Tyrosine 870 of TLR9 is critical for receptor maturation rather than phosphorylation-dependent ligand-induced signaling

PloS One
Chhanda BiswasEdward M Behrens

Abstract

Toll like receptors (TLRs) share a conserved structure comprising the N-terminal ectodomain, a transmembrane segment and a C-terminal cytoplasmic Toll/IL-1 receptor (TIR) domain. Proper assembly of the TIR domain is crucial for signal transduction; however, the contribution of individual motifs within the TIR domain to TLR trafficking and signaling remains unclear. We targeted a highly conserved tyrosine (Y870) located in the box 1 region of the TIR domain of most TLRs, including TLR9, previously described to be a critical site of phosphorylation in TLR4. We reconstituted bone marrow-derived dendritic cells (BMDC) from Tlr9-/- mice WT TLR9 or Y870F or Y870A mutants. Despite normal interactions with the luminal chaperones GRP94 and UNC93B1, Y870F conferred only partial responsiveness to CpG, and Y870A had no activity and functioned as a dominant negative inhibitor when coexpressed with endogenous TLR9. This loss of function correlated with reduction or absence, respectively, of the 80 kDa mature form of TLR9. In Y870F-expressing cells, CpG-dependent signaling correlated directly with levels of the mature form, suggesting that signaling did not require tyrosine phosphorylation but rather that the Y870F mutation conferred reduced ...Continue Reading

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Datasets Mentioned

BETA
GM130

Methods Mentioned

BETA
flow cytometry
ELISA
coimmunoprecipitation
glycosylation
immunoprecipitation
transfection
co-immunoprecipitation
FACS

Software Mentioned

Licor
FlowJo
FACSDiva
ImageJ
Prism

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