Discovery of Regulators of Receptor Internalization with High-Throughput Flow Cytometry
Discovery of Regulators of Receptor Internalization with High-Throughput Flow Cytometry
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DOI：
10.1124/mol.112.079897
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发表时间：
2012-10-01
影响因子：
3.6
通讯作者：
Sklar, Larry A.
中科院分区：
文献类型：
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作者：
Wu, Yang;Tapia, Phillip H.;Sklar, Larry A.
我们开发了一个平台，结合荧光激活蛋白（FAP）技术与高通量流式细胞术，以检测实时蛋白质运输到和从质膜活细胞。该混合平台有助于药物发现运输受体，如G蛋白偶联受体，并验证了β（2）-肾上腺素能受体（β（2）AR）系统。当一个含有类似于1200种非专利药物的化学库针对表达FAP标记的β 2 AR的细胞进行筛选时，库中所有33种已知的β 2 AR活性配体都被成功鉴定，以及许多可能以非传统方式调节受体内化的化合物。结果表明，无论相关的信号通路如何，该平台都能识别靶蛋白的配体;因此，这种方法提供了寻找偏倚受体调节剂的机会，并且适合于筛选多重靶点以提高效率。结果表明，配体可能偏向于相对于受体内化的速率或持续时间，并且受体内化可能独立于丝裂原活化蛋白激酶途径的活化。
We developed a platform combining fluorogen-activating protein (FAP) technology with high-throughput flow cytometry to detect real-time protein trafficking to and from the plasma membrane in living cells. The hybrid platform facilitates drug discovery for trafficking receptors such as G protein-coupled receptors and was validated with the beta(2)-adrenergic receptor (beta(2)AR) system. When a chemical library containing similar to 1200 off-patent drugs was screened against cells expressing FAP-tagged beta(2)ARs, all 33 known beta(2)AR-active ligands in the library were successfully identified, together with a number of compounds that might regulate receptor internalization in a nontraditional manner. Results indicated that the platform identified ligands of target proteins regardless of the associated signaling pathway; therefore, this approach presents opportunities to search for biased receptor modulators and is suitable for screening of multiplexed targets for improved efficiency. The results revealed that ligands may be biased with respect to the rate or duration of receptor internalization and that receptor internalization may be independent of activation of the mitogen-activated protein kinase pathway.