Modulation of Androgen Receptor Activation Function 2 by Testosterone and Dihydrotestosterone

Robert T. Gampe,Emily B. Askew,Elizabeth M. Wilson,Jonathan L. Faggart,Thomas B. Stanley

doi:10.1074/jbc.m703268200

Abstract

The androgen receptor (AR) is transcriptionally activated by high affinity binding of testosterone (T) or its 5alpha-reduced metabolite, dihydrotestosterone (DHT), a more potent androgen required for male reproductive tract development. The molecular basis for the weaker activity of T was investigated by determining T-bound ligand binding domain crystal structures of wild-type AR and a prostate cancer somatic mutant complexed with the AR FXXLF or coactivator LXXLL peptide. Nearly identical interactions of T and DHT in the AR ligand binding pocket correlate with similar rates of dissociation from an AR fragment containing the ligand binding domain. However, T induces weaker AR FXXLF and coactivator LXXLL motif interactions at activation function 2 (AF2). Less effective FXXLF motif binding to AF2 accounts for faster T dissociation from full-length AR. T can nevertheless acquire DHT-like activity through an AR helix-10 H874Y prostate cancer mutation. The Tyr-874 mutant side chain mediates a new hydrogen bonding scheme from exterior helix-10 to backbone protein core helix-4 residue Tyr-739 to rescue T-induced AR activity by improving AF2 binding of FXXLF and LXXLL motifs. Greater AR AF2 activity by improved core helix interactions is supported by the effects of melanoma antigen gene protein-11, an AR coregulator that binds the AR FXXLF motif and targets AF2 for activation. We conclude that T is a weaker androgen than DHT because of less favorable T-dependent AR FXXLF and coactivator LXXLL motif interactions at AF2.

Highlights

The androgen receptor (AR)2 is a member of the nuclear receptor superfamily of ligand-activated transcription factors
In two-hybrid assays, GAL-AR-(640 – 919) and GAL-AR-(658 –919) interacted to a greater extent than GAL-AR-(624 –919) with VP-AR-(1– 660) indicative of the AR NH2- and carboxyl-terminal (N/C) interaction and with VP-TIF2-(624 –1287) reflecting coactivator LXXLL motif binding to activation function 2 (AF2) (Fig. 1D) (4 – 8)
AR Activation by T and DHT—AR is unique among the family of steroid hormone receptors by having two biologically active high affinity hormones that differ in physiological potency

Summary

EXPERIMENTAL PROCEDURES

Plasmids—pCMVhAR vectors expressing full-length human AR with H874Y, E897K, and K720A mutations [6, 14, 24] and AR-(507–919) which lacks the NH2-terminal region [2] were described. Cells were incubated at 37 °C overnight, washed with PBS, and harvested in 0.25 ml of lysis buffer containing 1% Triton X-100, 2 mM EDTA, and 25 mM Tris phosphate, pH 7.8. The day cells were washed with PBS, and 1 ml/well serum-free medium lacking phenol red containing the indicated steroids was added and incubated overnight at 37 °C. Cultured cell incubations at 37 °C were terminated at different times, and cells were washed with PBS, harvested in 0.5 ml of lysis buffer containing 2% SDS, 10% glycerol, and 20 mM Tris, pH 6.8, and radioactivity was determined by scintillation counting. Purified AR LBD was eluted with buffer containing 10 ␮M T, 25 mM HEPES, pH 7.5, 0.15 M LiSO4, 10 mM DTT, 0.5 mM EDTA, and 10% glycerol and concentrated to 2– 4 mg/ml using a Centriprep centrifugal filter unit.

RESULTS

27 Ϯ 4 25 Ϯ 5

32 Ϯ 4 87 Ϯ 6

DISCUSSION