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The genetic design of signaling cascades to record receptor activation

机译:信号级联反应记录受体激活的遗传设计

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We have developed an experimental strategy to monitor protein interactions in a cell with a high degree of selectivity and sensitivity. A transcription factor is tethered to a membrane-bound receptor with a linker that contains a cleavage site for a specific protease. Activation of the receptor recruits a signaling protein fused to the protease that then cleaves and releases the transcription factor to activate reporter genes in the nucleus. This strategy converts a transient interaction into a stable and amplifiable reporter gene signal to record the activation of a receptor without interference from endogenous signaling pathways. We have developed this assay for three classes of receptors: G protein-coupled receptors, receptor tyrosine kinases, and steroid hormone receptors. Finally, we use the assay to identify a ligand for the orphan receptor GPR1, suggesting a role for this receptor in the regulation of inflammation.
机译:我们已经开发出一种实验策略,可以高度选择性和灵敏性地监测细胞中的蛋白质相互作用。转录因子通过含有特定蛋白酶切割位点的接头与膜结合受体结合。受体的激活募集与蛋白酶融合的信号蛋白,然后裂解并释放转录因子以激活细胞核中的报告基因。该策略将瞬时相互作用转化为稳定且可扩增的报道基因信号,以记录受体的激活,而不受内源性信号通路的干扰。我们已经针对三类受体开发了这种检测方法:G蛋白偶联受体,受体酪氨酸激酶和类固醇激素受体。最后,我们使用该测定法来鉴定孤儿受体GPR1的配体,表明该受体在炎症调节中的作用。

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