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.

The androgen receptor undergoes an androgen-specific NH 2 -and COOH-terminal interaction between NH 2 -terminal motif FXXLF and activation function 2 in the ligand binding domain. We demonstrated previously that activation function 2 forms overlapping binding sites for the androgen receptor FXXLF motif and the LXXLL motifs of p160 coactivators. Here we investigate the influence of the NH 2 -and COOH-terminal interaction on androgen receptor function. Specificity and relative potency of the motif interactions were evaluated by ligand dissociation rate and the stability of chimeras of transcriptional intermediary factor 2 with full-length and truncated androgen or glucocorticoid receptor. The results indicate that the androgen receptor activation function 2 interacts specifically and with greater avidity with the single FXXLF motif than with the LXXLL motif region of p160 coactivators, whereas this region of the glucocorticoid receptor interacts preferentially with the LXXLL motifs. Expression of the LXXLL motifs as a fusion protein with the glucocorticoid receptor resulted in loss of agonist-induced receptor destabilization and increased half-time of ligand dissociation. The NH 2 -and COOH-terminal interaction inhibited binding and activation by transcriptional intermediary factor 2. We conclude that the androgen receptor NH 2 -and COOH-terminal interaction reduces the dissociation rate of bound androgen, stabilizes the receptor, and inhibits p160 coactivator recruitment by activation function 2. The androgen receptor (AR)1 is a member of the steroid receptor family of nuclear receptors that act as ligand-dependent transcriptional regulators. The AR shares with other steroid receptors an overall structural arrangement that includes a COOH-terminal ligand binding domain, central DNA binding region, and a less well conserved NH 2 -terminal region (Fig. 1). Within these domains are two major transactivation regions, activation function 1 in the NH 2 -terminal region and activation function 2 (AF2) in the ligand binding domain. The NH 2 -terminal activation function 1 region, although not well defined, requires androgen binding for transcriptional activity and appears to be critical for AR-mediated gene activation. The AF2 region in the ligand binding domain forms a putative hydrophobic binding site for the LXXLL motifs of p160 coactivators (1-6), as recently revealed in the crystal structure of the AR ligand binding domain (7,8). The p160 group of transcriptional coregulators includes steroid receptor coactivator 1 (SRC1), transcriptional intermediary factor 2 (TIF2, SRC2), and the SRC3/TRAM1/AIB1/pCIP/ACTR/RAC3 group of activators (9, 10), which are associated with histone acetyltransferase activity and can recruit CREB-binding protein, pCAF, and other coactivators required for chromatin modification (11).The contribution of the AF2 region to AR-mediated transcriptional activity is unclear. Androgen-dependent transcriptional activity of an AR DNA and ligand binding domain fragment (AR-(507-919)) was only observed i...

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.

The agonist-induced androgen receptor NH 2 -and COOH-terminal (N/C) interaction is mediated by the FXXLF and WXXLF NH 2 -terminal motifs. Here we demonstrate that agonist-dependent transactivation of prostate-specific antigen (PSA) and probasin enhancer/promoter regions requires the N/C interaction, whereas the sex-limited protein gene and mouse mammary tumor virus long terminal repeat do not. Transactivation of PSA and probasin response regions also depends on activation function 1 (AF1) in the NH 2 -terminal region but can be increased by binding an overexpressed p160 coactivator to activation function 2 (AF2) in the ligand binding domain. The dependence of the PSA and probasin enhancer/promoters on the N/C interaction for transactivation allowed us to demonstrate that in the presence of androgen, the WXXLF motif with the sequence 433 WHTLF 437 contributes as an inhibitor to AR transactivation. We further show that like the FXXLF and LXXLL motifs, the WXXLF motif interacts in the presence of androgen with AF2 in the ligand binding domain. Sequence comparisons among species indicate greater conservation of the FXXLF motif compared with the WXXLF motif, paralleling the functional significance of these binding motifs. The data provide evidence for promoter-specific differences in the requirement for the androgen receptor N/C interaction and in the contributions of AF1 and AF2 in androgen-induced gene regulation.Steroid receptors are ligand-activated transcription factors that regulate gene activation through a series of events triggered by high affinity hormone binding and mediated by receptor binding to response element DNA and coactivators. At least two domains have been identified that mediate nuclear receptor interactions with coregulators. These are activation function 1 (AF1) 1 in the NH 2 -terminal region and activation function 2 (AF2) in the ligand binding domain. The AF2 binding surface in the ligand binding domain is comprised of helices 3, 4, and 12 and forms after hormone binding. For many nuclear receptors, transactivation depends on AF2 recruitment of p160 coactivator complexes that have histone acetyl transferase activity to modify chromatin structure (1). The p160 coactivators are a group of proteins that include steroid receptor coactivator 1 (SRC1), transcriptional intermediary protein 2 (TIF2, GRIP1 or SRC2), and the steroid receptor coactivator 3 subfamily (SRC3). Interaction with AF2 is mediated by the p160 coactivator LXXLL motif that forms an amphipathic ␣-helix and binds the AF2 hydrophobic binding surface in the nuclear receptor ligand binding domain (2-5). For the androgen receptor (AR), the functional importance of AF2 recruitment of p160 coactivators is unclear, with data implicating the AR NH 2 -terminal AF1 region in AR-mediated gene activation. The AF2 binding site in the AR ligand binding domain was shown to mediate the agonist-induced NH 2 -and COOH-terminal (N/C) interaction (6 -10). Agonist-induced N/C interdomain interactions are also reported for the estrogen (11) and progest...

The NH 2 -terminal sequence of steroid receptors is highly variable between different receptors and in the same receptor from different species. In this study, a primary sequence homology comparison identified a 14-amino acid NH 2 -terminal motif of the human androgen receptor (AR) that is common to AR from all species reported, including the lower vertebrates. The evolutionarily conserved motif is unique to AR, with the exception of a partial sequence in the glucocorticoid receptor of higher species. The presence of the conserved motif in AR and the glucocorticoid receptor and its absence in other steroid receptors suggests convergent evolution. The function of the AR NH 2 -terminal conserved motif was suggested from a yeast two-hybrid screen that identified the COOH terminus of the Hsp70-interacting protein (CHIP) as a binding partner. We found that CHIP functions as a negative regulator of AR transcriptional activity by promoting AR degradation. In support of this, two mutations in the AR NH 2 -terminal conserved motif previously identified in the transgenic adenocarcinoma of mouse prostate model reduced the interaction between CHIP and AR. Our results suggest that the AR NH 2 -terminal domain contains an evolutionarily conserved motif that functions to limit AR transcriptional activity. Moreover, we demonstrate that the combination of comparative sequence alignment and yeast two-hybrid screening using short conserved peptides as bait provides an effective strategy to probe the structure-function relationships of steroid receptor NH 2 -terminal domains and other intrinsically unstructured transcriptional regulatory proteins.Steroid receptors depend on multiple domains for their function as ligand-dependent transcriptional activators. The DNAand ligand-binding domains have been studied extensively and have a high degree of structural and functional conservation. In contrast, the largely unstructured NH 2 -terminal domains (1-3) are highly variable in size and sequence, and the molecular mechanisms that contribute to transactivation are not well understood. The size of the NH 2 -terminal region of steroid receptors increases with evolutionary expansion (4, 5), from estrogen receptor-␣ (185 amino acid residues) to the glucocorticoid receptor (GR 1 ; 420 residues), androgen receptor (AR; 558 residues), progesterone receptor (B form; 566 residues), and mineralocorticoid receptor (602 residues). In contrast, transcription factors such as p53, NF-B, and VP16 typically have transcriptional activation domains of Ͻ100 residues.