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 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.
Tyagi
R K
RK
Department of Cellular and Structural Biology, The University of Texas Health Science Center at San Antonio, 78284, USA.
Lavrovsky
Y
Y
Ahn
S C
SC
Song
C S
CS
Chatterjee
B
B
Roy
A K
AK
D000726:Androgen Antagonists / Q000494:pharmacology; D000813:Anilides / Q000494:pharmacology; D000818:Animals; D001693:Biological Transport, Active / Q000187:drug effects; D002451:Cell Compartmentation; D002460:Cell Line; D002467:Cell Nucleus / Q000187:drug effects / Q000378:metabolism*; D003534:Cyproterone / Q000494:pharmacology; D003593:Cytoplasm / Q000187:drug effects / Q000378:metabolism*; D013196:Dihydrotestosterone / Q000494:pharmacology; D004958:Estradiol / Q000494:pharmacology; D005231:Fatty Acids, Unsaturated / Q000494:pharmacology; D049452:Green Fluorescent Proteins; D006801:Humans; D007700:Kinetics; D008024:Ligands; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D009570:Nitriles; D011374:Progesterone / Q000494:pharmacology; D051381:Rats; D011944:Receptors, Androgen / Q000187:drug effects* / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D014105:Tosyl Compounds; D015533:Transcriptional Activation
D000726:Androgen Antagonists; D000813:Anilides; D005231:Fatty Acids, Unsaturated; D008024:Ligands; D008164:Luminescent Proteins; D009570:Nitriles; D011944:Receptors, Androgen; D011994:Recombinant Proteins; D014105:Tosyl Compounds; D013196:Dihydrotestosterone; D049452:Green Fluorescent Proteins; D011374:Progesterone; D004958:Estradiol; C053541:bicalutamide; D003534:Cyproterone; C038753:leptomycin B