Dynamics of intracellular movement and nucleocytoplasmic recycling of the ligand-activated androgen receptor in living cells

An expression construct containing the cDNA encoding a modified aequorea gr een fluorescent protein (GFP) ligated to the 5'-end of the rat androgen rec eptor (AR) cDNA (GFP-AR) was used to study the intracellular dynamics of th e receptor movement in living cells. In three different cell lines, be. PC3 , HeLa, and COS1, unliganded GFP-AR was seen mostly in the cytoplasm and ra pidly (within 15-60 min) moved to the nuclear compartment after androgen tr eatment. Upon androgen withdrawal, the labeled AR migrated back to the cyto plasmic compartment and maintained its ability to reenter the nucleus on su bsequent exposure to androgen. Under the condition of inhibited protein syn thesis by cycloheximide (50 mu g/ml), at least four rounds of receptor recy cling after androgen treatment and withdrawal were recorded. Two nonandroge nic hormones, 17 beta-estradiol and progesterone at higher concentrations ( 10(-7)/10(-6) M), were able to both transactivate the AR-responsive promote r and translocate the GFP-AR into the nucleus. Similarly, antiandrogenic li gands, cyproterone acetate and casodex, were also capable of translocating the cytoplasmic AR into the nucleus albeit at a slower rate than the androg en 5 alpha-dihydrotestosterone (DHT). All AR ligands with transactivation p otential, including the mixed agonist/antagonist cyproterone acetate, cause d translocation of the GFP-AR into a subnuclear compartment indicated by it s punctate intranuclear distribution. However, translocation caused by caso dex, a pure antagonist, resulted in a homogeneous nuclear distribution. Sub sequent exposure of the casodex-treated cell to DHT rapidly (15-30 min) alt ered the homogeneous to punctate distribution of the already translocated n uclear AR. When transported into the nucleus either by casodex or by DHT, G FP-AR was resistant to 2 M NaCl extraction, indicating that the homogeneous ly distributed AR is also associated with the nuclear matrix. Taken togethe r, these results demonstrate that AR requires ligand activation for its nuc lear translocation where occupancy by only agonists and partial agonists ca n direct it to a potentially functional subnuclear location and that one re ceptor molecule can undertake multiple rounds of hormonal signaling; this i ndicates that ligand dissociation/inactivation rather than receptor degrada tion may play a critical role in terminating hormone action.