Abstract
The androgen receptor (AR) regulates male sexual development. We have now investigated AR homodimerization, hormone-dependent monomerization and nuclear translocation in PC-3 and COS-1 cells, by utilizing mutations associated with the androgen insensitivity syndrome: Pro767Ala, Phe765Leu, Met743Val and Trp742Arg. AR wild type (WT) was expressed as a homodimer in the cytoplasm, while none of these mutants formed homodimers. Unlike AR WT which responded to 1 nM dihydrotestosterone (DHT) to dissociate and translocate into the nucleus, AR Pro767Ala and Phe765Leu mutants remain as the monomer in the cytoplasm. In the crystal structure of the AR LBD homodimer, Pro767 and Phe765 reside closely on a loop that constitutes the dimer interface; their sidechains interact with the Pro767 of the other monomer and with the DHT molecule in the ligand-binding pocket. These observations place Phe765 at a position to facilitate DHT binding to Pro767 and lead to dissociation of the AR homodimer in the cytoplasm. This Pro-Phe Met relay may constitute a structural switch that mediates androgen signaling and is conserved in other steroid hormone receptors.
Highlights
The androgen receptor (AR), a member of the nuclear steroid hormone receptor (NR3C) subfamily, regulates male sexual development[1]
Prostate cancer cell line PC-3 cells were expressed with AR wild type (WT) or AR P767A mutant and treated with 0.1% DMSO as vehicle or 0.01–10 nM DHT for luciferase reporter assay using the promoter of PSA gene which includes two AR binding motif[22]
We confirmed that AR P767A mutant required a 10-fold higher DHT concentration to activate PSA promoter-luciferase reporter gene compared to AR WT (Fig. 1B)
Summary
The androgen receptor (AR), a member of the nuclear steroid hormone receptor (NR3C) subfamily, regulates male sexual development[1]. The nuclear receptor CAR (NR1I3) has been found to form a homodimer through its D3L interface in the cytoplasm which dissociates upon activation and translocation into the nucleus[12,13]. We demonstrated that AR WT formed a homodimer that dissociated and nuclear translocated in response to dihydrotestosterone (DHT) in COS-1 cells With this background, AR mutants (Pro767Ala, Phe765Leu, Met743Val and Trp742Arg) were expressed in COS-1 cells to examine their roles in AR homodimerization. AR mutants (Pro767Ala, Phe765Leu, Met743Val and Trp742Arg) were expressed in COS-1 cells to examine their roles in AR homodimerization These residues are mapped to AR molecules based on its X-ray crystal structure to understand structure-function relationships. We present experimental observations in support of the hypothesis that the AR homodimer responds to androgen in the cytoplasm by dissociation and nuclear translocation, and, that an intramolecular Pro-Phe-Met relay regulates the androgen signal
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