The mammalian-membrane two-hybrid assay (MaMTH) for probing membrane-protein interactions in human cells
The mammalian-membrane two-hybrid assay (MaMTH) for probing membrane-protein interactions in human cells
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DOI：
10.1038/nmeth.2895
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发表时间：
2014-05-01
期刊：
影响因子：
48
通讯作者：
Stagljar, Igor
中科院分区：
文献类型：
--
作者：
Petschnigg, Julia;Groisman, Bella;Stagljar, Igor
细胞信号传导是细胞正常功能和疾病的关键过程之一，它是由细胞膜蛋白之间的许多相互作用协调的，这些相互作用响应于刺激而改变。我们提出了一种基于泛素裂解的方法，用于检测人类细胞中的膜蛋白-蛋白相互作用（PPI），称为膜双杂交试验（MaMTH）。我们表明，该技术检测刺激（激素或激动剂）依赖性和磷酸化依赖性PPI。MaMTH可以检测由突变（如致癌ErbB受体变体）或药物治疗（如酪氨酸激酶抑制剂厄洛替尼）引起的PPI变化。使用MaMTH作为筛选测定，我们鉴定了CRKII作为致癌EGFR（L 858 R）的相互作用物，并表明CRKII促进非小细胞肺癌细胞中异常信号传导的持续活化。MaMTH是研究人类整合膜蛋白动态相互作用组的有力工具。
Cell signaling, one of key processes in both normal cellular function and disease, is coordinated by numerous interactions between membrane proteins that change in response to stimuli. We present a split ubiquitin based method for detection of integral membrane protein-protein interactions (PPIs) in human cells, termed mammalian-membrane two-hybrid assay (MaMTH). We show that this technology detects stimulus (hormone or agonist)-dependent and phosphorylation-dependent PPIs. MaMTH can detect changes in PPIs conferred by mutations such as those in oncogenic ErbB receptor variants or by treatment with drugs such as the tyrosine kinase inhibitor erlotinib. Using MaMTH as a screening assay, we identified CRKII as an interactor of oncogenic EGFR(L858R) and showed that CRKII promotes persistent activation of aberrant signaling in non small cell lung cancer cells. MaMTH is a powerful tool for investigating the dynamic interactomes of human integral membrane proteins.