certain environments, however, Fas and other members of the TNFR family that contain death domains can stimulate alternative signaling pathways, which exert positive effects on cell survival and proliferation rather than triggering apoptosis (1).Endogenous FADD associates with Fas in an activation-dependent fashion (11), and TRADD and RIP (receptor-interacting protein) have been found in the activated TNFR1 complex (8). It has been postulated that TRADD acts as an ...

FADD self-association is required for stable interaction with an activated death receptor

“…DN-FADD tends to be effective only (Figure 2d) or when DN-FADD is present in significant excess over the quantity of DR in a given cell type. Thus, the FADD DED is required for stable association with an activated DR. Mutagenesis of the FADD DD and DED revealed not only the likely surfaces of the protein necessary for interaction with the receptor 4,18,19,23,26,30 and the initiator procaspases (Figure 3), but also suggested that part of the DED serves a 'universal' purpose for FADD function. This 'universal' surface encompasses portions of the second and third a helices, to include (Figure 4) and possibly Q34 and K35.…”

Extensive regions of the FADD death domain are required for binding to the TRAIL receptor DR5

“…[9][10][11][12] Both consist of six antiparallel alpha helices tethered together by a small linker peptide. DD interactions are mediated through charge-charge interactions whereas DED interactions appear to occur mostly through hydrophobic residues.…”

“…Several of these residues have previously been shown to be necessary for FADD binding to Fas and TRADD. 9,10,12,13 In addition we made mutations that were in regions not previously shown to be important for FADD binding to death receptors. To determine which mutations prevented FADD binding to the DR5 receptor, we performed a functional assay using a dominant negative version of FADD (FADD-DD).…”

“…Both involve the DED and an expanded binding surface on the DD that encompasses more than just helices 2 and 3 as previously thought. 9,10 Several residues in FADD identified as preventing interaction with DR5 (R113, R117A and D123) are essential for interaction with Fas. 9 Additionally R166, Q169, L172 and D175, which form a loop between helices 5 and 6 of FADD, were recently shown to contribute to the interaction with Fas.…”

“…9,10 Several residues in FADD identified as preventing interaction with DR5 (R113, R117A and D123) are essential for interaction with Fas. 9 Additionally R166, Q169, L172 and D175, which form a loop between helices 5 and 6 of FADD, were recently shown to contribute to the interaction with Fas. 13 FADD also uses this loop for binding to DR5 because mutations in two of these residues (R166E and D175R) inhibited binding to DR5.…”