Liu J, DeFranco D B
Department of Biological Sciences, University of Pittsburgh, Pennsylvania 15260, USA.
Mol Endocrinol. 1999 Mar;13(3):355-65. doi: 10.1210/mend.13.3.0258.
Unliganded glucocorticoid receptors (GRs) released from chromatin after hormone withdrawal remain associated with the nucleus within a novel subnuclear compartment that serves as a nuclear export staging area. We set out to examine whether unliganded nuclear receptors cycle between distinct subnuclear compartments or require cytoplasmic transit to regain hormone and chromatin-binding capacity. Hormone-withdrawn rat GrH2 hepatoma cells were permeabilized with digitonin to deplete cytoplasmic factors, and then hormone-binding and chromatin-binding properties of the recycled nuclear GRs were measured. We found that recycled nuclear GRs do not require cytosolic factors or ATP to rebind hormone. Nuclear GRs that rebind hormone in permeabilized cells target to high-affinity chromatin-binding sites at 30 C, but not 0 C, in the presence of ATP. Since geldanamycin, a heat shock protein-90 (hsp90)-binding drug, inhibits hormone binding to recycled nuclear GRs, hsp90 may be required to reassemble the receptor into a form capable of productive interactions with hormone. Geldanamycin also inhibits GR release from chromatin during hormone withdrawal, suggesting that hsp90 chaperone function may play multiple roles to facilitate chromatin recycling of GR.
激素撤除后从染色质释放的未结合配体的糖皮质激素受体（GRs）仍与细胞核相关联，存在于一个作为核输出暂存区的新型亚核区室中。我们着手研究未结合配体的核受体是否在不同的亚核区室之间循环，或者是否需要细胞质转运来恢复激素和染色质结合能力。用洋地黄皂苷使激素撤除的大鼠GrH2肝癌细胞透化以耗尽细胞质因子，然后测量再循环核GRs的激素结合和染色质结合特性。我们发现再循环核GRs重新结合激素不需要细胞质因子或ATP。在有ATP存在的情况下，在透化细胞中重新结合激素的核GRs在30℃而非0℃时靶向高亲和力染色质结合位点。由于格尔德霉素是一种与热休克蛋白90（hsp90）结合的药物，它抑制激素与再循环核GRs的结合，因此可能需要hsp90将受体重新组装成能够与激素进行有效相互作用的形式。格尔德霉素还抑制激素撤除期间GR从染色质的释放，这表明hsp90伴侣功能可能发挥多种作用以促进GR的染色质再循环。
