In this report, we demonstrate that 4-1BB and Ox40, two members of the TNF-NGF receptor family, can use TRAF molecules to trigger cytoplasmic signal transduction cascades. Both m4-1BBCP and mOx40CP interacted with TRAF2 in a yeast two-hybrid system. The specificity of this interaction was confirmed by coimmunoprecipitation experiments in mammalian cells. These results indicate that the TRAF2 interaction with the cytoplasmic tails of 4-1BB and Ox40 is specific. We did not obtain any other members of the TRAF family in the yeast two-hybrid library screens, possibly because m4-1BBCP and mOx40CP bind only to TRAF2 or because other TRAF cDNAs are underrepresented in the mouse T-cell cDNA library used in these screens. Therefore, we performed directed yeast two-hybrid assays with TRAF1, TRAF2, TRAF3, TRAF4 (CART1), and TRAF5. Both receptors displayed measurable and distinct interactions with TRAF1, TRAF2, and TRAF3. This finding is consistent with observations made in our laboratory and others that different members of the TNF-NGF receptor superfamily have different binding specificities for TRAF proteins (9
). It is possible that the differential abilities of 4-1BB and Ox40 to bind TRAF proteins result in differential intracellular signaling events in vivo. Neither 4-1BB nor Ox40 was found to interact with TRAF4 or TRAF5 (Fig. B). However, this does not exclude the possibility that these two TRAF molecules or other members of this growing protein family are involved in signaling by 4-1BB and/or Ox40, since TRAF proteins are capable of binding to each other. For example, TRAF5 has been found to interact with TRAF2 in a yeast two-hybrid system (6
Mutational analyses of the 4-1BB and Ox40 cytoplasmic domains revealed two independent TRAF binding sites in the cytoplasmic tail of 4-1BB and only one site in Ox40. This result suggests that the TRAF-dependent signaling complex assembled by 4-1BB in vivo may be dependent not only on the relative binding affinities of TRAF proteins for each of the two binding sites but also on the relative affinities of TRAF proteins for each other. Like previously identified TRAF binding sites, the TRAF binding sites in both 4-1BB and Ox40 receptors contain clusters of acidic amino acids. However, no consensus sequence for the specific binding of an individual TRAF protein is discernible from the data. Therefore, the in vivo binding of TRAF proteins to a specific receptor in the TNF receptor family may be influenced by their differential affinities for the receptors and the relative expression levels of the TRAF proteins. Furthermore, receptors like 4-1BB which contain two TRAF binding sites may facilitate the formation of specific heteromers of TRAF proteins.
Members of the TRAF family have been shown to be mediators of NF-κB activation in cells after binding to clustered TNF-NGF receptor family members (1
). To investigate whether 4-1BB and Ox40 could also induce NF-κB in mammalian cells, we transfected HEK293 cells with the chimeric CD28–m4-1BBCP or CD28-mOx40CP receptors and found a 10- to 20-fold induction of luciferase expression after cotransfection with the 2×NF-κB construct (Fig. and ). These results suggest that NF-κB may function as a downstream mediator for at least some of the reported effects of 4-1BB and Ox40 in activated T lymphocytes. The cytoplasmic tails of both 4-1BB and Ox40 can induce the activation of NF-κB when expressed transiently as a dimer but not when expressed as a monomer. NF-κB activation by each receptor was shown to be dependent on TRAF proteins, and NF-κB activation was inhibited by a dominant negative TRAF2 (Fig. B and C). Interestingly, Ox40 was found by yeast two-hybrid analysis to bind most avidly to TRAF3. However, when TRAF3 was cotransfected with the Ox40 signaling construct, NF-κB activation was reproducibly repressed (Fig. B). Therefore, TRAF proteins are not interchangeable adapter molecules. TRAF3 appears either to act as an inhibitor of Ox40 signal transduction or to play a role in activating an alternative signal transduction pathway through the receptor. Thus, the signaling properties of Ox40 appear to be regulated by the relative levels of intracellular TRAF proteins.
This report describes the ability of the cytoplasmic domains of two members of the TNF-NGF receptor family, 4-1BB and Ox40, to bind to proteins of the TRAF family of intracellular adapter molecules. Multimerization of the cytoplasmic domains of 4-1BB and Ox40 in transfected cells can activate the transcription factor NF-κB in a TRAF-dependent manner. Interestingly, increased expression of individual TRAF proteins can either positively or negatively affect the ability of these receptors to induce NF-κB activation. These data suggest that both the differential binding affinity and relative abundance of individual TRAF proteins can influence the cellular response to receptor cross-linking. These results provide a potential explanation for the variable effects that have been observed when members of TNF-NGF receptor family are cross-linked on activated T cells.