Dynamics of Intracellular Movement and Nucleocytoplasmic Recycling of the Ligand-Activated Androgen Receptor in Living Cells

Abstract

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.

Androgen receptor activity is inhibited in response to genotoxic agents in a p53-independent manner

“…This suggests that the mechanism involved in the inhibition of the AR is functioning within the nucleus. The partial nuclear translocation of the AR we observed is in contrast to a complete nuclear translocation previously reported by immunofluorescence studies in LNCaP cells (Tyagi et al, 2000). The reason for this difference is not clear but is most likely to be explained by the use of different techniques.…”

“…However, in conjunction with the etoposide-induced reduction of DHT bound in the ligand-binding assay, it can be speculated that the ligand binding of the AR is more unstable in the presence of etoposide, which could lead to a dissociation of the ligand from the AR after the nuclear translocation. It has been reported that AR is translocated to the nucleus within an hour of hormone stimulation, but when the ligand is washed off the cells, it takes 12 h for the AR to be relocalised to the cytoplasm (Tyagi et al, 2000). It is therefore possible that in the presence of etoposide, an equilibrium of ligand-bound and -unbound AR is found in the nucleus.…”

“…When testosterone, the main circulating androgen, enters prostate cells, it is converted into the more active hormone, dihydrotestosterone (DHT,, that binds to and activates the AR (Bruchovsky and Wilson, 1968;Feldman and Feldman, 2001). The activated AR is translocated to the nucleus where it binds to androgen response elements (AREs) in promoter regions of androgen-responsive genes (Riegman et al, 1991;Cleutjens et al, 1997;Tyagi et al, 2000). Recruitment of cofactors enables the AR to interact with the general transcription apparatus and through this either activate or repress the transcription of target genes (Rosenfeld and Glass, 2001;Nelson et al, 2002).…”

Silencing of androgen-regulated genes using a fusion of AR with the PLZF transcriptional repressor

“…Furthermore, intracellular localization of AR has been shown to be mediated by sequences located close to, or within the LBD (Zhou et al, 1994;Tyagi et al, 2000;Tomura et al, 2001). The lack of transcriptional repression by unliganded PLZF-AR may be due to its exclusion from the nucleus.…”

Suppression of androgen receptor-mediated gene expression by a sequence-specific DNA-binding polyamide

“…Ligand binding to AR initiates release from the cytoplasm, dimerization, binding to the androgen response elements (ARE) of target genes, and gene activation through interaction with coactivators and the general transcription machinery (2). Functional AREs, consensus sequence 5Ј-GGTACAnnnTGTTCT-3Ј (Fig.…”