Structural basis for androgen receptor interdomain and coactivator interactions suggests a transition in nuclear receptor activation function dominance

The androgen receptor (AR) is required for male sex development and contributes to prostate cancer cell survival. In contrast to other nuclear receptors that bind the LXXLL motifs of coactivators, the AR ligand binding domain is preferentially engaged in an interdomain interaction with the AR FXXLF motif. Reported here are crystal structures of the ligand-activated AR ligand binding domain with and without bound FXXLF and LXXLL peptides. Key residues that establish motif binding specificity are identified through comparative structure-function and mutagenesis studies. A mechanism in prostate cancer is suggested by a functional AR mutation at a specificity-determining residue that recovers coactivator LXXLL motif binding. An activation function transition hypothesis is proposed in which an evolutionary decline in LXXLL motif binding parallels expansion and functional dominance of the NH(2)-terminal transactivation domain in the steroid receptor subfamily.     

Novel FXXFF and FXXMF motifs in androgen receptor cofactors mediate high affinity and specific interactions with the ligand-binding domain

Upon hormone binding, a hydrophobic coactivator binding groove is induced in the androgen receptor (AR) ligand-binding domain (LBD). This groove serves as high affinity docking site for alpha-helical FXXLF motifs present in the AR N-terminal domain and in AR cofactors. Study of the amino acid requirements at position +4 of the AR FXXLF motif revealed that most amino acid substitutions strongly reduced or completely abrogated AR LBD interaction. Strong interactions were still observed following substitution of Leu+4 by Phe or Met residues. Leu+4 to Met or Phe substitutions in the FXXLF motifs of AR cofactors ARA54 and ARA70 were also compatible with strong AR LBD binding. Like the corresponding FXXLF motifs, interactions of FXXFF and FXXMF variants of AR and ARA54 motifs were AR specific, whereas variants of the less AR-selective ARA70 motif displayed increased AR specificity. A survey of currently known AR-binding proteins revealed the presence of an FXXFF motif in gelsolin and an FXXMF motif in PAK6. In vivo fluorescence resonance energy transfer and functional protein-protein interaction assays showed direct, efficient, and specific interactions of both motifs with AR LBD. Mutation of these motifs abrogated interaction of gelsolin and PAK6 proteins with AR. In conclusion, we have demonstrated strong interaction of FXXFF and FXXMF motifs to the AR coactivator binding groove, thereby mediating specific binding of a subgroup of cofactors to the AR LBD.     

Isolation and characterization of ARA160 as the first androgen receptor N-terminal-associated coactivator in human prostate cells.

The androgen receptor (AR) is a member of the steroid receptor superfamily that may require coactivators for proper or maximal transactivation. Using a purified AR N-terminal peptide as a probe to screen the human testis expression library, we identified an androgen-enhanced AR N-terminal-associated protein ARA160, which consists of 1,093 amino acids with an apparent molecular mass of 160 kDa. Sequence comparison in GenBank(TM) reveals that ARA160 shares an identical sequence with a HIV-1 TATA element modulatory factor, TMF. The far-Western blotting and co-immunoprecipitation assays demonstrate that the AR can interact directly with ARA160/TMF. Affinity gel pull-down and mammalian two-hybrid assays further suggest androgen can enhance significantly the interaction between AR and ARA160. Transient transfection assays demonstrated that ARA160 might function as a coactivator for AR-mediated transactivation in human prostate cancer PC-3 cells. Our data further suggest that this AR N-terminal coactivator can function cooperatively with AR C-terminal coactivator, ARA70, in PC-3 cells. Together, our data demonstrate that ARA160 might represent the first identified androgen-enhanced N-terminal coactivator for the AR.     

Steroid receptor coactivator 1 links the steroid and interferon gamma response pathways

We show here that steroid receptor coactivator 1 (SRC-1) is a coactivator of MHC class II genes that stimulates their interferon gamma (IFNgamma) and class II transactivator (CIITA)-mediated expression. SRC-1 interacts physically with the N-terminal activation domain of CIITA through two regions: one central [extending from amino acids (aa) 360-839] that contains the nuclear receptors binding region and one C-terminal (aa 1138-1441) that contains the activation domain 2. Using chromatin immunoprecipitation assays we show that SRC-1 recruitment on the class II promoter is enhanced upon IFNgamma stimulation. Most importantly, SRC-1 relieves the inhibitory action of estrogens on the IFNgamma-mediated induction of class II genes in transient transfection assays. We provide evidence that inhibition by estradiol is due to multiple events such as slightly reduced recruitment of CIITA and SRC-1 and severely inhibited assembly of the preinitiation complex.     

The linkage of Kennedy's neuron disease to ARA24, the first identified androgen receptor polyglutamine region-associated coactivator.

Although the linkage of polyglutamine (poly-Q) repeat expansion in the androgen receptor (AR) to Kennedy's disease (X-linked spinal and bulbar muscular atrophy) was a major step forward, the detailed molecular mechanism of how the change in poly-Q length contributes to the disease remains unclear. Here we report the identification of a nuclear G-protein, Ras-related nuclear protein/ARA24, as the first AR coactivator that can bind differentially with different lengths of poly-Q within AR. In the yeast and mammalian reciprocal interacting assays, our data suggested the interaction of AR N-terminal domain with ARA24 diminishes as the poly-Q length increases. The coactivation of ARA24 also diminishes with the poly-Q expansion within AR. Deletion of the acidic hexapeptide (DEDDDL) at the C terminus of ARA24 further enhances its AR coactivation. Together, our data suggest that poor interaction and weaker coactivation of ARA24 to the longer poly-Q AR in the X-linked spinal and bulbar muscular atrophied AR could contribute to the weaker transactivation of AR. The consequence of poor interaction and weak coactivation may eventually lead to the partial androgen insensitivity during the development of Kennedy's disease.     

The AF1 and AF2 domains of the androgen receptor interact with distinct regions of SRC1

The androgen receptor is unusual among nuclear receptors in that most, if not all, of its activity is mediated via the constitutive activation function in the N terminus. Here we demonstrate that p160 coactivators such as SRC1 (steroid receptor coactivator 1) interact directly with the N terminus in a ligand-independent manner via a conserved glutamine-rich region between residues 1053 and 1123. Although SRC1 is capable of interacting with the ligand-binding domain by means of LXXLL motifs, this interaction is not essential since an SRC1 mutant with no functional LXXLL motifs retains its ability to potentiate androgen receptor activity. In contrast, mutants lacking the glutamine-rich region are inactive, indicating that this region is both necessary and sufficient for recruitment of SRC1 to the androgen receptor. This recruitment is in direct contrast to the recruitment of SRC1 to the estrogen receptor, which requires interaction with the ligand-binding domain.