Phenotypic knockout of the high-affinity human interleukin 2 receptor by intracellular single-chain antibodies against the alpha subunit of the receptor.
Phenotypic knockout of the high-affinity human interleukin 2 receptor by intracellular single-chain antibodies against the alpha subunit of the receptor.
复制标题
通过针对受体 α 亚基的细胞内单链抗体对高亲和力人白细胞介素 2 受体进行表型敲除。
DOI：
10.1073/pnas.92.8.3137
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发表时间：
1995
影响因子：
11.1
通讯作者：
Marasco,WA
中科院分区：
文献类型：
--
作者：
Richardson,JH;Sodroski,JG;Waldmann,TA;Marasco,WA
肽生长激素及其细胞受体的实验操作是理解细胞信号传导和生长控制途径的核心。先前的工作已经表明，靶向内质网（ER）的细胞内抗体可用于捕获进入ER的特定蛋白质，阻止其转运到细胞表面。在这里，我们使用这种技术来抑制高亲和力白细胞介素2受体（IL-2 R α）α亚基的细胞表面表达。抗Tac单克隆抗体的单链可变区片段与信号肽和C-末端ER保留信号一起构建。发现单链抗体的细胞内表达完全消除了刺激的Jurkat T细胞中IL-2 R α的细胞表面表达。在Jurkat细胞内可检测到IL-2 R α，其为未成熟的40-kDa形式，对内切糖苷酶H敏感，与其保留在前高尔基体或早期高尔基体隔室中一致。缺乏ER滞留信号的单链抗体也能够抑制IL-2 R α的细胞表面表达，尽管该机制似乎涉及ER内受体链的快速降解。这些细胞内抗体将提供用于检查IL-2 R α在T细胞活化、IL-2信号转导和过度表达IL-2 R α的白血病细胞的失调生长中的作用的有价值的工具。
The experimental manipulation of peptide growth hormones and their cellular receptors is central to understanding the pathways governing cellular signaling and growth control. Previous work has shown that intracellular antibodies targeted to the endoplasmic reticulum (ER) can be used to capture specific proteins as they enter the ER, preventing their transport to the cell surface. Here we have used this technology to inhibit the cell surface expression of the alpha subunit of the high-affinity interleukin 2 receptor (IL-2R alpha). A single-chain variable-region fragment of the anti-Tac monoclonal antibody was constructed with a signal peptide and a C-terminal ER retention signal. Intracellular expression of the single-chain antibody was found to completely abrogate cell surface expression of IL-2R alpha in stimulated Jurkat T cells. IL-2R alpha was detectable within the Jurkat cells as an immature 40-kDa form that was sensitive to endoglycosidase H, consistent with its retention in a pre- or early Golgi compartment. A single-chain antibody lacking the ER retention signal was also able to inhibit cell surface expression of IL-2R alpha although the mechanism appeared to involve rapid degradation of the receptor chain within the ER. These intracellular antibodies will provide a valuable tool for examining the role of IL-2R alpha in T-cell activation, IL-2 signal transduction, and the deregulated growth of leukemic cells which overexpress IL-2R alpha.
影响因子：
64.8
作者：
LEONARD, WJ;DEPPER, JM;GREENE, WC
通讯作者：
GREENE, WC