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

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 receptor levels due to defective processing or trafficking. Microscopy revealed targeting of the mutant protein to an autophagolysosome-like structure for likely degradation. Collectively we postulate that the conserved Y870 in the TIR domain does not participate in phosphorylation-induced signaling downstream of ligand recognition, but rather is crucial for proper TIR assembly and ER egress, resulting in maturation-specific stabilization of TLR9 within endolysosomes and subsequent pro-inflammatory signaling.

TLR9 signaling requires ligand-induced phosphorylation of two specific tyrosine residues by EGFR and Syk

“…These studies clearly indicate the EGFR requirement for endosomal TLRs. A structural role of Y 870 for TLR9 has also been shown independent of Tyr phosphorylation (35). Mutation of Y 870 causes immature processing of TLR9, leading to impaired downstream signaling.…”

The transmembrane adapter SCIMP recruits tyrosine kinase Syk to phosphorylate Toll-like receptors to mediate selective inflammatory outputs

“…Ligand-induced tyrosine phosphorylation of the TLR TIR domains provides an initial mechanism for downstream adapter recruitment and intracellular signal transduction ( 31 , 32 ). Syk and Src family kinase Lyn are both known to be responsible for TLR phosphorylation ( 11 , 13 ).…”