Lakhasly

TLRs contain a conserved TIR domain with regions of varying homology. The tyrosine located in the box 1 region of the TIR domain is conserved among most TLRs (Fig 1). Curiously, the receptors that heterodimerize to function–TLR1, TLR6, and TLR12 –contain a phenylalanine instead of a tyrosine, but the TLRs with which these molecules pair all have a tyrosine at that position. The remaining TLRs that harbor a tyrosine form homodimers. Thus, all TLR dimers have at least one box 1 region containing the conserved tyrosine, suggesting the importance of this residue for TLR function. It was previously reported that the box 1 tyrosine was critical for TLR4 signaling [14], but how it regulated signaling or whether it also functioned in other TLRs was not determined. To more comprehensively understand the molecular components in TLR9 function, we questioned whether Y870 was important for full receptor activation in TLR9. Here, we demonstrate that substitution of Y870 for alanine results in a TLR9 variant whose maturation and subsequent signaling are abolished. Consistent with a prior report in bone marrow derived macrophages [25], we find that tyrosine at position 870 is necessary for full TLR9 stabilization and normal trafficking to endosomal compartments. We expand upon these ideas by showing that Y870 mutants can interact with the co-expressed wild type protein and that mutants harboring an alanine at this position are consumed by autophagy. It is likely that dimerization with the mutant and partially misfolded TLR9 results in some degradation of wild type molecules, therefore likely explaining the dominant negative effect of the mutant. While activated TLR9 has been shown to be targeted for autophagy upon B-cell receptor signaling in B cells [26], we now demonstrate that a mutant TLR9 can be similarly targeted in myeloid cells independent of any signaling.

Mutagenesis of the analogous N-terminal box 1 tyrosine residue in TLR4 (Y674A) abolished LPS-induced signaling in a heterologous expression system in 293T cells [14]. While the investigators attributed the defective signaling to decreased LPS-induced receptor phosphorylation, our data suggest the alternative possibility that the receptor did not traffic normally to the plasma membrane to access ligand. In fact, our observation that TLR9 Y870F signals as effectively as WT TLR9 in response to CpG in dendritic cells, after correcting for differences in levels of mature TLR9 (Fig 4B), corroborates previously published data in bone marrow derived macrophages [25] and indicates that a tyrosine at the N-terminal box 1 position is not strictly necessary for CpG-induced signaling, but rather supports generating a functional receptor. Furthermore, these observations indicate that receptor phosphorylation at this site is not necessary for function, as was suggested for TLR4 [14]. Importantly, the TLR4 report did not investigate signaling with a phenylalanine mutant. Therefore, it is possible that in their report, as we see here for TLR9, TLR4 was retained in a compartment without access to ligand, explaining the apparent loss of activity. Our data support this notion for TLR9 and suggest that the conserved residue is necessary for receptor stabilization. Alternatively, this residue may be important for interaction with cytoplasmic chaperone molecules important for TLR folding and/or localization. Since Y870 is within the cytoplasmic TIR domain of TLR9, our data are consistent with a requirement for heretofore undiscovered cytoplasmic chaperones in TLR folding, analogous to the requirement for both ER-resident and cytoplasmic chaperones for folding of the cystic fibrosis transporter CFTR [27]. Hsp70 family members play an important role in cytoplasmic folding for CFTR and could be playing a similar unappreciated role in TLR folding as well.

The cytoplasmic TIR domain of all TLRs is best known for its ability to interact with the adaptor molecules TRIF/TRAM and/or MYD88/TIRAP to transduce downstream signaling following ligand engagement [28]. An additional role for localization was attributed to this region, as a TLR9 molecule with deletions of several amino acids in the TIR domain downstream of Y870 was mislocalized to the cell surface [29]. In contrast, our data suggest that point mutation of the N-terminal tyrosine 870 in the Box 1 of the TIR domain prevents normal TLR9 egress from the ER. One possible explanation for this discrepancy could be attributed to the cell types used for experimentation. We conducted our studies in BMDCs, which more closely resemble physiologically relevant TLR-responsive innate immune cell types than do HeLa cells that were used in other studies [29]. It is possible that non-physiologic, cell line-specific machinery contributed to the trafficking of overexpressed TLR9 in the HeLa experiments. Nonetheless, both pieces of evidence support the concept that TIR domain assembly is important for the correct sub-cellular localization and, thus, function of TLR9.

Previous work has suggested that some residues of the box 1 region of the TIR domain create a binding pocket required for TIR-TIR domain interactions between the TLR and its adaptor Mal/TIRAP [15]. Our data are consistent with a model in which the N-terminal tyrosine of this domain is critical for proper TIR domain assembly and ER egress. Accordingly, the more conservative Y870F mutation has an intermediate phenotype, while the more divergent Y870A mutant completely disrupts proper assembly. Interestingly, the Y870A variant forms homodimers, the interaction for which is based on the N-terminal ectodomain [16], and associates with its appropriate ER chaperone molecules GRP94 and UNC93B1. These data suggest that altered ER egress does not reflect misfolding in the lumenal space. Rather, these data are consistent with a model in which misfolding of the TIR domain is recognized by a cytoplasmic quality control system [30] and is sufficient to prevent normal ER egress. A properly folded TIR domain is likely partially stabilized by growth at 30°C, permitting some degree of normal ER exit—even for the mutant forms—to eventually reach endosomes. Therefore, we propose a model of TLR processing that requires proper TIR domain assembly prior to ER exit and subsequent subcellular localization. Interestingly, the observed dimerization of mutant TLR9 must take place in the ER since the mutant TLR9 is unable to exit the ER to endosomes. This suggests that TLR9 dimerization via the luminal domain is a ligand independent process, as these molecules would not be expected to have access to their ligands within the ER.

It is of interest that the Y870A TLR9 molecule is consumed by autophagy for disposal, rather than exploiting the endoplasmic-reticulum-associated protein degradation (ERAD) pathway. ERAD makes use of ubiquitinylation and proteosomal degradation rather than autophagy. It has been proposed that proteins that form large aggregates, too large for the pore of the proteasome machinery, are instead degraded by autophagy [31, 32]. Disposal of improperly folded transmembrane proteins by autophagy has been described [31] but is relatively poorly understood. The fact that the Y870A mutant binds to its luminal chaperones, and forms TLR9 dimers suggests that it has retained at least its luminal integrity. Perhaps the properly folded and dimerized luminal domain is unable to be unfolded by the ER quality control machinery—a requirement for retrotranslocation of ERAD substrates into the cytosol for proteosomal degradation—obviating the need for a distinct disposal mechanism, much like an aggregate.

While traditionally associated with ligand-induced signaling, we show here that the TIR domain is important for stability and normal receptor trafficking. This is the first report, to our knowledge, implicating this region in receptor stability in the ER. It will be of interest to investigate how this conserved tyrosine residue impacts the stability and trafficking of other TLRs and adaptor molecules, as many of these molecules have specific subcellular localization requirements. Furthermore, the Y870A mutant TLR9 model may provide an interesting platform to better characterize the autophagosomal degradation pathway of integral membrane protein disposal.