CD40-TUMOR NECROSIS FACTOR RECEPTOR-ASSOCIATED FACTOR (TRAF) INTERACTIONS - REGULATION OF CD40 SIGNALING THROUGH MULTIPLE TRAF BINDING-SITES AND TRAF HETERO-OLIGOMERIZATION

CD40 is a TNF receptor superfamily member that provides activation sig nals in antigen-presenting cells such as B cells, macrophages, and den dritic cells. Multimerization of CD40 by its ligand initiates signalin g by recruiting TNF receptor-associated factors (TRAFs) to the CD40 cy toplasmic domain. Recombinant human TRAF proteins overexpressed in ins ect cells were biochemically characterized and used to finely map TRAF binding regions in the human CD40 cytoplasmic domain. TRAF1, TRAF2, T RAF3, and TRAF6, but not TRAF4 or TRAF5, bound directly to the CD40 cy toplasmic domain. CD40 interactions with TRAF2 and TRAF3 were stronger than the interactions with TRAF1 and TRAF6. Full-length TRAF3 and TRA F5 formed hetero-oligomers, presumably through their predicted isoleuc ine zippers. TRAF3-TRAF5 hetero-oligomers interacted with CD40, indica ting that TRAF5 can be indirectly recruited to the CD40 cytoplasmic do main. Overlapping peptides synthesized on cellulose membranes were use d to map each TRAF interaction region. TRAF1, TRAF2, and TRAF3 interac ted with the same region. The recognition site for TRAF6 was a nonover lapping membrane proximal region. Using peptides with progressive dele tions, a minimal TRAF1, TRAF2, and TRAF3 binding region was mapped to the PVQET sequence in the CD40 cytoplasmic domain. The minimal region for TRAF6 binding was the sequence QEPQEINF. These studies demonstrate that the CD40 cytoplasmic domain contains two nonoverlapping TRAF bin ding regions and suggest that TRAF1, TRAF2, and TRAF3 could bind compe titively to one site. Relative affinities and competition of individua l and hetero-oligomeric TRAF proteins for CD40 binding sites may contr ibute to receptor specificity and cell-type selectivity in CD40-depend ent signaling.