The FXXLF Motif Mediates Androgen Receptor-specific Interactions with Coregulators

Bin He,John T Minges,Lori W Lee,Elizabeth M Wilson

doi:10.1074/jbc.m111975200

Bin He, John T Minges + Show 2 more

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https://doi.org/10.1074/jbc.m111975200

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Abstract

The androgen receptor (AR) activation function 2 region of the ligand binding domain binds the LXXLL motifs of p160 coactivators weakly, engaging instead in an androgen-dependent, interdomain interaction with an FXXLF motif in the AR NH(2) terminus. Here we show that FXXLF motifs are present in previously reported AR coactivators ARA70/RFG, ARA55/Hic-5, and ARA54, which account for their selection in yeast two-hybrid screens. Mammalian two-hybrid assays, ligand dissociation rate studies, and glutathione S-transferase adsorption assays indicate androgen-dependent selective interactions of these FXXLF motifs with the AR ligand binding domain. Mutagenesis of residues within activation function 2 indicates distinct but overlapping binding sites where specificity depends on sequences within and flanking the FXXLF motif. Mutagenesis of the FXXLF motifs eliminated interaction with the ligand binding domain but only modestly reduced AR coactivation in transcription assays. The studies indicate that the FXXLF binding motif is specific for the AR and mediates interactions both within the AR and with coregulatory proteins.

Highlights

The androgen receptor (AR)1 belongs to the steroid receptor subfamily of hormone-dependent nuclear receptor transcriptional regulators
This report identifies FXXLF motifs in three previously reported AR coactivators, which provides a molecular explanation for their cloning in yeast two-hybrid screens using the AR ligand binding domain as probe
FXXLF motifs in ARA54, ARA55, and ARA70 interact to different degrees with the activation function 2 (AF2) binding surface of the AR ligand binding domain

Summary

Introduction

The androgen receptor (AR)1 belongs to the steroid receptor subfamily of hormone-dependent nuclear receptor transcriptional regulators. Interaction of the AR coactivator FXXLF motifs with the AR ligand binding domain was tested in the absence and presence of 10 nM DHT using two-hybrid peptide assays (29) in HeLa or HepG2 cells with GAL4-peptide fusions. Androgen-dependent interactions were detected which increased luciferase activity by 8 – 48-fold in twohybrid assays between VPAR507–919 (AR DNA and ligand binding domain) and the GAL4-FXXLF fusion peptides with sequences from AR (GAL-AR-(16 –36)), ARA54 (GAL-ARA54(447– 465)), and ARA70 (GAL-ARA70-(321–340)) (Fig. 2B).

Results

Conclusion