Optical probes to identify the glucocorticoid receptor ligands in living cells
Optical probes to identify the glucocorticoid receptor ligands in living cells
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DOI：
10.1016/j.steroids.2007.08.006
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发表时间：
2007-12-01
期刊：
影响因子：
2.7
通讯作者：
Umezawa, Yoshio
中科院分区：
文献类型：
--
作者：
Awais, Muhammad;Sato, Moritoshi;Umezawa, Yoshio
糖皮质激素通过糖皮质激素受体(GR)发挥作用，用于治疗多种疾病。配体诱导的辅调节蛋白(S)共激活/辅抑制子募集到GR是GR转录激活/抑制的第一步。在此，我们描述了用于在单个活细胞中筛选天然和合成糖皮质激素的基因编码的荧光探针。GR配体结合区通过柔性接头序列与辅活化子或辅阻遏子蛋白的GR相互作用肽序列相连。该融合蛋白被夹在青色和黄色荧光蛋白(分别为CFP和YFP)之间，以完成探针的构建。该结构作为一个光学探针，用于成像活细胞中糖皮质激素受体和辅助调节蛋白(GLUCOCOR)之间的配体诱导的相互作用。GR LBD与GLUCOCOR中的辅助调节肽相互作用使CFP靠近YFP，从而诱导荧光共振能量从CFP转移到YFP。GLUCOCORs可以在高通量筛选中快速、方便地识别具有功能活性的GR配体；并能够在完整的活细胞中区分GR激动剂、拮抗剂和选择性GR调节剂。因此，本方法对开发临床使用的新型糖皮质激素具有重要意义。(C)2007 Elsevier Inc.保留所有权利。
Glucocorticoids act through glucocorticoid receptor (GR) and are used for the treatment of several diseases. Ligand-induced recruitment of coregulator protein(s), coactivator/corepressor, to GR is an initial step in transcriptional activation/inhibition of GR. We describe herein genetically encoded fluorescent probes for screening of glucocorticoids, natural and synthetic, in single living cells. The GR ligand binding domain was connected to the GR interacting peptide sequence from coactivator or corepressor protein via a flexible linker sequence. This fusion protein was sandwiched between cyan and yellow fluorescent proteins (CFP and YFP, respectively) to complete the construct of the probe. This construct functions as an optical probe for imaging ligand-induced interaction between the glucocorticoid receptor and the coregulator protein (GLUCOCOR) in live cells. The interaction between GR LBD and coregulator peptide within GLUCOCOR brings CFP in close proximity of YFP to induce fluorescence resonance energy transfer from CFP to YFP. The GLUCOCORs can identify functionally active GR ligands, rapidly and conveniently, in a high-throughput screen; and are capable of distinguishing GR agonists, antagonists, and selective GR modulators in intact living cells. Therefore, the present method may play a significant role in developing new glucocorticoids for clinical use. (c) 2007 Elsevier Inc. All rights reserved.