P45 forms a complex with FADD.

(A) Schematic representation of the domains in the mouse p75, p45 and FADD (SP, signal peptide; TNFR, tumor necrosis factor receptor; ECD, extracellular domain; TM, transmembrane domain; ICD, intracellular domain; DD, death domain, PDZ, post synaptic density protein, Drosophila disc large tumor suppressor and zonula occludens-1 protein domain; DED, death effector domain) (B) Full-length FADD is essential for the p45-FADD interaction. V5-tagged full length FADD, FADD-ΔDED or FADD-ΔDD (data not shown) was co-transfected with Flag-tagged p45 into HEK293 cells. The lysates were immunorecipitated by an anti-Flag M2 antibody and immunoblotted with an anti-V5 antibody. (C) Both FADD dimerization and FADD-Caspase-8 (CSP-8) interaction are critical for p45-FADD interaction. A series of FADD point mutants were created according to reports in the literature. Schematic representation of the domains in FADD is shown (DED, death-effector domain; DD, death domain). Point-mutated amino acid in FADD is indicated above for its protein-binding ability or individual function (CSP-8, caspase-8/Flice; PhosP, phosphorylation sites). Interaction between FADD and FAS and the serine phosphorylations of FADD are not essential for p45-FADD interaction. In contrast, the binding sites for FADD dimerization and FADD-Caspase-8 interaction, which are located in the α3 helix in the FADD death effector domain, are critical for p45-FADD interaction, suggesting that FADD dimerization or FADD-Caspase-8 interaction might be essential for sequential p45-FADD interaction. (C, D).