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首页> 外文期刊>Molecular and Cellular Biology >ATP-dependent release of glucocorticoid receptors from the nuclear matrix.
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ATP-dependent release of glucocorticoid receptors from the nuclear matrix.

机译：ATP依赖性糖皮质激素受体从核基质中释放。

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Glucocorticoid receptors (GRs) have the capacity to shuttle between the nuclear and cytoplasmic compartments, sharing that trait with other steroid receptors and unrelated nuclear proteins of diverse function. Although nuclear import of steroid receptors, like that of nearly all other karyophilic proteins examined to date, requires ATP, there appear to be different energetic requirements for export of proteins, including steroid receptors, from nuclei. In an attempt to reveal which steps, if any, in the nuclear export pathway utilized by steroid receptors require ATP, we have used indirect immunofluorescence to visualize GRs within cells subjected to a reversible ATP depletion. Under conditions which lead to >95% depletion of cellular ATP levels within 90 min, GRs remain localized within nuclei and do not efflux into the cytoplasm. Under analogous conditions of ATP depletion, transfected progesterone receptors are also retained within nuclei. Importantly, GRs which accumulate within nuclei of ATP-depleted cells are distinguished from nuclear receptors in metabolically active cells by their resistance to in situ extraction with a hypotonic, detergent-containing buffer. GRs in ATP-depleted cells are not permanently trapped in this nuclear compartment, as nuclear receptors rapidly regain their capacity to be extracted upon restoration of cellular ATP, even in the absence of de novo protein synthesis. More extensive extraction of cells with high salt and detergent, coupled with DNase I digestion, established that a significant fraction of GRs in ATP-depleted cells are associated with an RNA-containing nuclear matrix. Quantitative Western blot (immunoblot) analysis confirmed the dramatic increase in GR binding to the nuclear matrix of ATP-depleted cells, while confocal microscopy revealed that GRs are bound to the matrix throughout all planes of the nucleus. ATP depletion does not lead to wholesale collapse of nuclear proteins onto the matrix, as the interaction of a subpopulation of simian virus 40 large tumor antigen with the nuclear matrix is not quantitatively altered in ATP-depleted Cos-1 cells. Nuclear GRs which are not bound to the nuclear matrix of metabolically active cells (i.e., a DNA-binding domain deletion mutant and a beta-galactosidase chimera possessing the GR nuclear localization signal sequence) are not recruited to the matrix upon depletion of cellular ATP. Thus, it appears that ATP depletion does not expose the GR to nuclear matrix interactions which are not normally encountered in cells but merely alters the dynamics of such interactions. The dynamic association of steroid receptors with the nuclear matrix may provide a mechanism which is utilized by these regulable transcription factors to facilitate their efficient scanning of the genome.
机译：糖皮质激素受体（GRs）具有在核和细胞质区室之间穿梭的能力，与其他类固醇受体和功能多样的无关核蛋白共享该特性。尽管类固醇受体的核输入需要ATP，就像迄今为止检查的几乎所有其他亲核蛋白一样，它们都需要ATP，但是从核中输出包括类固醇受体的蛋白质似乎存在着不同的能量需求。为了揭示类固醇受体利用的核输出途径中哪些步骤（如果有的话）需要ATP，我们使用了间接免疫荧光技术来观察遭受可逆ATP消耗的细胞内的GRs。在导致90分钟内细胞ATP含量消耗> 95％的条件下，GR仍位于细胞核内，不会外流到细胞质中。在ATP耗竭的类似条件下，转染的孕激素受体也保留在细胞核内。重要的是，积累在ATP耗尽的细胞核内的GRs与代谢活性细胞中的核受体不同，在于它们对低渗，含去污剂缓冲液的原位提取具有抵抗力。耗尽ATP的细胞中的GR不会永久地困在该核区隔中，因为即使没有从头合成的蛋白质，核受体也会在恢复细胞ATP后迅速恢复其提取能力。用高盐和去污剂对细胞进行更广泛的提取，再结合DNase I消化，可以确定ATP耗尽的细胞中显着的GRs与含RNA的核基质有关。定量Western印迹（免疫印迹）分析证实，GR与耗尽ATP的细胞的核基质的结合显着增加，而共聚焦显微镜显示GRs在整个核平面上均与基质结合。 ATP耗竭不会导致核蛋白完全塌陷到基质上，因为在ATP耗竭的Cos-1细胞中，猿猴病毒40大肿瘤抗原的亚群与核基质的相互作用并未得到定量改变。没有与代谢活性细胞的核基质结合的核GR（即具有GR核定位信号序列的DNA结合结构域缺失突变体和β-半乳糖苷酶嵌合体）在细胞ATP耗尽后不会被募集到基质中。因此，看来ATP耗竭不会使GR暴露于细胞中通常不会遇到的核基质相互作用，而仅改变了这种相互作用的动力学。类固醇受体与核基质的动态缔合可以提供一种机制，这些可调节的转录因子利用该机制促进它们对基因组的有效扫描。

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