Tyagi R K, Lavrovsky Y, Ahn S C, Song C S, Chatterjee B, Roy A K
Department of Cellular and Structural Biology, The University of Texas Health Science Center at San Antonio, 78284, USA.
Mol Endocrinol. 2000 Aug;14(8):1162-74. doi: 10.1210/mend.14.8.0497.
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.
一个表达构建体，其包含编码修饰的水母绿色荧光蛋白（GFP）的cDNA，该cDNA连接到大鼠雄激素受体（AR）cDNA的5'端（GFP-AR），用于研究活细胞中受体运动的细胞内动力学。在三种不同的细胞系中，即PC3、HeLa和COS1，未结合配体的GFP-AR大多位于细胞质中，在雄激素处理后迅速（15 - 60分钟内）转移到核区室。雄激素撤除后，标记的AR迁移回细胞质区室，并在随后再次暴露于雄激素时保持其重新进入细胞核的能力。在环己酰亚胺（50μg/ml）抑制蛋白质合成的条件下，记录到雄激素处理和撤除后至少四轮受体循环。两种非雄激素激素，高浓度（10⁻⁷/10⁻⁶M）的17β-雌二醇和孕酮，既能反式激活AR反应性启动子，又能将GFP-AR转运到细胞核中。同样，抗雄激素配体醋酸环丙孕酮和比卡鲁胺，也能够将细胞质中的AR转运到细胞核中，尽管其速率比雄激素5α-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，都会导致GFP-AR转运到核内一个点状分布所指示的亚核区室。然而，由纯拮抗剂比卡鲁胺引起的转运导致核内均匀分布。随后将用比卡鲁胺处理的细胞暴露于DHT，迅速（15 - 30分钟）使已经转运到核内的AR的均匀分布变为点状分布。当通过比卡鲁胺或DHT转运到细胞核中时，GFP-AR对2M NaCl提取具有抗性，表明均匀分布的AR也与核基质相关。综上所述，这些结果表明AR的核转位需要配体激活，只有激动剂和部分激动剂占据时才能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/失活而非受体降解可能在终止激素作用中起关键作用。
