Conserved Features in the Extracellular Domain of Human Toll-like Receptor 8 Are Essential for pH-dependent Signaling

Abstract

Toll-like receptor (TLR) 8 has an important role in initiating immune responses to viral single-stranded RNA and the antiviral compound resiquimod. Together with TLR3, -7, and -9, it forms a subgroup of the TLRs that are localized intracellularly and signal in response to pathogen-derived nucleic acids. In this work, we have used site-directed mutagenesis to identify regions of the TLR8 extracellular domain that are required for stimulusinduced signal transduction. We have shown that a cysteinerich sequence predicted to form a loop projecting from the solenoidal ectodomain structure at leucine-rich repeat 8 is essential for signaling in response to both single-stranded RNA and resiquimod. A second region, centered on an aspartic acid residue in leucine-rich repeat 17, is also required for TLR8 function. The corresponding residue in TLR9 is known to be important for pH-dependent binding and signaling in response to unmethylated CpG DNA, suggesting that the TLR7/8/9 subgroups share a common signaling mechanism. We have also shown that TLR8 is localized predominantly in the endoplasmic reticulum but that signaling is completely abolished by an inhibitor of vesicle-type H ؉ ATPases. This indicates that TLR8 is present at low levels in an acidified compartment and that a lowered pH is required for receptor function. We propose that pH-dependent changes in the ligand facilitate activation of the receptor. The protonated form of resiquimod, a cell-permeable weak base, is likely to concentrate significantly (ϳ100؋) in acidified compartments, and this may potentiate low affinity interactions with either the receptor or a specific binding protein.

TLR7 Is Involved in Sequence-Specific Sensing of Single-Stranded RNAs in Human Macrophages

“…Furthermore, the previous demonstration that transient overexpression of hTLR8 in HEK293 and U20S cells reconstitutes responsiveness to ssRNAs via activation of NF-B (11,31) indicates that hTLR8 does not require hTLR7 for signaling when expressed artificially. The discrepancies between these results and the data presented here are likely related to overexpression of TLRs in these studies and the saturation doses of ssRNAs used (ϳ3-fold higher than the highest dose used here).…”

Identification of RNA Sequence Motifs Stimulating Sequence-Specific TLR8-Dependent Immune Responses

“…Structural analysis of TLR8 will probably result in more information of a potential ligand binding site and further help to explain functional differences among vertebrates. Initial mutagenesis studies identified regions in the human TLR8 extracellular domain most probably involved in signaling (48). Specific residues in LRR8 and LRR17 appear to be absolutely required for human TLR8 activation (48), and mutation of a residue in LRR17 of human TLR9 abolished specific binding to CpG DNA (30), indicating a role for LRR17 in ligand recognition for TLR7, 8, and 9 due to the similarities between these TLRs.…”

“…Initial mutagenesis studies identified regions in the human TLR8 extracellular domain most probably involved in signaling (48). Specific residues in LRR8 and LRR17 appear to be absolutely required for human TLR8 activation (48), and mutation of a residue in LRR17 of human TLR9 abolished specific binding to CpG DNA (30), indicating a role for LRR17 in ligand recognition for TLR7, 8, and 9 due to the similarities between these TLRs. Unfortunately, nothing is known yet about the potential function of LRR3, but our data suggest that this region plays a role in species-specific recognition of RNA by TLR8.…”

Chicken TLR21 Is an Innate CpG DNA Receptor Distinct from Mammalian TLR9

“…These data suggest that DNA binding and responsiveness are distinct events and that intrinsic structural differences between mammalian TLR9 contribute to the species-specific response to ODNs. Recent evidence shows that fulllength TLR9 (i.e., not the TLR chimeric protein) targeted to the cell surface using a yeast sorting sequence is unresponsive, and that the receptor requires a low pH (31) and protease activity (12) in the endolysosomes to gain functionality. As the uptake of CpG DNA via endocytosis is not DNA sequence specific (4), these results suggest that the physical properties of the (polyanionic) ligand at low pH (35), possible regulatory molecules (39), and/or intracellular processing of TLR9 (11,12) are important for the species-specific response to CpG DNA (7).…”