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

Abstract

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.

Mechanisms of androgen receptor activation in advanced prostate cancer: differential co-activator recruitment and gene expression

“…In the presence of OHF the reverse was found for mutant receptors, with interaction only evident between these and the phenylalanine motifs. A similar trend was reported for AR T877A by Ozers et al (2007) who showed, using fluorescence resonance energy transfer, that CPA antagonized its interaction with an FxxLF-containing peptide, while promoting its interaction with an LxxLL-containing peptide.…”

Systematic Structure-Function Analysis of Androgen Receptor Leu701 Mutants Explains the Properties of the Prostate Cancer Mutant L701H

“…For example, LXXLL peptides are shifted in the AR groove toward Lys 720 compared with FXXLF peptides (38 -41). In addition, AR T877A bound to cyproterone acetate (CPA) strongly interacts with LXXLL motifs but less with FXXLF motifs, whereas the opposite is observed if it is bound to hydroxyflutamide (42,43). We therefore determined the interaction capacities of distinct peptides containing FXXLF, FXXFF, FXXMF, and LXXLL sequences with the different AR Leu 701 mutants in the presence of steroids from our panel.…”

Quantification of Ligand-Regulated Nuclear Receptor Corepressor and Coactivator Binding, Key Interactions Determining Ligand Potency and Efficacy for the Thyroid Hormone Receptor

“…The studies of androgen receptors also revealed the effect of mutations on the agonist/antagonist balance of various ligands (38). The role of coactivator binding affinity as a co-determinant of ligand potency, however, has thus far not been studied in a coherent fashion.…”