The androgen receptor T877A mutant recruits LXXLL and FXXLF peptides differently than wild-type androgen receptor in a time-resolved fluorescence resonance energy transfer assay.
The interactions of the ligand binding domain (LBD) of androgen receptor (AR) and the AR T877A mutant, found in prostate cancer, with peptides from coactivator and corepressor proteins or random phage display peptides were investigated using in vitro time-resolved fluorescence resonance energy transfer (TR-FRET). Interaction of wild-type AR LBD with the random phage display peptide D11FxxLF was observed with dihydrotestosterone (DHT), testosterone, R1881, estradiol, spironolactone, progesterone, and cortisol resulting in distinct dose dependency (EC50) values for each ligand and correlating well with the reported rank order potency of these agonists. Increasing concentrations of cyproterone acetate and mifepristone resulted in more complete disruption of the DHT-mediated AR-D11FxxLF peptide interaction, while flutamide, hydroxyflutamide, and bicalutamide caused only partial disruption of the complex. The mutant AR T877A LBD exhibited increased binding affinities for all ligands tested except for bicalutamide, mifepristone, DHT, and R1881 in a competitive binding assay as compared to wild-type AR LBD. This mutation was also characterized by increased ligand potency for agonist-induced peptide recruitment. Although usually an antagonist, hydroxyflutamide was more potent in the recruitment of D11FxxLF or an SRC3-1 LXXLL motif to AR T877A LBD than AR LBD. The antagonist cyproterone acetate behaved as a full antagonist of D11FxxLF recruitment to AR LBD and AR T877A LBD but as a more potent agonist in the recruitment of SRC3-1 to AR T877A LBD. These results suggest that the AR T877A mutation affects both ligand affinity and ligand dose dependency for peptide recruitment and may explain in part the altered responses of antagonists and increased transcriptional activation reported in androgen-independent prostate cancers.
Ozers
Mary Szatkowski
MS
Invitrogen Discovery Sciences, 501 Charmany Drive, Madison, Wisconsin 53719, USA.
Marks
Bryan D
BD
Gowda
Krishne
K
Kupcho
Kevin R
KR
Ervin
Kerry M
KM
De Rosier
Therese
T
Qadir
Naveeda
N
Eliason
Hildegard C
HC
Riddle
Steven M
SM
Shekhani
Mohammed Saleh
MS
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000726:Androgen Antagonists / Q000494:pharmacology; D000813:Anilides / Q000494:pharmacology; D000818:Animals; D017373:Cyproterone Acetate / Q000494:pharmacology; D013196:Dihydrotestosterone / Q000378:metabolism; D005452:Fluoresceins / Q000737:chemistry; D031541:Fluorescence Resonance Energy Transfer; D005485:Flutamide / Q000031:analogs & derivatives / Q000494:pharmacology; D008024:Ligands; D015735:Mifepristone / Q000494:pharmacology; D009570:Nitriles; D009842:Oligopeptides / Q000737:chemistry*; D051381:Rats; D011944:Receptors, Androgen / Q000235:genetics* / Q000378:metabolism*; D014105:Tosyl Compounds
D000726:Androgen Antagonists; D000813:Anilides; D005452:Fluoresceins; D008024:Ligands; D009570:Nitriles; D009842:Oligopeptides; D011944:Receptors, Androgen; D014105:Tosyl Compounds; D013196:Dihydrotestosterone; C014290:hydroxyflutamide; D015735:Mifepristone; D017373:Cyproterone Acetate; D005485:Flutamide; C053541:bicalutamide