Molecular mechanism of intracellular trafficking of TLR4

天然免疫系统的基本组成部分是TLRs，它识别被称为病原体相关分子模式(PAMPs)的微生物产物。PAMP识别导致先天免疫系统的激活，进而激活获得性免疫。然而，TLR4转运到细胞表面并被激活的机制仍有待确定。在本研究中，我们试图阐明决定TLR4细胞定位的一些重要基序。针对L1基序，我们对TLR4进行了各种截断突变。将TLR4截短突变和md2基因导入HEK293T细胞并在其上表达，用荧光素酶比色法比较内毒素诱导的NF-κB活性。在转染1-826TLR4突变体和野生型TLR4的细胞中观察到脂多糖诱导的NF-κB活性。然而，在1-815和更短的TLR4截断突变体转染的细胞中没有观察到它们。此外，TLR4突变体1-826的定位与野生型相似。然而，在1-815和更短的TLR4的截断突变体中没有观察到细胞表面的表达。这些结果表明，TLR4在815位附近存在决定其定位的基序。接下来，我们在TLR4的813、815、816和817位上进行了一系列的单截断突变。我们发现TLR4突变体(L815A)没有观察到细胞表面的表达。在没有κ的情况下，未观察到脂多糖诱导的NF-CD14B活性。而在CD14存在的情况下，它们被恢复了。因此，我们确定了决定TLR4细胞定位的一个重要区域。

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.