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.

Analysis of ligand-dependent recruitment of coactivator peptides to estrogen receptor using fluorescence polarization.

Ligand-dependent recruitment of coactivators to estrogen receptor (ER) plays an important role in transcriptional activation of target genes. Agonist-bound ER has been shown to adopt a favorable conformation for interaction with the LXXLL motifs of the coactivator proteins. To further examine the affinity and ligand dependence of the ER-coactivator interaction, several fluorescently tagged short peptides bearing an LXXLL motif (LXXLL peptide) from either natural coactivator sequences or random phage display sequences were used with purified ERalpha or ERbeta in an in vitro high-throughput fluorescence polarization assay. In the presence of saturating amounts of ligand, several LXXLL peptides bound to ERalpha and ERbeta with affinity ranging from 20-500 nm. The random phage display LXXLL peptides exhibited a higher affinity for ER than the natural single-LXXLL coactivator sequences tested. These studies indicated that ER agonists, such as 17beta-estradiol or estrone, promoted the interaction of ER with the coactivator peptides, whereas antagonists such as 4-hydroxytamoxifen or ICI-182,780 did not. Different LXXLL peptides demonstrated different affinities for ER depending on which ligand was bound to the receptor, suggesting that the peptides were recognizing different receptor conformations. Using the information obtained from direct measurement of the affinity of the ER-LXXLL peptide interaction, the dose dependency (EC50) of various ligands to either promote or disrupt this interaction was also determined. Interaction of ER with the LXXLL peptide was observed with ligands such as 17beta-estradiol, estriol, estrone, and genistein but not with ICI-182,780, 4-hydroxytamoxifen, clomiphene, or tamoxifen, resulting in distinct EC50 values for each ligand and correlating well with the ligand biological function as an agonist or antagonist. Ligand-dependent recruitment of the LXXLL peptide to ERbeta was observed in the presence of the ERbeta-selective agonist diarylpropionitrile, but not the ERalpha-selective ligand propyl pyrazole triol. This assay could be used to classify unknown ligands as agonists, antagonists, or partial modulators, based on either the receptor-coactivator peptide affinities or the dose dependency of this interaction in comparison with known compounds.