Journal of Biological Chemistry
Volume 280, Issue 46, 18 November 2005, Pages 38133-38145
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Mechanisms of Signal Transduction
Recognition of Double-stranded RNA by Human Toll-like Receptor 3 and Downstream Receptor Signaling Requires Multimerization and an Acidic pH*

https://doi.org/10.1074/jbc.M507163200Get rights and content
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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 Ca2+ 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 Ca2+ 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.

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1

These authors contributed equally to this paper.

2

Present address: IFR128-BioSciences Lyon-Gerland, 21 avenue Tony Garnier, 69365 Lyon cedex 07, France.

3

Recipient of a grant from the Fondation Marcel Mérieux (Lyon, France). Present address: Ludwig Institute for Cancer Research, Chemin des Boveresses 155, 1066 Epalinges, Switzerland.

*

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

4

Present address: Infections and Cancer Biology, IARC, W.H.O., 150 Cours Albert Thomas, Lyon 69008, France.

5

Present address: Laboratory of Parasitic Diseases, NIAID, National Institutes of Health, Bldg. 50, Rm. 6150, 50 S. Dr., MSC 8003, Bethesda, MD 20892-8003.