Molecular mechanism of intracellular trafficking of TLR4

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Essential Components of the innate immune system are TLRs, which recognize microbial products termed pathogen-associated molecular patterns (PAMPs). PAMP recognition leads to activation of the innate immune system, which in turn activates adaptive immunity. However, it remains to be determined that the mechanisms by which TLR4 were transported to the cell surface and activated. In this study, we tried to elucidate some important motifs which determined cellular localization of TLR4.Focused on LL motif, we made various truncation mutations of TLR4. These TLR4 truncation mutations and MD2 were transfected into and expressed on HEK293T cells, and LPS-induced activities of NF-κB were compared by a luciferase assay. LPS-induced NF-κB activities were observed in cells transfected with 1-826 TLR4 mutant as well as wild type TLR4. However, they were not observed in cells transfected with 1-815 and shorter TLR4 truncation mutants. In addition, localization of 1-826 TLR4 mutant was similar to that of wild type TLR4. However, cell surface expression was not observed in truncation mutants of 1-815 and shorter TLR4. These results indicated that there were some motifs which determined localization of TLR4 around position of 815. Next, we made a series of single truncation mutants of TLR4 at the positions of 813, 815, 816, and 817. We found that cell surface expression of a TLR4 mutant (L815A) was not observed. LPS-induced NF-κB activities were not observed in cells transfected with a TLR4 mutant (L815A) in the absence of CD14. While, they were reinstated in the presence of CD14. Thus, we identified an important region which determines cellular localization of TLR4.