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Recognition of double-stranded RNA by human toll-like receptor 3 and downstream receptor signaling requires multimerization and an acidic pH.

Identifieur interne : 000204 ( France/Analysis ); précédent : 000203; suivant : 000205

Recognition of double-stranded RNA by human toll-like receptor 3 and downstream receptor signaling requires multimerization and an acidic pH.

Auteurs : Odette De Bouteiller [France] ; Estelle Merck ; Uzma A. Hasan ; Sylvain Hubac ; Barbara Benguigui ; Giorgio Trinchieri ; Elizabeth E M. Bates ; Christophe Caux

Source :

RBID : pubmed:16144834

Descripteurs français

English descriptors

Abstract

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.

DOI: 10.1074/jbc.M507163200
PubMed: 16144834


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<term>Antigens, CD (chemistry)</term>
<term>Antirheumatic Agents (pharmacology)</term>
<term>Base Sequence</term>
<term>Binding Sites</term>
<term>Blotting, Western</term>
<term>Calcium (metabolism)</term>
<term>Cell Line</term>
<term>Cell Membrane (metabolism)</term>
<term>Cell Separation</term>
<term>Chloroquine (chemistry)</term>
<term>Cross-Linking Reagents (pharmacology)</term>
<term>Cysteine (chemistry)</term>
<term>Cysteine (metabolism)</term>
<term>Cytokines (metabolism)</term>
<term>DNA (metabolism)</term>
<term>Dendritic Cells (metabolism)</term>
<term>Dimerization</term>
<term>Dose-Response Relationship, Drug</term>
<term>Endosomes (metabolism)</term>
<term>Enzyme Inhibitors (pharmacology)</term>
<term>Flow Cytometry</term>
<term>Genes, Dominant</term>
<term>Genes, Reporter</term>
<term>Glycosylation</term>
<term>Humans</term>
<term>Hydrogen-Ion Concentration</term>
<term>Leucine (chemistry)</term>
<term>Leukocytes, Mononuclear (metabolism)</term>
<term>Ligands</term>
<term>Luciferases (metabolism)</term>
<term>Lysosomes (chemistry)</term>
<term>Lysosomes (metabolism)</term>
<term>Macrolides (pharmacology)</term>
<term>Molecular Sequence Data</term>
<term>Mutagenesis, Site-Directed</term>
<term>Mutation</term>
<term>NF-kappa B (metabolism)</term>
<term>Phagosomes (chemistry)</term>
<term>Protein Binding</term>
<term>Protein Structure, Tertiary</term>
<term>Receptors, IgG (biosynthesis)</term>
<term>Receptors, IgG (chemistry)</term>
<term>Recombinant Fusion Proteins (chemistry)</term>
<term>Recombinant Fusion Proteins (metabolism)</term>
<term>Sequence Homology, Amino Acid</term>
<term>Signal Transduction</term>
<term>Time Factors</term>
<term>Toll-Like Receptor 3 (chemistry)</term>
<term>Toll-Like Receptor 3 (metabolism)</term>
<term>Transfection</term>
<term>Tyrosine (chemistry)</term>
<term>U937 Cells</term>
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<term>ADN (métabolisme)</term>
<term>Agranulocytes (métabolisme)</term>
<term>Antienzymes (pharmacologie)</term>
<term>Antigènes CD ()</term>
<term>Antirhumatismaux (pharmacologie)</term>
<term>Calcium (métabolisme)</term>
<term>Cellules U937</term>
<term>Cellules dendritiques (métabolisme)</term>
<term>Chloroquine ()</term>
<term>Concentration en ions d'hydrogène</term>
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<term>Cytokines (métabolisme)</term>
<term>Cytométrie en flux</term>
<term>Dimérisation</term>
<term>Données de séquences moléculaires</term>
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<term>Facteur de transcription NF-kappa B (métabolisme)</term>
<term>Facteurs temps</term>
<term>Glycosylation</term>
<term>Gènes dominants</term>
<term>Gènes rapporteurs</term>
<term>Humains</term>
<term>Leucine ()</term>
<term>Liaison aux protéines</term>
<term>Ligands</term>
<term>Lignée cellulaire</term>
<term>Luciferases (métabolisme)</term>
<term>Lysosomes ()</term>
<term>Lysosomes (métabolisme)</term>
<term>Macrolides (pharmacologie)</term>
<term>Membrane cellulaire (métabolisme)</term>
<term>Mutagenèse dirigée</term>
<term>Mutation</term>
<term>Phagosomes ()</term>
<term>Protéines de fusion recombinantes ()</term>
<term>Protéines de fusion recombinantes (métabolisme)</term>
<term>Relation dose-effet des médicaments</term>
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<term>Récepteur de type Toll-3 (métabolisme)</term>
<term>Récepteurs du fragment Fc des IgG ()</term>
<term>Récepteurs du fragment Fc des IgG (biosynthèse)</term>
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<term>Sites de fixation</term>
<term>Structure tertiaire des protéines</term>
<term>Séparation cellulaire</term>
<term>Séquence d'acides aminés</term>
<term>Séquence nucléotidique</term>
<term>Technique de Western</term>
<term>Transduction du signal</term>
<term>Transfection</term>
<term>Tyrosine ()</term>
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<term>Receptors, IgG</term>
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<keywords scheme="MESH" type="chemical" qualifier="chemistry" xml:lang="en">
<term>Antigens, CD</term>
<term>Chloroquine</term>
<term>Cysteine</term>
<term>Leucine</term>
<term>Receptors, IgG</term>
<term>Recombinant Fusion Proteins</term>
<term>Toll-Like Receptor 3</term>
<term>Tyrosine</term>
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<term>Calcium</term>
<term>Cysteine</term>
<term>Cytokines</term>
<term>DNA</term>
<term>Luciferases</term>
<term>NF-kappa B</term>
<term>Recombinant Fusion Proteins</term>
<term>Toll-Like Receptor 3</term>
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<keywords scheme="MESH" type="chemical" qualifier="pharmacology" xml:lang="en">
<term>Antirheumatic Agents</term>
<term>Cross-Linking Reagents</term>
<term>Enzyme Inhibitors</term>
<term>Macrolides</term>
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<term>Cystéine</term>
<term>Cytokines</term>
<term>Endosomes</term>
<term>Facteur de transcription NF-kappa B</term>
<term>Luciferases</term>
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<term>Protéines de fusion recombinantes</term>
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<term>Antienzymes</term>
<term>Antirhumatismaux</term>
<term>Macrolides</term>
<term>Réactifs réticulants</term>
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<term>Base Sequence</term>
<term>Binding Sites</term>
<term>Blotting, Western</term>
<term>Cell Line</term>
<term>Cell Separation</term>
<term>Dimerization</term>
<term>Dose-Response Relationship, Drug</term>
<term>Flow Cytometry</term>
<term>Genes, Dominant</term>
<term>Genes, Reporter</term>
<term>Glycosylation</term>
<term>Humans</term>
<term>Hydrogen-Ion Concentration</term>
<term>Ligands</term>
<term>Molecular Sequence Data</term>
<term>Mutagenesis, Site-Directed</term>
<term>Mutation</term>
<term>Protein Binding</term>
<term>Protein Structure, Tertiary</term>
<term>Sequence Homology, Amino Acid</term>
<term>Signal Transduction</term>
<term>Time Factors</term>
<term>Transfection</term>
<term>U937 Cells</term>
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<term>Antigènes CD</term>
<term>Cellules U937</term>
<term>Chloroquine</term>
<term>Concentration en ions d'hydrogène</term>
<term>Cystéine</term>
<term>Cytométrie en flux</term>
<term>Dimérisation</term>
<term>Données de séquences moléculaires</term>
<term>Facteurs temps</term>
<term>Glycosylation</term>
<term>Gènes dominants</term>
<term>Gènes rapporteurs</term>
<term>Humains</term>
<term>Leucine</term>
<term>Liaison aux protéines</term>
<term>Ligands</term>
<term>Lignée cellulaire</term>
<term>Lysosomes</term>
<term>Mutagenèse dirigée</term>
<term>Mutation</term>
<term>Phagosomes</term>
<term>Protéines de fusion recombinantes</term>
<term>Relation dose-effet des médicaments</term>
<term>Récepteur de type Toll-3</term>
<term>Récepteurs du fragment Fc des IgG</term>
<term>Similitude de séquences d'acides aminés</term>
<term>Sites de fixation</term>
<term>Structure tertiaire des protéines</term>
<term>Séparation cellulaire</term>
<term>Séquence d'acides aminés</term>
<term>Séquence nucléotidique</term>
<term>Technique de Western</term>
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<term>Transfection</term>
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<front>
<div type="abstract" xml:lang="en">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.</div>
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<name sortKey="Trinchieri, Giorgio" sort="Trinchieri, Giorgio" uniqKey="Trinchieri G" first="Giorgio" last="Trinchieri">Giorgio Trinchieri</name>
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<region name="Auvergne-Rhône-Alpes">
<name sortKey="De Bouteiller, Odette" sort="De Bouteiller, Odette" uniqKey="De Bouteiller O" first="Odette" last="De Bouteiller">Odette De Bouteiller</name>
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