Dynamics of intracellular movement and nucleocytoplasmic recycling of the ligand-activated androgen receptor in living cells.
Dynamics of intracellular movement and nucleocytoplasmic recycling of the ligand-activated androgen receptor in living cells.
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DOI：
10.1210/mend.14.8.0497
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发表时间：
2000-08
影响因子：
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通讯作者：
R. Tyagi;Y. Lavrovsky;S. Ahn;C. Song;B. Chatterjee;A. Roy
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文献类型：
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作者：
R. Tyagi;Y. Lavrovsky;S. Ahn;C. Song;B. Chatterjee;A. Roy
将编码修饰的水母绿色荧光蛋白（GFP）的cDNA连接到大鼠雄激素受体（AR）cDNA的5 '端（GFP-AR），构建了表达载体，用于研究受体在活细胞内运动的细胞内动力学。在三个不同的细胞系，即。在PC 3、HeLa和COS 1中，未配体的GFP-AR主要见于细胞质中，并在雄激素处理后迅速（在15-60分钟内）移动到核室。雄激素戒断后，标记的AR迁移回细胞质区室，并保持其能力，重新进入细胞核后，随后暴露于雄激素。在放线菌酮（50 μ g/ml）抑制蛋白质合成的条件下，雄激素处理和停药后至少记录了4轮受体再循环。两种非雄激素，17 β-雌二醇和孕酮在较高浓度（10（-7）/10（-6）M）下，能够反式激活AR反应启动子并将GFP-AR转运到细胞核中。类似地，抗雄激素配体醋酸环丙孕酮和casodex也能够将细胞质AR转运到细胞核中，尽管速率比雄激素5 α-二氢睾酮（DHT）慢。所有具有反式激活潜力的AR配体，包括混合激动剂/拮抗剂醋酸环丙孕酮，引起GFP-AR易位到由其点状核内分布指示的亚核隔室中。然而，由casodex（一种纯拮抗剂）引起的易位导致了均匀的核分布。随后暴露的casodex处理的细胞迅速（15-30分钟）改变了均匀的点状分布已经易位的核AR。当运输到细胞核无论是casodex或DHT，GFP-AR耐2 M NaCl提取，表明均匀分布的AR也与核基质。总之，这些结果表明，AR需要配体激活其核转位，其中仅激动剂和部分激动剂的占用可以将其引导到潜在的功能性亚核位置，并且一个受体分子可以进行多轮激素信号传导;这表明配体解离/失活而不是受体降解可能在终止激素作用中起关键作用。
An expression construct containing the cDNA encoding a modified aequorea green fluorescent protein (GFP) ligated to the 5'-end of the rat androgen receptor (AR) cDNA (GFP-AR) was used to study the intracellular dynamics of the receptor movement in living cells. In three different cell lines, ie. PC3, HeLa, and COS1, unliganded GFP-AR was seen mostly in the cytoplasm and rapidly (within 15-60 min) moved to the nuclear compartment after androgen treatment. Upon androgen withdrawal, the labeled AR migrated back to the cytoplasmic compartment and maintained its ability to reenter the nucleus on subsequent exposure to androgen. Under the condition of inhibited protein synthesis by cycloheximide (50 microg/ml), at least four rounds of receptor recycling after androgen treatment and withdrawal were recorded. Two nonandrogenic hormones, 17beta-estradiol and progesterone at higher concentrations (10(-7)/10(-6) M), were able to both transactivate the AR-responsive promoter and translocate the GFP-AR into the nucleus. Similarly, antiandrogenic ligands, cyproterone acetate and casodex, were also capable of translocating the cytoplasmic AR into the nucleus albeit at a slower rate than the androgen 5alpha-dihydrotestosterone (DHT). All AR ligands with transactivation potential, including the mixed agonist/antagonist cyproterone acetate, caused translocation of the GFP-AR into a subnuclear compartment indicated by its punctate intranuclear distribution. However, translocation caused by casodex, a pure antagonist, resulted in a homogeneous nuclear distribution. Subsequent exposure of the casodex-treated cell to DHT rapidly (15-30 min) altered the homogeneous to punctate distribution of the already translocated nuclear AR. When transported into the nucleus either by casodex or by DHT, GFP-AR was resistant to 2 M NaCl extraction, indicating that the homogeneously distributed AR is also associated with the nuclear matrix. Taken together, these results demonstrate that AR requires ligand activation for its nuclear translocation where occupancy by only agonists and partial agonists can direct it to a potentially functional subnuclear location and that one receptor molecule can undertake multiple rounds of hormonal signaling; this indicates that ligand dissociation/inactivation rather than receptor degradation may play a critical role in terminating hormone action.