The genetic design of signaling cascades to record receptor activation
The genetic design of signaling cascades to record receptor activation
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DOI：
10.1073/pnas.0710487105
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发表时间：
2008-01-08
影响因子：
11.1
通讯作者：
Lee, Kevin J.
中科院分区：
文献类型：
--
作者：
Barnea, Gilad;Strapps, Walter;Lee, Kevin J.
我们开发了一种实验策略，能够以高度的选择性和敏感性监测细胞内的蛋白质相互作用。一种转录因子通过一个含有特定蛋白酶切割位点的连接子连接到膜结合受体上。受体的激活会招募一个与蛋白酶融合的信号蛋白，该蛋白酶随后切割并释放转录因子，从而激活细胞核中的报告基因。这种策略将一种瞬时相互作用转化为一种稳定且可放大的报告基因信号，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，我们利用该检测方法鉴定了孤儿受体GPR1的一种配体，这表明该受体在炎症调节中具有作用。
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.