Roles for LPS-dependent interaction and relocation of TLR4 and TRAM in TRIF-signaling

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Abstract

Toll-like receptor 4 (TLR4) activates two distinct signaling pathways inducing production of proinflammatory cytokines or type I interferons (IFNs), respectively. MyD88 and TIRAP/Mal are essential adaptor molecules for the former but not for the latter pathway. In contrast, TRIF/TICAM-1 and TRAM/TICAM-2 are essential for both. TIRAP is a sorting adaptor molecule recruiting MyD88 to activated TLR4 in the plasma membrane. TRAM is thought to bridge between TLR4 and TRIF by physical association. Little is known, however, how TRAM interacts with TLR4 or with TRIF during LPS response. Here, we show that TRAM recruits TRIF to the plasma membrane. Moreover, LPS induces upregulation of TLR4-association with TRAM and their subsequent translocation into endosome/lysosome. The internalized signaling complex consisting of TLR4 and TRAM colocalizes with TRAF3, a signaling molecule downstream of TRIF, in endosome/lysosome. These results suggest that TLR4 activates TRIF-signaling in endosome/lysosome after relocation from the cell surface.

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Materials and methods

Expression constructs and stable transfectants. Ba/F3 cells were cultured as described previously [9]. The cDNAs encoding muTLR4, muMD-2, and muCD14 were cloned as described previously [14]. The C-terminus of TLR4 was tagged with GFP (TLR4-GFP). The cDNAs encoding muTRAM, muTRIF, and muTRAF3 were cloned into retrovirus vector pMX-puro. The C-terminus of TRAM was tagged with GFP or DsRed monomer (TRAM-GFP or TRAM-DsRed). To mutate the RHIM in muTRIF was replaced with alanines by QuikChange site

CD14-dependent TLR4-internalization to endosome/lysosome

We previously reported that TLR4-clustering is followed by TLR4-internalization [9]. Lipid A induced TLR4-internalization in Ba/F3 cells expressing TLR4/MD-2 with CD14, but not in those expressing TLR4/MD-2 without CD14 (Fig. 1A). Upon stimulation with lipid A, cell surface TLR4 was gradually downregulated and disappeared from the plasma membrane within 120 min. To determine the subcellular distribution of internalized TLR4, TLR4-GFP was expressed in Ba/F3 cells together with MD-2 and CD14. Most

Discussion

TLRs can be divided into two groups based on their specificity and subcellular location. TLR1/2/4/6 recognize microbial membrane components and reside on the plasma membrane, whereas nucleic acid-sensing TLR3/7/8/9 are intracellular and signal from endosome/lysosome [2]. Cell surface TLRs are distinct from intracellular TLRs in adaptor usage. TIRAP has a phosphatidylinositol 4,5-bisphosphate (PIP2) binding domain, which mediates TIRAP recruitment to the plasma membrane [10]. TIRAP then

Acknowledgments

The authors wish to thank Drs Jun-ichiro Inoue and George Mosialos for cDNAs encoding TRAF3. We also thank Mr. Yuji Motoi for technical assistance, and Dr. Toshihiko Kobayashi for critical discussions. This study was supported by Research Fellowships of the Japan Society for the Promotion of Science for Young Scientists, Strategic cooperation to control emerging and reemerging infections funded by The Special Coordination Funds for Promoting Science and Technology of Ministry of Education,

References (22)

  • R. Shimazu et al.

    MD-2, a molecule that confers lipopolysaccharide responsiveness on Toll-like receptor 4

    J. Exp. Med.

    (1999)
  • Cited by (0)

    1

    These authors contributed equally to this study.

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