Quantification of integrin receptor agonism by fluorescence lifetime imaging
Quantification of integrin receptor agonism by fluorescence lifetime imaging
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DOI：
10.1242/jcs.018440
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发表时间：
2008-02-01
影响因子：
4
通讯作者：
Humphries, Martin J.
中科院分区：
文献类型：
--
作者：
Parsons, Maddy;Messent, Anthea J.;Humphries, Martin J.
目前，粘附信号传导的时空分析和整合素受体的药理学抑制剂的开发都缺乏测量整合素效应物结合以及这些相互作用对拮抗剂的反应的测定方法。事实上，由于激动活性引起的继发副作用，抗整联蛋白化合物在临床上失败了。在这里，我们在活细胞中表达了整合素-GFP和效应子-mRFP对，并使用荧光寿命成像显微镜（FLIM）测量荧光共振能量转移（FRET）来量化它们的关联。 talin 与 β 1 整合素以及桩蛋白与 α 4 整合素的结合依赖于配体和受体的激活状态，并且分别对小分子 RGD 和 LDV 模拟物的抑制敏感。该测定的改进揭示了这些小分子的激动活性，从而证明这些化合物可能通过整合素激活在体内诱导次级效应。这项研究深入了解了小分子抗整合素化合物的活性对受体构象的依赖性，并为验证潜在的整合素拮抗剂提供了一种新的定量测定方法。
Both spatiotemporal analyses of adhesion signalling and the development of pharmacological inhibitors of integrin receptors currently suffer from the lack of an assay to measure integrin-effector binding and the response of these interactions to antagonists. Indeed, anti-integrin compounds have failed in the clinic because of secondary side effects resulting from agonistic activity. Here, we have expressed integrin-GFP and effector-mRFP pairs in living cells and quantified their association using fluorescence lifetime imaging microscopy (FLIM) to measure fluorescence resonance energy transfer (FRET). Association of talin with beta 1 integrin and paxillin with alpha 4 integrin was dependent on both the ligand and receptor activation state, and was sensitive to inhibition with small molecule RGD and LDV mimetics, respectively. An adaptation of the assay revealed the agonistic activity of these small molecules, thus demonstrating that these compounds may induce secondary effects in vivo via integrin activation. This study provides insight into the dependence of the activity of small molecule anti-integrin compounds upon receptor conformation, and provides a novel quantitative assay for the validation of potential integrin antagonists.