Recognition of double-stranded RNA by human toll-like receptor 3 and downstream receptor signaling requires multimerization and an acidic pH.
Studies involving Toll-like receptor 3 (TLR3)-deficient mice suggest that this receptor binds double-stranded RNA. In the present study, we analyzed ligand/receptor interactions and receptor-proximal events leading to TLR3 activation. The mutagenesis approach showed that certain cysteine residues and glycosylation in TLR3 amino-terminal leucine-rich repeats were necessary for ligand-induced signaling. Furthermore, inactive mutants had a dominant negative effect, suggesting that the signaling module is a multimer. We constructed a chimeric molecule fusing the amino-terminal ectodomain of TLR3 to the transmembrane and carboxyl terminal domains of CD32a containing an immunoreceptor tyrosine-based motif. Expression of TLR3-CD32 in HEK293T cells and the myeloid cell line U937 resulted in surface localization of the receptor, whereas the nonrecombinant molecule was intracellularly localized. The synthetic double-stranded RNAs poly(I-C) and poly(A-U) induced calcium mobilization in a TLR3-CD32 stably transfected U937 clone but not in control cells transfected with other constructs. An anti-TLR3 antibody also induced Ca(2+) flux but only when cross-linked by a secondary anti-immunoglobulin antibody, confirming that multimerization by the ligand is a requirement for signaling. The inhibitors of lysosome maturation, bafilomycin and chloroquine, inhibited the poly(I-C)-induced biological response in immune cells, showing that TLR3 interacted with its ligand in acidic subcellular compartments. Furthermore, TLR3-CD32 activation with poly(I-C) was only observed within a narrow pH window (pH 5.7-6.7), whereas anti-TLR3-mediated Ca(2+) flux was pH-insensitive. The importance of an acidic pH for TLR3-ligand interaction becomes critical when using oligomeric poly(I-C) (15-40-mers). These observations demonstrate that engagement of TLR3 by poly(I-C) at an acidic pH, probably in early phagolysosomes or endosomes, induces receptor aggregation leading to signaling.
de Bouteiller
Odette
O
Laboratory for Immunological Research, Schering-Plough Research Institute, 27 Chemin des Peupliers, 69571 Dardilly Cedex, France.
Merck
Estelle
E
Hasan
Uzma A
UA
Hubac
Sylvain
S
Benguigui
Barbara
B
Trinchieri
Giorgio
G
Bates
Elizabeth E M
EE
Caux
Christophe
C
D000595:Amino Acid Sequence; D015703:Antigens, CD / Q000737:chemistry; D018501:Antirheumatic Agents / Q000494:pharmacology; D001483:Base Sequence; D001665:Binding Sites; D015153:Blotting, Western; D002118:Calcium / Q000378:metabolism; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D002469:Cell Separation; D002738:Chloroquine / Q000737:chemistry; D003432:Cross-Linking Reagents / Q000494:pharmacology; D003545:Cysteine / Q000737:chemistry / Q000378:metabolism; D016207:Cytokines / Q000378:metabolism; D004247:DNA / Q000378:metabolism*; D003713:Dendritic Cells / Q000378:metabolism; D019281:Dimerization; D004305:Dose-Response Relationship, Drug; D011992:Endosomes / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005434:Flow Cytometry; D005799:Genes, Dominant; D017930:Genes, Reporter; D006031:Glycosylation; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007930:Leucine / Q000737:chemistry; D007963:Leukocytes, Mononuclear / Q000378:metabolism; D008024:Ligands; D008156:Luciferases / Q000378:metabolism; D008247:Lysosomes / Q000737:chemistry / Q000378:metabolism; D018942:Macrolides / Q000494:pharmacology; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D016328:NF-kappa B / Q000378:metabolism; D010588:Phagosomes / Q000737:chemistry; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D017452:Receptors, IgG / Q000096:biosynthesis / Q000737:chemistry; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D015398:Signal Transduction; D013997:Time Factors; D051196:Toll-Like Receptor 3 / Q000737:chemistry / Q000378:metabolism*; D014162:Transfection; D014443:Tyrosine / Q000737:chemistry; D020298:U937 Cells
D015703:Antigens, CD; D018501:Antirheumatic Agents; D003432:Cross-Linking Reagents; D016207:Cytokines; D004791:Enzyme Inhibitors; C080568:Fc gamma receptor IIA; D008024:Ligands; D018942:Macrolides; D016328:NF-kappa B; D017452:Receptors, IgG; D011993:Recombinant Fusion Proteins; D051196:Toll-Like Receptor 3; C057620:bafilomycin A; D014443:Tyrosine; D002738:Chloroquine; D004247:DNA; D008156:Luciferases; D007930:Leucine; D003545:Cysteine; D002118:Calcium