The androgen receptor (AR) encoding gene can undergo mutations during the development and treatment of prostate cancer. Even in hormone-independent stages, mutations in the receptor paradoxically seem to result in an increased AR function. Two such point mutations have been described in the part of the AR involved in DNA binding and nuclear translocation, namely the hinge region. Despite a decreased nuclear translocation, these mutant ARs display increased transactivating potencies. Through detailed analysis of the hinge region, we found that deletion of residues 629 to 636 resulted in a stronger androgen response on different reporters, although this mutant displays an extremely low in vitro affinity for androgen response elements. This superactivity is independent of nuclear localization and can be inhibited by antiandrogens. Surprisingly, the AR activation functions, AF1 and AF2, are not dramatically affected when the inhibitory region (629-RKLKKLGN-636) is deleted, although cotransfected p160 coactivator TIF2 had a stronger potentiating effect in the absence of this motif. The liganddependent interaction between the amino-terminal domain and the ligand-binding domain (N/C interaction) plays an important role in transactivation by the AR. We found that this interaction is strongly enhanced by deletion of the inhibitory region. In conclusion, the description of prostate cancer mutations has led to the discovery of a complex role of the hinge region in nuclear localization, DNA binding, coactivator recruitment, and N/C interaction of the AR. [Cancer Res 2007;67(9):4514-23]

The androgen receptor (AR) plays a key role in prostate cancer development, as well as its treatments, even for the hormonerefractory state. Here, we report that an earlier described lysine-to-arginine mutation at position 179 in AR leads to a more potent AR. We show that two activation domains (Tau-1 and Tau-5) are necessary and sufficient for the full activity of AR and the intrinsic activity of the AR-NTD. Two A-helices surrounding the Lys 179 define the core of Tau-1, which can act as an autonomous activation function, independent of p160 coactivators. Furthermore, we show that although the recruitment of p160 coactivators is mediated through Tau-5, this event is attenuated by core Tau-1. This better definition of the mechanisms of action of both Tau-1 and Tau-5 is instrumental for the design of alternative therapeutic strategies against prostate cancer.

Androgens influence transcription of their target genes through the activation of the androgen receptor (AR) that subsequently interacts with specific DNA motifs in these genes. These DNA motifs, called androgen response elements (AREs), can be classified in two classes: the classical AREs, which are also recognized by the other steroid hormone receptors; and the AR-selective AREs, which display selectivity for the AR. For in vitro interaction with the selective AREs, the androgen receptor DNA-binding domain is dependent on specific residues in its second zinc-finger. To evaluate the physiological relevance of these selective elements, we generated a germ-line knockin mouse model, termed SPARKI (SPecificity-affecting AR KnockIn), in which the second zinc-finger of the AR was replaced with that of the glucocorticoid receptor, resulting in a chimeric protein that retains its ability to bind classical AREs but is unable to bind selective AREs. The reproductive organs of SPARKI males are smaller compared with wild-type animals, and they are also subfertile. Intriguingly, however, they do not display any anabolic phenotype. The expression of two testis-specific, androgen-responsive genes is differentially affected by the SPARKI mutation, which is correlated with the involvement of different types of response elements in their androgen responsiveness. In this report, we present the first in vivo evidence of the existence of two functionally different types of AREs and demonstrate that AR-regulated gene expression can be targeted based on this distinction.DNA-binding domain ͉ fertility ͉ Rhox5 ͉ transcription

The androgen receptor interacts with the p160 coactivators via two surfaces, one in the ligand binding domain and one in the amino-terminal domain. The ligand binding domain interacts with the nuclear receptor signature motifs, whereas the amino-terminal domain has a high affinity for a specific glutamine-rich region in the p160s. We here describe the implication of two conserved motifs in the latter interaction. The amino-terminal domain of the androgen receptor is a very strong activation domain constituent of Tau5, which is mainly active in the absence of the ligand binding domain, and Tau1, which is only active in the presence of the ligand binding domain. Both domains are, however, implicated in the recruitment of the p160s. Mutation analysis of the p160s has shown that the relative contribution of the two recruitment mechanisms via the signature motifs or via the glutamine-rich region depend on the nature of the enhancers tested. We propose, therefore, that the androgen receptor-coactivator complex has several alternative conformations, depending partially on the context of the enhancer. The androgen receptor (AR)1 is a member of the steroid receptor family of transcription factors. Steroid receptors are ligand-inducible sequence-specific transcription factors with highly conserved DNA binding domains (DBDs), moderately conserved ligand binding domains (LBDs), and divergent amino-terminal domains (NTD) (1-4). Two transactivating functions (AFs) have been characterized, AF1 in the NTD and AF2 in the LBD. For the AR, AF1 has strong constitutive activity, since deletion of the LBD results in a molecule that can activate a reporter gene to the same extent as the full-length receptor in the presence of ligand, whereas AF2 appears to be weak (5-8). This is in contrast to what occurs in most other nuclear receptors, for example for the estrogen receptor (ER), in which AF2 is the major activation domain (9). The precise residues and mechanisms that contribute to the AF1 activity of the AR have not been conclusively established. Almost the entire NTD is required for full transcriptional activity of the full-length receptor, whereas a core region located between residues 101 and 360 (Tau1) contributes 50% of activity (10). When a constitutively active AR mutant lacking an LBD is studied, the region necessary for transcriptional activation shifts to the region 370 -494 (Tau5) (10).Binding of the appropriate hormones to the steroid hormone receptors causes a translocation of the receptors to enhancer elements in the promoters of target genes. Transcriptional coactivators are recruited to the promotor through proteinprotein interaction with the receptor (11-14). Most known coactivators are complex proteins that harbor multiple activation domains and receptor-interacting domains (15-16). The best studied group of coactivators is the p160 family of 160-kDa proteins. Three family members have been identified. The first p160 coactivators cloned were the human steroid receptor coactivator 1 (SRC1) and the transcription inte...

No abstract