Molecular genetic analysis of an endotoxin nonresponder mutant cell line: a point mutation in a conserved region of MD-2 abolishes endotoxin-induced signaling.
Molecular genetic analysis of an endotoxin nonresponder mutant cell line: a point mutation in a conserved region of MD-2 abolishes endotoxin-induced signaling.
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DOI：
10.1084/jem.194.1.79
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发表时间：
2001-07-02
期刊：
影响因子：
--
通讯作者：
Golenbock DT
中科院分区：
文献类型：
--
作者：
Schromm AB;Lien E;Henneke P;Chow JC;Yoshimura A;Heine H;Latz E;Monks BG;Schwartz DA;Miyake K;Golenbock DT
体细胞诱变是表征受体系统的有力工具。我们之前报道了两个互补组来自cd14转染的中国仓鼠卵巢k1成纤维细胞的突变细胞系对细菌内毒素的反应有缺陷。两类突变体均表达toll样受体（TLR）4的正常基因产物，并对肿瘤坏死因子(TNF)-α或白细胞介素(IL)-1β的刺激完全应答。我们在MD-2基因的一个互补组中发现了病变，MD-2是一种假定的TLR4辅助受体。该突变体的无应答表型通过转染MD-2被逆转。从无应答细胞系中克隆MD-2发现在一个高度保守区域发生点突变，导致C95Y氨基酸交换。转染后，两种形式的MD-2都与TLR4在细胞表面共定位，但只有野生型cDNA恢复了脂多糖（LPS）无应答表型。此外，可溶性MD-2，而非可溶性MD-2C95Y，能够在表达TLR4的细胞中激活LPS反应。因此，MD-2是LPS信号复合物的必需组分，可以作为不表达它的细胞的可溶性受体发挥作用。我们假设MD-2构象影响TLR4的细胞外结构域，可能导致对LPS的亲和力改变或作为TLR4真正配体的一部分起作用。
Somatic cell mutagenesis is a powerful tool for characterizing receptor systems. We reported previously two complementation groups of mutant cell lines derived from CD14-transfected Chinese hamster ovary–K1 fibroblasts defective in responses to bacterial endotoxin. Both classes of mutants expressed a normal gene product for Toll-like receptor (TLR)4, and fully responded to stimulation by tumor necrosis factor (TNF)-α or interleukin (IL)-1β. We identified the lesion in one of the complementation groups in the gene for MD-2, a putative TLR4 coreceptor. The nonresponder phenotype of this mutant was reversed by transfection with MD-2. Cloning of MD-2 from the nonresponder cell line revealed a point mutation in a highly conserved region resulting in a C95Y amino acid exchange. Both forms of MD-2 colocalized with TLR4 on the cell surface after transfection, but only the wild-type cDNA reverted the lipopolysaccharide (LPS) nonresponder phenotype. Furthermore, soluble MD-2, but not soluble MD-2C95Y, functioned to enable LPS responses in cells that expressed TLR4. Thus, MD-2 is a required component of the LPS signaling complex and can function as a soluble receptor for cells that do not otherwise express it. We hypothesize that MD-2 conformationally affects the extracellular domain of TLR4, perhaps resulting in a change in affinity for LPS or functioning as a portion of the true ligand for TLR4.
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影响因子：
32.4
作者：
Kawai, T;Adachi, O;Akira, S
通讯作者：
Akira, S
影响因子：
15.9
作者：
Lien, E;Means, TK;Golenbock, DT
通讯作者：
Golenbock, DT
影响因子：
32.4
作者：
PUGIN, J;HEUMANN, D;ULEVITCH, RJ
通讯作者：
ULEVITCH, RJ
DOI：
10.1084/jem.176.2.485
发表时间：
1992-08-01
期刊：
The Journal of experimental medicine
影响因子：
--
作者：
Kitchens RL;Ulevitch RJ;Munford RS
通讯作者：
Munford RS
影响因子：
10.5
作者：
Lomaga, MA;Yeh, WC;Mak, TW
通讯作者：
Mak, TW