Steroid hormone receptor dynamics in neural cells

我们使用绿色荧光蛋白（GFP）的融合蛋白检测了糖皮质激素受体（GR）和盐皮质激素受体（MR）在活神经元和非神经细胞中的细胞内运输。皮质酮（Corticosterone，CORT）诱导GFP-MR在细胞核内迅速聚集，而在无配体的情况下，GFP-MR在大多数转染细胞中分布于细胞质和细胞核内。我们还发现微管不参与这些受体的核转位，但热休克蛋白90在这些受体的核转位中起重要作用。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配体的调节是重要的。为了检测单个活细胞内MR和GR的同时运输，我们使用了GFP的不同光谱变体，分别与MR和GR连接的黄色荧光蛋白（YFP）和青色荧光蛋白（CFP）， ...更多信息 伊利。在不表达内源性皮质类固醇受体的COS-1细胞中，在10 μ M CORT存在下，YFP MR嵌合体在细胞核中的积累速度比CFP-GR嵌合体快<-9>，而在10 μ M CORT存在下，细胞核积累速率没有显著差异<-6>。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CORT存在下，YFP-MR嵌合体和CFP-GR嵌合体的核积累速率几乎相同。这些结果表明，皮质激素诱导的核转位的MR和GR表现出不同的模式，这取决于配体浓度或细胞类型。此外，我们还用同样的融合蛋白方法研究了importin α与这些皮质类固醇受体的关系。当YFP-输入蛋白α单独转染COS细胞时，它们在转染细胞的主要群体中存在于细胞质和细胞核中，并且YFP输入蛋白α在没有任何刺激的情况下易位到细胞核中。然而，YFP-输入蛋白α和CFP-GR共转染到COS细胞中，配体处理后YFP-输入蛋白α在细胞核中的积累更强烈。这些结果表明，该系统成功地检测了活的完整细胞中的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