Recognition of double-stranded RNA by human toll-like receptor 3 and downstream receptor signaling requires multimerization and an acidic pH.
Identifieur interne : 002E85 ( Main/Exploration ); précédent : 002E84; suivant : 002E86Recognition 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 CauxSource :
- The Journal of biological chemistry [ 0021-9258 ] ; 2005.
Descripteurs français
- KwdFr :
- ADN (métabolisme), Agranulocytes (métabolisme), Antienzymes (pharmacologie), Antigènes CD (), Antirhumatismaux (pharmacologie), Calcium (métabolisme), Cellules U937, Cellules dendritiques (métabolisme), Chloroquine (), Concentration en ions d'hydrogène, Cystéine (), Cystéine (métabolisme), Cytokines (métabolisme), Cytométrie en flux, Dimérisation, Données de séquences moléculaires, Endosomes (métabolisme), Facteur de transcription NF-kappa B (métabolisme), Facteurs temps, Glycosylation, Gènes dominants, Gènes rapporteurs, Humains, Leucine (), Liaison aux protéines, Ligands, Lignée cellulaire, Luciferases (métabolisme), Lysosomes (), Lysosomes (métabolisme), Macrolides (pharmacologie), Membrane cellulaire (métabolisme), Mutagenèse dirigée, Mutation, Phagosomes (), Protéines de fusion recombinantes (), Protéines de fusion recombinantes (métabolisme), Relation dose-effet des médicaments, Réactifs réticulants (pharmacologie), Récepteur de type Toll-3 (), Récepteur de type Toll-3 (métabolisme), Récepteurs du fragment Fc des IgG (), Récepteurs du fragment Fc des IgG (biosynthèse), Similitude de séquences d'acides aminés, Sites de fixation, Structure tertiaire des protéines, Séparation cellulaire, Séquence d'acides aminés, Séquence nucléotidique, Technique de Western, Transduction du signal, Transfection, Tyrosine ().
- MESH :
- biosynthèse : Récepteurs du fragment Fc des IgG.
- métabolisme : ADN, Agranulocytes, Calcium, Cellules dendritiques, Cystéine, Cytokines, Endosomes, Facteur de transcription NF-kappa B, Luciferases, Lysosomes, Membrane cellulaire, Protéines de fusion recombinantes, Récepteur de type Toll-3.
- pharmacologie : Antienzymes, Antirhumatismaux, Macrolides, Réactifs réticulants.
- Antigènes CD, Cellules U937, Chloroquine, Concentration en ions d'hydrogène, Cystéine, Cytométrie en flux, Dimérisation, Données de séquences moléculaires, Facteurs temps, Glycosylation, Gènes dominants, Gènes rapporteurs, Humains, Leucine, Liaison aux protéines, Ligands, Lignée cellulaire, Lysosomes, Mutagenèse dirigée, Mutation, Phagosomes, Protéines de fusion recombinantes, Relation dose-effet des médicaments, Récepteur de type Toll-3, Récepteurs du fragment Fc des IgG, Similitude de séquences d'acides aminés, Sites de fixation, Structure tertiaire des protéines, Séparation cellulaire, Séquence d'acides aminés, Séquence nucléotidique, Technique de Western, Transduction du signal, Transfection, Tyrosine.
English descriptors
- KwdEn :
- Amino Acid Sequence, Antigens, CD (chemistry), Antirheumatic Agents (pharmacology), Base Sequence, Binding Sites, Blotting, Western, Calcium (metabolism), Cell Line, Cell Membrane (metabolism), Cell Separation, Chloroquine (chemistry), Cross-Linking Reagents (pharmacology), Cysteine (chemistry), Cysteine (metabolism), Cytokines (metabolism), DNA (metabolism), Dendritic Cells (metabolism), Dimerization, Dose-Response Relationship, Drug, Endosomes (metabolism), Enzyme Inhibitors (pharmacology), Flow Cytometry, Genes, Dominant, Genes, Reporter, Glycosylation, Humans, Hydrogen-Ion Concentration, Leucine (chemistry), Leukocytes, Mononuclear (metabolism), Ligands, Luciferases (metabolism), Lysosomes (chemistry), Lysosomes (metabolism), Macrolides (pharmacology), Molecular Sequence Data, Mutagenesis, Site-Directed, Mutation, NF-kappa B (metabolism), Phagosomes (chemistry), Protein Binding, Protein Structure, Tertiary, Receptors, IgG (biosynthesis), Receptors, IgG (chemistry), Recombinant Fusion Proteins (chemistry), Recombinant Fusion Proteins (metabolism), Sequence Homology, Amino Acid, Signal Transduction, Time Factors, Toll-Like Receptor 3 (chemistry), Toll-Like Receptor 3 (metabolism), Transfection, Tyrosine (chemistry), U937 Cells.
- MESH :
- chemical , biosynthesis : Receptors, IgG.
- chemical , chemistry : Antigens, CD, Chloroquine, Cysteine, Leucine, Receptors, IgG, Recombinant Fusion Proteins, Toll-Like Receptor 3, Tyrosine.
- chemical , metabolism : Calcium, Cysteine, Cytokines, DNA, Luciferases, NF-kappa B, Recombinant Fusion Proteins, Toll-Like Receptor 3.
- chemical , pharmacology : Antirheumatic Agents, Cross-Linking Reagents, Enzyme Inhibitors, Macrolides.
- chemistry : Lysosomes, Phagosomes.
- metabolism : Cell Membrane, Dendritic Cells, Endosomes, Leukocytes, Mononuclear, Lysosomes.
- Amino Acid Sequence, Base Sequence, Binding Sites, Blotting, Western, Cell Line, Cell Separation, Dimerization, Dose-Response Relationship, Drug, Flow Cytometry, Genes, Dominant, Genes, Reporter, Glycosylation, Humans, Hydrogen-Ion Concentration, Ligands, Molecular Sequence Data, Mutagenesis, Site-Directed, Mutation, Protein Binding, Protein Structure, Tertiary, Sequence Homology, Amino Acid, Signal Transduction, Time Factors, Transfection, U937 Cells.
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
Affiliations:
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Le document en format XML
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<author><name sortKey="Hubac, Sylvain" sort="Hubac, Sylvain" uniqKey="Hubac S" first="Sylvain" last="Hubac">Sylvain Hubac</name>
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<author><name sortKey="Benguigui, Barbara" sort="Benguigui, Barbara" uniqKey="Benguigui B" first="Barbara" last="Benguigui">Barbara Benguigui</name>
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<profileDesc><textClass><keywords scheme="KwdEn" xml:lang="en"><term>Amino Acid Sequence</term>
<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>
</keywords>
<keywords scheme="KwdFr" xml:lang="fr"><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>
<term>Cystéine ()</term>
<term>Cystéine (métabolisme)</term>
<term>Cytokines (métabolisme)</term>
<term>Cytométrie en flux</term>
<term>Dimérisation</term>
<term>Données de séquences moléculaires</term>
<term>Endosomes (métabolisme)</term>
<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>
<term>Réactifs réticulants (pharmacologie)</term>
<term>Récepteur de type Toll-3 ()</term>
<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>
<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>
<term>Transduction du signal</term>
<term>Transfection</term>
<term>Tyrosine ()</term>
</keywords>
<keywords scheme="MESH" type="chemical" qualifier="biosynthesis" xml:lang="en"><term>Receptors, IgG</term>
</keywords>
<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>
</keywords>
<keywords scheme="MESH" type="chemical" qualifier="metabolism" xml:lang="en"><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>
</keywords>
<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>
</keywords>
<keywords scheme="MESH" qualifier="biosynthèse" xml:lang="fr"><term>Récepteurs du fragment Fc des IgG</term>
</keywords>
<keywords scheme="MESH" qualifier="chemistry" xml:lang="en"><term>Lysosomes</term>
<term>Phagosomes</term>
</keywords>
<keywords scheme="MESH" qualifier="metabolism" xml:lang="en"><term>Cell Membrane</term>
<term>Dendritic Cells</term>
<term>Endosomes</term>
<term>Leukocytes, Mononuclear</term>
<term>Lysosomes</term>
</keywords>
<keywords scheme="MESH" qualifier="métabolisme" xml:lang="fr"><term>ADN</term>
<term>Agranulocytes</term>
<term>Calcium</term>
<term>Cellules dendritiques</term>
<term>Cystéine</term>
<term>Cytokines</term>
<term>Endosomes</term>
<term>Facteur de transcription NF-kappa B</term>
<term>Luciferases</term>
<term>Lysosomes</term>
<term>Membrane cellulaire</term>
<term>Protéines de fusion recombinantes</term>
<term>Récepteur de type Toll-3</term>
</keywords>
<keywords scheme="MESH" qualifier="pharmacologie" xml:lang="fr"><term>Antienzymes</term>
<term>Antirhumatismaux</term>
<term>Macrolides</term>
<term>Réactifs réticulants</term>
</keywords>
<keywords scheme="MESH" xml:lang="en"><term>Amino Acid Sequence</term>
<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>
</keywords>
<keywords scheme="MESH" xml:lang="fr"><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>
<term>Transduction du signal</term>
<term>Transfection</term>
<term>Tyrosine</term>
</keywords>
</textClass>
</profileDesc>
</teiHeader>
<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>
</front>
</TEI>
<affiliations><list><country><li>France</li>
</country>
<region><li>Auvergne-Rhône-Alpes</li>
<li>Rhône-Alpes</li>
</region>
<settlement><li>Dardilly</li>
</settlement>
</list>
<tree><noCountry><name sortKey="Bates, Elizabeth E M" sort="Bates, Elizabeth E M" uniqKey="Bates E" first="Elizabeth E M" last="Bates">Elizabeth E M. Bates</name>
<name sortKey="Benguigui, Barbara" sort="Benguigui, Barbara" uniqKey="Benguigui B" first="Barbara" last="Benguigui">Barbara Benguigui</name>
<name sortKey="Caux, Christophe" sort="Caux, Christophe" uniqKey="Caux C" first="Christophe" last="Caux">Christophe Caux</name>
<name sortKey="Hasan, Uzma A" sort="Hasan, Uzma A" uniqKey="Hasan U" first="Uzma A" last="Hasan">Uzma A. Hasan</name>
<name sortKey="Hubac, Sylvain" sort="Hubac, Sylvain" uniqKey="Hubac S" first="Sylvain" last="Hubac">Sylvain Hubac</name>
<name sortKey="Merck, Estelle" sort="Merck, Estelle" uniqKey="Merck E" first="Estelle" last="Merck">Estelle Merck</name>
<name sortKey="Trinchieri, Giorgio" sort="Trinchieri, Giorgio" uniqKey="Trinchieri G" first="Giorgio" last="Trinchieri">Giorgio Trinchieri</name>
</noCountry>
<country name="France"><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>
</region>
</country>
</tree>
</affiliations>
</record>
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