Jee Y Chung Young Chul Park Hong Ye and Hao Wu

Department of Biochemistry, Weill Medical College of Cornell University, New York, New York

Unlike receptors with intrinsic kinase activity in their intracellular domains or in direct association with intracellular enzymes, members of the tumor necrosis factor (TNF) receptor superfamily and the interleukin-1 (IL-1) receptor/ Toll-like receptor (IL-1R/TLR) superfamily use adapter proteins to couple to enzymatic activation and signal amplification. The TNF-receptor-associated factors (TRAFs), which currently consist of six members (TRAF1-6) in mammals [1-8], have emerged to be the major adapter proteins for these receptors. Through versatile protein-protein interactions, TRAFs link receptor activation to downstream kinase activation and eventually the stimulation of nuclear factor kB (NF-kB) and AP-1 transcriptional activity. Collectively, a wide range of cellular effects including cell survival, proliferation, and differentiation may be elicited by TRAF signaling.

The TRAFs participate in receptor signal transduction by either direct association with receptors or indirect interaction through additional adapter proteins, in at least three distinct pathways (Fig. 1). Members of the TNF receptor superfamily that do not contain intracellular death domains, such as TNF-R2 and CD40, recruit TRAFs directly via short sequences in their intracellular tails [1,2,5,7]. The Epstein-Barr virus transforming protein LMP1 has also been shown to directly recruit TRAFs for viral survival and cell transformation [3]. Receptors that contain an intracellular death domain, such as TNF-R1, first recruit an adapter protein TRADD via a death domain-death domain interaction [9]. TRADD then serves as a central platform of the TNF-R1 signaling complex, which assembles TRAF2 [10] and RIP [11,12] for survival signaling and Fas-associated death domain (FADD) and caspase-8 for inducing apoptosis [10,13]. Members of the IL-1R/TLR superfamily contain a protein interaction module known as the TIR domain, which recruits sequentially MyD88 [14], a TIR-domain- and death-domain-containing protein, and IRAK [15], an adapter Ser/Thr kinase with a death domain. Oligomerization of IRAK appears to result in its association with intracellular TRAF6 to elicit signaling [8].

Members of the TRAF family are characterized by the presence of a novel TRAF domain at the C terminus which in turn consists of a coiled-coil domain followed by a conserved TRAF-C domain 1 (Fig. 2). The TRAF domain plays an important role in TRAF function by mediating self-association, receptor interaction, and interactions with other signaling proteins such as TRADD and IRAK [8,10,16]. The N-terminal portion of most of the TRAF proteins contains a RING finger and several (five to seven) zinc finger motifs, which is important for downstream signaling events [16,17]. The presence of TRAFs is conserved genetically in other multi-cellular organisms such as Drosophila [18], Caenorhabditis elegans [19], and Dictyostelium discoideum [4].

Crystal structures and biochemical characterizations have revealed a conserved trimeric association of TRAFs that is mediated by both the coiled-coil domain and the TRAF-C domain (Fig. 3 A) [20-22]. This trimeric stoichiometry of TRAFs provides a structural basis for the signal transduc-tion across the cellular membrane after receptor trimeriza-tion by trimeric extracellular ligands in the tumor necrosis factor (TNF) superfamily [23]. Interestingly, recent studies suggest that specific ligand-induced receptor trimerization may be primed by nonsignaling receptor preassociation prior to ligand binding [24]. Thermodynamic characterization reveals the low-affinity nature of monomeric TRAF2-receptor interactions, thereby confirming the importance of oligomerization-based affinity enhancement or avidity in receptor-mediated TRAF recruitment [25].

Figure 1 Membrane-proximal events in TRAF signaling, showing direct and indirect TRAF recruitment to post-receptor signaling complexes.

Figure 2 Domain organization of TRAFs.

Receptor sequences bind symmetrically to the surface groove on the TRAF-C domain of TRAF2 in an extended conformation (Fig. 3A) [20,22]. It makes main-chain hydrogen bonding interactions with the edge of the P-sandwich structure of the TRAF-C domain. Specific side-chain interactions observed in multiple TRAF2-receptor complexes have led to the establishment of short TRAF2-binding motifs [26]. Residues on the TRAF2 surface used for receptor interaction is generally conserved in TRAFs 1, 3, and 5, suggesting that these TRAFs interact with similar receptor sequences. However, a somewhat different binding mode has been observed for the interaction of TRAF3 with the same sequence from CD40, which forms a hairpin on the TRAF3 surface [27].

The mode of TRAF2 recruitment by TRADD has been revealed by the crystal structure of the TRAF2-TRADD complex (Fig. 3B) [28]. The more extensive TRAF2-TRADD interface overlaps spatially and therefore potentially competes with TRAF2-receptor-peptide interactions. Biochemical characterization of the interaction has shown that TRAF2 has a significantly higher affinity for TRADD than for the peptide motifs in direct receptor interactions, which leads to more efficient initiation of TRAF2 signaling by TRADD. In addition, TRADD interacts with only TRAF1 and TRAF2, but not other members of the TRAF family. It appears that TRAF1 and TRAF2 work in conjunction with associated caspase inhibitors, cIAP-1 and cIAP-2, to fully suppress TNF-induced apoptosis in the TNF-R1 signaling complex [28,29]. This leads to a dominance of survival signaling for TNF-R1 under most circumstances.

The TRAFs appears to undergo specific intracellular trafficking upon receptor stimulation, from either a diffuse or punctate cytoplasmic distribution to a cell-surface relocaliza-tion [3,30]. Accumulating evidence suggests that the recruitment of TRAFs to membrane microdomains or rafts is a crucial step for the initiation of TRAF signaling events [31-33]. The formation of TRAF-containing rafts may stabilize the receptor-signaling complex and bring TRAFs to the

Figure 3 TRAF structures. (A) Mushroom-shaped trimeric structure of the TRAF domain of TRAF2 in complex with TNF-R2 [20], shown with the three-fold axis vertical. The coiled-coil region (stalk) is in yellow. The P-strands of the three TRAF-C domains are shown, respectively, in blue, green, and purple. Bound peptides from TNF-R2 are shown as orange arrows indicating the direction of the peptide chains. (B) Ribbon diagram of the complex between TRADD and TRAF2 [28], shown with the three-fold axis vertical. TRAF2: blue, green, and purple; TRADD: magenta, red, and yellow.

Figure 3 TRAF structures. (A) Mushroom-shaped trimeric structure of the TRAF domain of TRAF2 in complex with TNF-R2 [20], shown with the three-fold axis vertical. The coiled-coil region (stalk) is in yellow. The P-strands of the three TRAF-C domains are shown, respectively, in blue, green, and purple. Bound peptides from TNF-R2 are shown as orange arrows indicating the direction of the peptide chains. (B) Ribbon diagram of the complex between TRADD and TRAF2 [28], shown with the three-fold axis vertical. TRAF2: blue, green, and purple; TRADD: magenta, red, and yellow.

proximity of kinases and other signaling proteins. Because many different receptors recruit the same TRAFs, the availability of intracellular TRAFs can be a limiting factor in TRAF-mediated signal transduction. It has been shown that receptor activation can lead to depletion of the cytoplasmic pool of TRAF2 via relocalization to insoluble fractions [34] and/or TRAF degradation [35,36]. This suggests a competitive nature of TRAF signaling by different receptors and adds to the potential complexity of TRAF-mediated signal regulation.


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