Inhibition of the Dihydrotestosterone-Activated Androgen Receptor by Nuclear Receptor Corepressor

Abstract Nuclear receptor corepressor (NCoR) mediates transcriptional repression by unliganded nuclear receptors and certain steroid hormone receptors (SHRs) bound to nonphysiological antagonists, but has not been found to regulate SHRs bound to their natural ligands. This report demonstrates that NCoR interacts directly with the androgen receptor (AR) and represses dihydrotestosterone-stimulated AR transcriptional activity. The NCoR C terminus, containing the receptor interacting domains, was necessary for repression, which was ablated by mutations in the corepressor nuclear receptor (CoRNR) boxes. In contrast, the NCoR N terminus, containing domains that can recruit histone deacetylases, was not necessary for repression. Binding studies in vitro with a series of glutathione-S-transferase-NCoR and -AR fusion proteins demonstrated a direct interaction that was similarly dependent upon the NCoR corepressor nuclear receptor boxes and AR ligand binding domain and was independent of ligand and helix 12 in the AR ligand binding domain. This NCoR-AR interaction was further demonstrated in mammalian two-hybrid assays and by coimmunoprecipitation of the endogenous proteins from a prostate cancer cell line. Finally, AR transcriptional activity could be enhanced in vivo by sequestration of endogenous NCoR with unliganded thyroid hormone receptor. These results demonstrate that AR, in contrast to other SHRs, is regulated by NCoR and suggest the possibility of developing selective AR modulators that enhance this interaction.

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Novel Mechanism of Nuclear Receptor Corepressor Interaction Dictated by Activation Function 2 Helix Determinants

Molecular and Cellular Biology ◽

10.1128/mcb.22.19.6831-6841.2002 ◽

2002 ◽

Vol 22 (19) ◽

pp. 6831-6841 ◽

Cited By ~ 69

Author(s):

Anna N. Moraitis ◽

Vincent Giguère ◽

Catherine C. Thompson

Keyword(s):

Retinoic Acid ◽

Thyroid Hormone ◽

Ligand Binding ◽

Nuclear Receptor ◽

Thyroid Hormone Receptor ◽

Activation Function ◽

Binding Domain ◽

Ligand Binding Domain ◽

Cerebellar Development ◽

Nuclear Receptor Corepressor

ABSTRACT Transcriptional regulation by nuclear receptors is controlled by the concerted action of coactivator and corepressor proteins. The product of the thyroid hormone-regulated mammalian gene hairless (Hr) was recently shown to function as a thyroid hormone receptor corepressor. Here we report that Hr acts as a potent repressor of transcriptional activation by RORα, an orphan nuclear receptor essential for cerebellar development. In contrast to other corepressor-nuclear receptor interactions, Hr binding to RORα is mediated by two LXXLL-containing motifs, a mechanism associated with coactivator interaction. Mutagenesis of conserved amino acids in the ligand binding domain indicates that RORα activity is ligand-dependent, suggesting that corepressor activity is maintained in the presence of ligand. Despite similar recognition helices shared with coactivators, Hr does not compete for the same molecular determinants at the surface of the RORα ligand binding domain, indicating that Hr-mediated repression is not simply through displacement of coactivators. Remarkably, the specificity of Hr corepressor action can be transferred to a retinoic acid receptor by exchanging the activation function 2 (AF-2) helix. Repression of the chimeric receptor is observed in the presence of retinoic acid, demonstrating that in this context, Hr is indeed a ligand-oblivious nuclear receptor corepressor. These results suggest a novel molecular mechanism for corepressor action and demonstrate that the AF-2 helix can play a dynamic role in controlling corepressor as well as coactivator interactions. The interaction of Hr with RORα provides direct evidence for the convergence of thyroid hormone and RORα-mediated pathways in cerebellar development.

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The retinoblastoma protein-associated transcription repressor RBaK interacts with the androgen receptor and enhances its transcriptional activity

Journal of Molecular Endocrinology ◽

10.1677/jme.0.0310583 ◽

2003 ◽

Vol 31 (3) ◽

pp. 583-596 ◽

Cited By ~ 11

Author(s):

K Hofman ◽

JV Swinnen ◽

F Claessens ◽

G Verhoeven ◽

W Heyns

Keyword(s):

Androgen Receptor ◽

Ligand Binding ◽

Transcriptional Activity ◽

Zinc Finger Protein ◽

Retinoblastoma Protein ◽

Binding Domain ◽

Ligand Binding Domain ◽

Binding Studies ◽

Two Hybrid

In search of potential androgen receptor coregulators we performed a yeast two-hybrid screening using the androgen receptor ligand-binding domain as bait and a human prostate cDNA library as prey and found that the carboxy-terminal domain of retinoblastoma-associated Kruppel protein (RbaK), a member of the Kruppel zinc finger protein family, interacts in a ligand-dependent way with the ligand-binding domain of the androgen receptor. RBaK was recently identified as a transcriptional regulator that interacts with the retinoblastoma protein and thereby influences E2F regulated transcription. The interaction of RBaK with the androgen receptor was further documented using mammalian two-hybrid experiments, in vitro binding studies and coimmunoprecipitation. Finally, we demonstrated that both RBaK and the retinoblastoma protein coactivate androgen receptor-mediated transcription in cotransfection experiments. In conclusion, our data show that RBaK interacts with the androgen receptor and increases its transcriptional activity. Moreover, the double interaction of RBaK with the retinoblastoma protein and with the androgen receptor provides a novel link between the androgen receptor and the regulation of the cell cycle.

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Preserved Male Fertility Despite Decreased Androgen Sensitivity Caused by a Mutation in the Ligand-Binding Domain of the Androgen Receptor Gene1

The Journal of Clinical Endocrinology & Metabolism ◽

10.1210/jcem.85.6.6626 ◽

2000 ◽

Vol 85 (6) ◽

pp. 2253-2259 ◽

Cited By ~ 2

Author(s):

Aleksander Giwercman ◽

Thomas Kledal ◽

Marianne Schwartz ◽

Yvonne Lundberg Giwercman ◽

Henrik Leffers ◽

...

Keyword(s):

Androgen Receptor ◽

Ligand Binding ◽

Male Fertility ◽

Receptor Gene ◽

Androgen Receptor Gene ◽

Binding Domain ◽

Ligand Binding Domain ◽

Binding Studies ◽

Mutant Receptor ◽

Activation Assay

Mutations in the androgen receptor gene are considered as incompatible with preservation of fertility and have been suggested as a cause of male infertility. Two adult brothers, referred because of gynecomastia and hormonal levels in serum indicating androgen insensitivity (high sex hormone-binding globulin, and LH levels, despite extremely high testosterone concentration), turned out to be relatives to a third young man, referred independently of the two others and exhibiting identical clinical and hormonal stigmata. In all three men, we found a C→A substitution at position 2470 (exon 7) in the androgen receptor gene, leading to a Gln824Lys mutation in the ligand-binding domain of the receptor. Exploring the family history revealed that their grandfathers, on their mothers’ side, were brothers; and the Gln824Lys mutation was also found in the one of them who was still alive. Binding studies with the mutant receptor in transfected COS-7 cells, with mibolerone as ligand, exhibited equal Kd (0.7 vs. 1.0 nmol/L), IC50 (0.8 vs. 1.1 nmol/L), and maximum binding (7.1 vs. 8.9 fmol/106 cells), as compared with the wild-type (WT) receptor. In a chloramphenicol acetyl transferase trans-activation assay, the activity of the mutant receptor was identical to that of the WT, when the synthetic androgen R1881 was used as a ligand; but with dihydrotestosterone, in concentrations up to 10 nmol/L, the activity of Gln824Lys mutated receptor was 10–62% of the WT variant. Thus, Gln824Lys mutation was found, both in vivo and in vitro, to cause slight impairment of receptor function but was compatible with preservation of male fertility. The patients inherited the mutation from their grandfathers through their mothers, and one of the young men possessing the mutation has fathered a daughter.

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Androgen Receptor Ligand-Binding Domain Interaction and Nuclear Receptor Specificity of FXXLF and LXXLL Motifs as Determined by L/F Swapping

Molecular Endocrinology ◽

10.1210/me.2005-0348 ◽

2006 ◽

Vol 20 (8) ◽

pp. 1742-1755 ◽

Cited By ~ 25

Author(s):

Hendrikus J. Dubbink ◽

Remko Hersmus ◽

Ashley C. W. Pike ◽

Michel Molier ◽

Albert O. Brinkmann ◽

...

Keyword(s):

Androgen Receptor ◽

Ligand Binding ◽

Nuclear Receptor ◽

Binding Domain ◽

Ligand Binding Domain ◽

Amino Acid Residues ◽

Systematic Analysis ◽

Core Motif ◽

Charged Residues ◽

Binding Groove

Abstract The androgen receptor (AR) ligand-binding domain (LBD) binds FXXLF motifs, present in the AR N-terminal domain and AR-specific cofactors, and some LXXLL motifs of nuclear receptor coactivators. We demonstrated that in the context of the AR FXXLF motif many different amino acid residues at positions +2 and +3 are compatible with strong AR LBD interaction, although a preference for E at +2 and K or R at +3 was found. Pairwise systematic analysis of F/L swaps at +1 and +5 in FXXLF and LXXLL motifs showed: 1) F to L substitutions in natural FXXLF motifs abolished AR LBD interaction; 2) binding of interacting LXXLL motifs was unchanged or increased upon L to F substitutions; 3) certain noninteracting LXXLL motifs became strongly AR-interacting FXXLF motifs; whereas 4) other nonbinders remained unaffected by L to F substitutions. All FXXLF motifs, but not the corresponding LXXLL motifs, displayed a strong preference for AR LBD. Progesterone receptor LBD interacted with some FXXLF motifs, albeit always less efficiently than corresponding LXXLL motifs. AR LBD interaction of most FXXLF and LXXLL peptides depended on classical charge clamp residue K720, whereas E897 was less important. Other charged residues lining the AR coactivator-binding groove, K717 and R726, modulated optimal peptide binding. Interestingly, these four charged residues affected binding of individual peptides independent of an F or L at +1 and +5 in swap experiments. In conclusion, F residues determine strong and selective peptide interactions with AR. Sequences flanking the core motif determine the specific mode of FXXLF and LXXLL interactions.

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