The biological functions fulfilled by members of the TNFR family rely on distinct signaling pathways for which recruitment of different TRAF proteins plays key roles. In this study, we identified an uncharacterized TRAF binding site spanning amino acid 345 to 368 of human LTβR. We showed that this region was as important as the triad D390
of LTβR for the recruitment of TRAF2 or TRAF3 in GST pulldown experiments (29
). Of note, the primary sequence of amino acids 345 to 368 did not reveal any features of canonical TRAF2 or TRAF3 binding sites. Interestingly, other unconventional TRAF binding sites have also been characterized for TNFR2 (PLGVPDAGMKPS) and NIK (ISIIAQA), for which the recruitment of TRAF2 and TRAF3 appeared to be indirect and direct, respectively (16
). Our data revealed that the affinity of the two TRAF binding sites of LTβR might fluctuate according to the oligomerization status of LTβR. Indeed, deletion of one of the two TRAF binding sites was sufficient to disrupt the recruitment of TRAF2 and TRAF3 to LTβR if expressed as a GST fusion protein. However, when LTβR was expressed as a native protein in eukaryotic cells, the region 345 to 368 retained some TRAF3 binding activity despite the deletion of the other TRAF binding site. It is reasonable to suggest that aggregation of LTβR increases the local concentration of LTβR, allowing it to increase its avidity toward TRAF3. The outcome of TRAF proteins following recruitment to TNFR varies from one receptor to another, involving degradation through either the proteasome or into lysosomes, as well as cellular relocalization to restricted cellular compartments (48
). Recently, it was proposed that LTβR-mediated TRAF3 proteasomal degradation was required for stabilizing and accumulating NIK (41
). However, under conditions for which overexpressed LTβR solely induced p100 processing, we did not observe an accumulation of K48-linked polyubiquitinated TRAF3, while the pool of K48-linked polyubiquitinated NIK was strongly reduced. Furthermore, it was shown that LTβR-mediated depletion of TRAF3 was required not only for the induction of the alternative pathway but also for the classical NF-κB pathway in MEFs, as well as in some colon epithelial cell lines (4
). Thus, proteasomal degradation of TRAF3 is associated not only with NIK stabilization. This statement can be also extended to signaling pathways downstream of CD40. Indeed, CD40-induced K48-linked polyubiquitination and proteasomal degradation of TRAF3 are strictly dependent on TRAF2 and c-IAP1/2 (21
). Based on these findings, a model had been proposed in which activated CD40 recruits TRAF2/TRAF3–c-IAP1/2 at the cell surface for promoting TRAF3 proteasomal degradation and NIK stabilization (52
). However, CD40-mediated K48-linked polyubiquitination and proteasomal degradation of TRAF3 are also required prior to cell membrane release of a MEKK1-containing complex that activates Jun N-terminal protein kinase (JNK) (34
). Again, c-IAP1/2-mediated TRAF3 polyubiquitination is engaged in two distinct pathways involving MEKK1 and NIK. Therefore, assessing K48-linked TRAF3 polyubiquitination is not a readout strictly associated with an activation of NIK. Overall, TRAF3 appears to be a multitask protein that acts mainly as an inhibitor. It is likely that different pools of TRAF3-containing complexes exist, and according to the cell type and the duration of stimulation, TRAF3 is recruited and degraded at different locations to activate distinct pathways. We further observed that lymphotoxin-induced TRAF2 and TRAF3 degradation also occurred in the lysosomal compartment. However, potent inhibition of TNFR-mediated lysosomal TRAF degradation did not alter the extent of p100 processing, suggesting that this type of degradation is likely secondary to NIK stabilization.
Internalization of TNFR has been mainly considered a mechanism participating in recycling and/or degradation. In this study, we identified a new function assigned to LTβR trafficking, that is, the activation of the alternative NF-κB pathway. We found that toward lymphotoxin α1β2-induced mesenteric lymph node stromal cell maturation, internalization of LTβR correlated with RelB-induced MAdCAM-1 expression.
Similarly, ligand-induced down-modulation of cell surface LTβR has been observed in other settings, such as in myeloid dendritic cell (DC) homeostasis, which is strictly dependent on the alternative pathway, or SCS (subcapsular sinus) macrophage differentiation (23
). Interestingly, we found that internalization of LTβR uncouples the activation of the alternative NF-κB pathway from the classical NF-κB pathway, probably reflecting the requirement of different adaptor proteins. This is reminiscent of the signaling pathways emerging from TLR4 for which TIRAP/MyD88 and TRAM/TRIF complexes regulate the proinflammatory and type I interferon responses from the plasma membrane and endosomes, respectively (24
Other biological functions have been assigned to internalized TNFR family members. Indeed, TNFR1 also activates the classical NF-κB from the cell surface, but its internalization is rather associated with TNF-α-induced cell death (43
). This process relies on a cytosolic region named the TRID domain, which contains a consensus YXXΦ motif that is targeted by the adaptor complex AP2 for sorting activated TNFR1 to endosomes (43
). We have also detected an interaction between AP2 and activated LTβR. Unlike TNFR1, our bioinformatics analyses of LTβR did not reveal any consensus YXXΦ motif. Nevertheless, we found a dileucine motif at position 299 of human LTβR, which probably recruits AP2 through the binding of the σ2 subunit (27
). However, we showed that deletion of the region encompassing this AP2 site did not alter the induction of the processing of p100, which is consistent with the fact that clathrin is dispensable for the activation of the alternative NF-κB pathway. Thus, AP2 may control LTβR internalization for the control of its half-life at the cell surface, its recycling, or a yet-undefined function.
The signaling pathways linked to TNFR internalization have been probably overlooked. Indeed, internalization of CD40 was reported to regulate the transcription of BAFF but also to allow the interaction with c-Rel for promoting B cell lymphoma proliferation (31
). Whether LTβR internalization controls nuclear functions is a possible scenario and is under investigation. Interestingly, ligand-dependent or -independent LTβR internalization might be linked to cancer progression. Indeed, we showed that dynamin-dependent internalization of LTβR is required for NIK stabilization, and exacerbated accumulation of NIK has been reported in hematopoietic as well as nonhematopoietic cancer cells (50
). For instance, liver-specific LTα/LTβ expression leads to hepatitis-induced hepatocellular carcinoma development and this phenotype can be reversed when mice are treated with Fc-LTβR recombinant decoy receptors (18
). Conversely, in LTβR-expressing melanoma cells, activation of NIK is driven in a ligand-independent way (12
). In this particular case, the use of decoy LTβR would be useless and other strategies should be envisioned for preventing LTβR-mediated cell proliferation. The development of molecules that specifically block LTβR internalization, or other receptors, might be a promising research avenue for inflammatory disorders and cancer treatment.