Roles for LPS-dependent interaction and relocation of TLR4 and TRAM in TRIF-signaling
Section snippets
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,
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These authors contributed equally to this study.