Conserved features in the extracellular domain of human toll-like receptor 8 are essential for pH-dependent signaling.
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 stimulus-induced 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 (approximately 100x) in acidified compartments, and this may potentiate low affinity interactions with either the receptor or a specific binding protein.
Gibbard
Rebecca J
RJ
Biochemistry Department, University of Cambridge, 80 Tennis Court Road, Cambridge CB2 1GA, United Kingdom.
Morley
Peter J
PJ
Gay
Nicholas J
NJ
D000595:Amino Acid Sequence; D045744:Cell Line, Tumor; D018899:CpG Islands; D004721:Endoplasmic Reticulum / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D017386:Sequence Homology, Amino Acid; D015398:Signal Transduction; D051200:Toll-Like Receptor 8 / Q000737:chemistry* / Q000378:metabolism; D051217:Toll-Like Receptor 9 / Q000378:metabolism
D051200:Toll-Like Receptor 8; D051217:Toll-Like Receptor 9