Visualization of glucocorticoid receptor and mineralocorticoid receptor interactions in living cells with GFP-based fluorescence resonance energy transfer
Visualization of glucocorticoid receptor and mineralocorticoid receptor interactions in living cells with GFP-based fluorescence resonance energy transfer
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DOI：
10.1523/jneurosci.5495-03.2004
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发表时间：
2004-05-26
影响因子：
5.3
通讯作者：
Kawata, M
中科院分区：
文献类型：
--
作者：
Nishi, M;Tanaka, M;Kawata, M
肾上腺皮质类固醇很容易进入大脑并发挥显着不同的作用，包括通过盐皮质激素受体（MR）和糖皮质激素受体（GR）这两个受体系统在目标神经细胞中产生应激反应。研究表明，GR 和 MR 在海马体中高度共定位。鉴于海马区 MR 和 GR 的不同作用，阐明这些受体在皮质类固醇反应中如何相互作用非常重要。我们利用基于绿色荧光蛋白的荧光共振能量转移（FRET）显微镜以时空方式研究了活细胞中 MR 和 GR 的异二聚化。 FRET通过三种方式进行评估：（1）比率成像； (2)发射光谱； (3)受体光漂白。 FRET分析表明，皮质酮（CORT）处理后，青色荧光蛋白GR和黄色荧光蛋白MR在培养的海马神经元和COS-1细胞的细胞核中形成异二聚体，而在细胞质中不形成异二聚体。在培养的海马神经元和COS-1细胞中，10(-6)M CORT时GR-MR异二聚体的含量高于10(-9)M CORT时的GR-MR异二聚体含量，并在CORT处理60分钟后达到最高水平。异二聚体在培养的海马神经元细胞核中的分布模式比 COS-1 细胞中的分布模式更受限制。目前在核定位信号中使用突变融合蛋白的研究表明，即使在用配体处理后，这些皮质类固醇受体也不会以异二聚体的形式易位到细胞核中，因此不允许在细胞质中发生异二聚化。 FRET 分析获得的这些结果为 GR 和 MR 异二聚化的位点、时间过程以及配体浓度的影响提供了新的见解。
Adrenal corticosteroids readily enter the brain and exert markedly diverse effects, including stress responses in the target neural cells via two receptor systems, the mineralocorticoid receptor (MR) and the glucocorticoid receptor (GR). It has been shown that the GR and MR are highly colocalized in the hippocampus. Given the differential action of the MR and GR in the hippocampal region, it is important to elucidate how these receptors interact with each other in response to corticosteroids. We investigated the heterodimerization of the MR and GR with green fluorescent protein-based fluorescence resonance energy transfer ( FRET) microscopy in living cells with spatiotemporal manner. FRET was evaluated in three ways: ( 1) ratio imaging; ( 2) emission spectra; and ( 3) acceptor photobleaching. FRET analysis demonstrated that cyan fluorescent protein - GR and yellow fluorescent protein - MR form heterodimers after corticosterone ( CORT) treatment both in the nucleus of cultured hippocampal neurons and COS-1 cells, whereas they do not form heterodimers in the cytoplasm. The content of the GR - MR heterodimer was higher at 10(-6) M CORT than at 10(-9) M CORT and reached a maximum level after 60 min of CORT treatment in both cultured hippocampal neurons and COS-1 cells. The distribution pattern of heterodimers in the nucleus of cultured hippocampal neurons was more restricted than that in COS-1 cells. The present study using mutant fusion proteins in nuclear localization signal showed that these corticosteroid receptors are not translocated into the nucleus in the form of heterodimers even after treatment with ligand and thus allow no heterodimerization to take place in the cytoplasm. These results obtained with FRET analyses give new insights into the sites, time course, and effects of ligand concentration on heterodimersization of the GR and MR.