title: 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.
authors.affiliation: Department of Immunology, Osaka Medical Center for Cancer and Cardiovascular Diseases, Higashinari-ku, Osaka 537-8511, Japan.
authors.forename: Kenji
authors.initials: K
authors.lastname: Funami
authors.forename: Misako
authors.initials: M
authors.lastname: Matsumoto
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Oshiumi
authors.forename: Takashi
authors.initials: T
authors.lastname: Akazawa
authors.forename: Akitsugu
authors.initials: A
authors.lastname: Yamamoto
authors.forename: Tsukasa
authors.initials: T
authors.lastname: Seya
mesh_terms: D033942:Adaptor Proteins, Vesicular Transport / Q000378:metabolism*; D019943:Amino Acid Substitution / Q000235:genetics*; D002478:Cells, Cultured; D003713:Dendritic Cells / Q000378:metabolism*; D015870:Gene Expression; D006801:Humans; D008562:Membrane Glycoproteins / Q000235:genetics / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed / Q000235:genetics*; D011485:Protein Binding / Q000235:genetics; D011956:Receptors, Cell Surface / Q000235:genetics / Q000378:metabolism*; D017384:Sequence Deletion / Q000235:genetics*; D015398:Signal Transduction / Q000235:genetics; D051196:Toll-Like Receptor 3; D051193:Toll-Like Receptors
chemical_list: D033942:Adaptor Proteins, Vesicular Transport; D008562:Membrane Glycoproteins; D011956:Receptors, Cell Surface; C470409:TICAM1 protein, human; C495344:TLR3 protein, human; D051196:Toll-Like Receptor 3; D051193:Toll-Like Receptors