Direct Binding of Fas-associated Death Domain (FADD) to the Tumor Necrosis Factor-related Apoptosis-inducing Ligand Receptor DR5 Is Regulated by the Death Effector Domain of FADD

Abstract

Members of the tumor necrosis factor superfamily of receptors induce apoptosis by recruiting adaptor molecules through death domain interactions. The central adaptor molecule for these receptors is the death domain-containing protein Fas-associated death domain (FADD). FADD binds a death domain on a receptor or additional adaptor and recruits caspases to the activated receptor. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) signals apoptosis through two receptors, DR4 and DR5. Although there is much interest in TRAIL, the mechanism by which FADD is recruited to the TRAIL receptors is not clear. Using a reverse two-hybrid system we previously identified mutations in the death effector domain of FADD that prevented binding to Fas/CD95. Here we show that these mutations also prevent binding to DR5. FADD-deficient Jurkat cells stably expressing these FADD mutations did not transduce TRAIL or Fas/CD95 signaling. Second site compensating mutations that restore binding to and signaling through Fas/CD95 and DR5 were also in the death effector domain. We conclude that in contrast to current models where the death domain of FADD functions independently of the death effector domain, the death effector domain of FADD comes into direct contact with both TRAIL and Fas/CD95 receptors. Members of the tumor necrosis factor (TNF)1 superfamily of receptors induce a variety of cellular responses including apoptosis, cellular differentiation, and proliferation. A subfamily of these receptors contains a death domain (DD) that is essential for transducing the apoptotic signal. Fas/CD95 is the best characterized member of this family. Binding of Fas ligand (FasL) to a preformed Fas/CD95 trimer (1) results in dimerization of two trimers (2) and higher levels of oligomerization (3). These activated receptors signal the apoptotic response by recruiting FADD to the cytoplasmic DD of the receptor to form the deathinducing signaling complex (DISC). FADD consists of two distinct domains, a DD that binds to the DD of Fas/CD95 and a death effector domain (DED) that binds to DEDs on caspase-8 and caspase-10 (4). The DD and DED have similar structural folds consisting of six anti-parallel ␣-helices, and both form globular protein structures whose only known function is to interact with other proteins (5). Thus, it is thought that binding of ligand to Fas/CD95 results in the recruitment of FADD through DD interactions followed by caspases through DED interactions. The induced proximity of two or more initiator caspases in a complex with FADD and the receptor results in their dimerization and activation (6, 7). Proteolytic processing leads to a fully processed, active form of the caspase that can dissociate from the receptor complex. Once activated, these caspases can then cleave and activate effector caspases such as caspase-3 and other substrates to induce the characteristic phenotypes associated with apoptosis.Activation of TNF receptor 1 requires an additional adaptor protein, TRADD. Binding of TNF␣ to TNF receptor 1 results in...

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

“…13 In addition, although the isolated DD of FADD is sufficient for binding, regions surrounding helix 5 of the FADD DED also contribute to the interaction of FADD with both Fas and DR5. 14,15 Together, these data indicate that FADD binding to Fas is not mediated just by the residues in helices 2 and 3 of the DD but instead involves a much larger region of the DD and regions in the DED.…”

“…15 To address this question, we mapped the DR5-binding surface of the FADD DD and identified residues within helices 2-6 of the DD that when mutated, prevented binding to DR5.…”

“…15 Therefore, full-length versions of each FADD mutation identified in the functional screen were tested for interaction with the cytoplasmic domain of DR5 in a directed yeast twohybrid assay. Wild-type FADD showed strong interaction with DR5 whereas empty vector and most of the FADD mutations did not (Figure 1c).…”

Apoptosis and autophagy: regulatory connections between two supposedly different processes

“…Upon ligand binding, death receptors recruit an adapter protein called FADD (56)(57)(58), which brings caspase-8 to the DISC where it is dimerized and catalytically activated (59). Active caspase-8 can then directly activate effector caspases such as caspase-3 or can activate the intrinsic apoptosis pathway by cleaving the BH3-only protein Bid resulting in its translocation to the mitochondria (60).…”

Quercetin sensitizes human hepatoma cells to TRAIL‐induced apoptosis via Sp1‐mediated DR5 up‐regulation and proteasome‐mediated c‐FLIPS down‐regulation

“…Binding of TRAIL to either DR4 or DR5, two death receptors of TRAIL, leads to oligomerization and clustering of their intracellular death domains. The subsequent interaction of DR4 or DR5 with the adaptor molecule, FADD (Fas-associated death domain), via their respective death domains leads to recruitment and activation of caspase-8 [ Thomas et al, 2004]. Finally, caspase-8 activates the executioner caspases (e.g., caspase-3 and caspase-7), leading to apoptotic cell death [Srivastava, 2001].…”