Steroid hormone receptor dynamics in neural cells

我们使用绿色荧光蛋白（GFP）的融合蛋白检测了活神经元和非神经细胞中糖皮质激素受体（GR）和矿皮质激素受体（MR）的细胞内运输。皮质酮（CORT）诱导GFP-MR在细胞核中快速积累，而在没有配体的情况下，大多数转染细胞中GFP-MR分布在细胞质和细胞核中。我们还发现微管不参与这些受体的核易位，但hsp90在这些受体的核易位中起重要作用。鉴于MR和GR在中枢神经系统中的不同作用，阐明这些受体在细胞质和细胞核之间的运输如何受到配体的调节是很重要的。为了研究MR和GR在单个活细胞内的同时运输，我们使用了不同的光谱变体GFP，分别与MR和GR连接的黄色荧光蛋白（YFP）和青色荧光蛋白（CFP）。在不表达内源性皮质类固醇受体的COS-1细胞中，当存在10^<-9>M CORT时，YFP MR嵌合体在细胞核内的积累速度比CFP-GR嵌合体快，而当存在10^<-6>M CORT时，细胞核积累速度无显著差异。另一方面，在原代培养的表达内源性受体的海马神经元中，两种浓度的CORT存在时，YFP-MR嵌合体和CFP-GR嵌合体的核积累率几乎相同。这些结果表明，cort诱导的MR和GR的核易位表现出不同的模式，这取决于配体浓度或细胞类型。此外，我们使用相同的融合蛋白方法研究了输入蛋白α与这些皮质类固醇受体的关系。在COS细胞中单独转染YFP-importin α后，它们在转染细胞的主要群体中同时存在于细胞质和细胞核中，并且YFP-importin α在没有任何刺激的情况下易位到细胞核中。而将YFP-importin α和CFP-GR共转染到COS细胞中，经配体处理后，YFP-importin α在细胞核内的积累更为强烈。这些结果表明，该系统成功地检测了完整活细胞中载体蛋白和核受体的核输入过程。少

We examined the intracellular trafficking of glucocorticoid receptor (GR) and mineralocorticoid receptor (MR) in living neurons and non-neural cells using a fusion protein of green fluorescent protein (GFP). Corticosterone (CORT) induced a rapid nuclear accumulation of GFP-MR, whereas in the absence of ligand, GFP-MR was distributed in both cytoplasm and nucleus in the majority of transfected cells. We also showed that microtubules are not involved in the nuclear translocation of these receptors, but hsp 90 plays important roles for nuclear translocation of these receptors. Given the differential action of MR and GR in the central nervous system, it is important to elucidate how the trafficking of these receptors between cytoplasm and nucleus is regulated by ligand. To examine the simultaneous trafficking of MR and GR within single living cells, we use different spectral variants of GFP, yellow fluorescent protein (YFP) and cyan fluorescent protein (CFP), linked to MR and GR, respectiv … More ely. In COS-1 cells, expressing no endogenous corticosteroid receptors, YFP MR chimera was accumulated in the nucleus faster than CFP-GR chimera in the presence of 10^<-9>M CORT, while there is no significant difference in the nuclear accumulation rates in the presence of 10^<-6>M CORT. On the other : hand, in primary cultured hippocampal neurons, expressing endogenous receptors, the nuclear accumulation rates of YFP-MR chimera and CFP-GR chimera were nearly the same in the presence of both concentrations of CORT. These results suggest that CORT-induced nuclear translocation of MR and GR exhibits differential patterns depending on ligand concentrations or cell types. Furthermore, we investigated the relationships of importin α and these corticosteroid receptors using the same fusion protein methods. When YFP-importin α is singly transfected in COS cells, they reside in both cytoplasm and nucleus in major populations of the transfected cells, and YFP importin α is translocated into the nucleus without any stimulation. However, YFP-importin α and CFP-GR are co transfected into COS cells, YFP-importin α is more strongly accumulated in the nucleus after ligand treatment. These results demonstrate that this system successfully detects a nuclear import process of carrier proteins and nuclear receptors in living intact cells. Less