Recognition of double-stranded RNA by human Toll-like receptor 3 and downstream receptor signaling requires multimerization and an acidic pH
Recognition of double-stranded RNA by human Toll-like receptor 3 and downstream receptor signaling requires multimerization and an acidic pH
复制标题
DOI：
10.1074/jbc.m507163200
复制
发表时间：
2005-11-18
影响因子：
4.8
通讯作者：
Caux, C
中科院分区：
文献类型：
--
作者：
de Bouteiller, O;Merck, E;Caux, C
涉及toll样受体3 （TLR3）缺陷小鼠的研究表明，该受体结合双链RNA。在本研究中，我们分析了导致TLR3激活的配体/受体相互作用和受体近端事件。诱变方法表明，TLR3氨基末端富亮氨酸重复序列中的某些半胱氨酸残基和糖基化对于配体诱导的信号传导是必要的。此外，失活突变体具有显性的负作用，这表明信号模块是一个多定时器。我们构建了一个嵌合分子，将TLR3的氨基末端外结构域与CD32a的跨膜和羧基末端结构域融合，其中包含一个基于酪氨酸的免疫受体基序。TLR3-CD32在HEK293T细胞和髓系U937中的表达导致受体表面定位，而非重组分子则在细胞内定位。合成的双链rna poly（I-C）和poly（a - u）在稳定转染TLR3-CD32的U937克隆中诱导钙动员，而在转染其他构建体的对照细胞中则没有。抗tlr3抗体也诱导Ca2+通量，但只有当二级抗免疫球蛋白抗体交联时，证实配体的多聚是信号传导的必要条件。溶酶体成熟抑制剂巴菲霉素和氯喹抑制免疫细胞中poly（I-C）诱导的生物反应，表明TLR3在酸性亚细胞区室中与其配体相互作用。此外，TLR3-CD32与poly（I-C）的激活仅在狭窄的pH窗口（pH 5.7-6.7）内观察到，而抗tlr3介导的Ca2+通量对pH不敏感。当使用低聚聚（I-C） （15-40-mers）时，酸性pH值对tlr3 -配体相互作用的重要性变得至关重要。这些观察结果表明，在酸性pH下，可能在早期吞噬溶酶体或内体中，poly（I-C）参与TLR3，诱导受体聚集导致信号传导。
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.