The cytoplasmic 'linker region' in Toll-like receptor 3 controls receptor localization and signaling

Abstract

Toll-like receptor 3 (TLR3) recognizes double-stranded RNA and transmits signals to activate NF-kappaB and the interferon (IFN)-beta promoter via the newly identified adaptor, TICAM-1. The extracellular LRR domain of TLR3 is engaged in the ligand recognition, while the intracellular TIR domain is crucial for the adaptor binding and signal transduction upon ligand stimulation. Here, we analyzed TLR3 localization in human monocyte-derived immature dendritic cells (iDCs) and stable transfectants expressing human TLR3 by immunofluorescence staining and confocal microscopy. TLR3 was predominantly localized in specific but as yet unidentified intracellular vesicles where TLR3 signaling was initiated. Expression analysis of TLR3-tail-truncated mutants revealed that the cytoplasmic 'linker' region (residues 730-755) determines the intracellular localization of TLR3. Site-directed mutagenesis of the linker region allowed us to identify the relevant determinants as Arg(740) and Val(741) residues for intracellular expression of TLR3. Furthermore, alanine scanning of the linker region demonstrated that the Phe(732), Leu(742) and Gly(743) in the TLR3 cytoplasmic linker region are essential for ligand-induced NF-kappaB and IFN-beta promoter activation. Thus, the cytoplasmic linker region of TLR3 regulates receptor retention inside the organelle and signaling, which may be closely linked to TLR3 function in DCs.

Toll-like receptor 3 regulates Mx expression in rare minnow Gobiocypris rarus after viral infection

“…We amplified 22 cycles for β-actin, and 32 cycles for other genes. M, 1-7 stands for DNA marker (DL2000), zygote, dome, shield, 1-somite, prim-5, protruding mouth, and adult stages, respectively Funami et al 2004;Sarkar et al 2003), and the TIR domain might be considered as a cassette (Xu et al 2000). Most of the conserved residues were located in the hydrophobic core.…”

“…The TIR domain of GrTLR3s was short of two β-sheets and two helices, which caused the hydrophobic core to be exposed outside. Funami et al (2004) identified three amino acid residues essential for ligand-inducing NF-κB and IFN-β promoter activation (Phe 732 , Leu 742 , and Gly 743 ) in human TLR3, all of which were present in the GrTLR3 sequence (Phe 734 , Leu 744 , and Gly 745 ). Sarkar et al (2003) identified specific tyrosine residues in the TIR domain as being essential for the intracellular signaling in human TLR3.…”

“…Of these, only Tyr 756 was conserved in the rare minnow sequence (Tyr 759 ). Funami et al (2004) identified two amino acids important for the intracellular localization in human TLR3, Arg 740 and Val 741 . The arginine residue was conserved in the rare minnow sequence (Arg 742 ).…”

Mycoplasma lipoproteins and Toll-like receptors

“…In contrast, TLR3, TLR7, TLR8, and TLR9 have been shown to be expressed in intracellular fractions, such as endosomes (Heil et al, 2003;Matsumoto et al, 2003;Latz et al, 2004). It has been reported that TLR3-, TLR7-, or TLR9-mediated recognition of their ligands requires endosomal maturation (Heil et al, 2004;Funami et al, 2004). Whether the TLR is on or in the cell, studies have shown that either phagosomal/lysosomal or endosomal/ lysomal compartments may be the main sites for TLR recognition of microbial components (Latz et al, 2004;Leifer et al, 2004;Underhill et al, 1999).…”

Characterization and expression analysis of Toll-like receptor 3 cDNA from Atlantic salmon (Salmo salar)

“…In general, TLRs are represented by an extracellular N-terminus with a leucine-rich repeat region (LRR) and a conserved cytoplasmic C-terminus with a Toll-interleukin-1 receptor (TIR) domain (Matsushima et al, 2007). The TIR-conserved amino acid regions have been shown to be implicated in the signaling and localization of TLRs (Sarkar et al, 2003;Funami et al, 2004), while the LRR has been implicated in pathogen detection (Bell et al, 2003). TLR family components are expressed by cells of the immune system and specialized cells, such as neutrophils, macrophages, dendritic cells, vascular endothelial cells, and intestinal epithelial cells (Skjaeveland et al, 2008).…”