Dominant negative mutants of TRAF3 reveal an important role for the coiled coil domains in cell death signaling by the lymphotoxin-β receptor

Ligation of the lymphotoxin-beta receptor (LT beta R) recruits tumor necrosis factor receptor-associated factor-3 (TRAF3) and initiates cell death in HT29 adenocarcinoma cells. The minimal receptor binding domain (TRAF-C) defined by two hybrid analyses is not sufficient for direct recruitment to the ligated receptor. A series of TRAF3 deletion mutants reveal that a subregion of the coiled coil motif is required for efficient recruitment to the LT beta R. Furthermore, the ability of TRAF3 to self-associate maps to an adjacent subregion, A TRAF3 deletion mutant that lacks the N-terminal zinc RING and zinc finger motifs, but retains the coiled coil and TRAF-C motifs, competitively displaces endogenous TRAF3 from the LT beta R. A second TRAF3 mutant that lacks the receptor binding domain, yet contains the TRAF3 self association domain, prevents TRAF3 homodimers from being recruited to the LT beta R. Both of these mutants have a dominant negative effect on cell death and demonstrate that the recruitment of TRAF3 oligomers is necessary to initiate signal transduction that activates the cell death pathway.