Nora Van Tilborgh

Diverse roles of androgen receptor (AR) domains in AR-mediated signaling

Androgens control male sexual development and maintenance of the adult male phenotype. They have very divergent effects on their target organs like the reproductive organs, muscle, bone, brain and skin. This is explained in part by the fact that different cell types respond differently to androgen stimulus, even when all these responses are mediated by the same intracellular androgen receptor. To understand these tissue- and cell-specific readouts of androgens, we have to learn the many different steps in the transcription activation mechanisms of the androgen receptor (NR3C4). Like all nuclear receptors, the steroid receptors have a central DNA-binding domain connected to a ligand-binding domain by a hinge region. In addition, all steroid receptors have a relatively large amino-terminal domain. Despite the overall structural homology with other nuclear receptors, the androgen receptor has several specific characteristics which will be discussed here. This receptor can bind two types of androgen response elements (AREs): one type being similar to the classical GRE/PRE-type elements, the other type being the more divergent and more selective AREs. The hormone-binding domain has low intrinsic transactivation properties, a feature that correlates with the low affinity of this domain for the canonical LxxLL-bearing coactivators. For the androgen receptor, transcriptional activation involves the alternative recruitment of coactivators to different regions in the amino-terminal domain, as well as the hinge region. Finally, a very strong ligand-induced interaction between the amino-terminal domain and the ligand-binding domain of the androgen receptor seems to be involved in many aspects of its function as a transcription factor. This review describes the current knowledge on the structure-function relationships within the domains of the androgen receptor and tries to integrate the involvement of different domains, subdomains and motifs in the functioning of this receptor as a transcription factor with tissue- and cell-specific readouts.

Interplay between two hormone-independent activation domains in the androgen receptor.

The androgen receptor (AR) plays a key role in prostate cancer development, as well as its treatments, even for the hormone-refractory 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 alpha-helices surrounding the Lys179 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.

A 629RKLKK633 motif in the hinge region controls the androgen receptor at multiple levels

The androgen receptor protein has specific domains involved in DNA binding, ligand binding, and transactivation, whose activities need to be integrated during transcription activation. The hinge region, more particular a 629RKLKK633 motif, seems to play a crucial role in this process. Indeed, although the motif is not part of the DNA-binding domain, its positive residues are involved in optimal DNA binding and nuclear translocation as shown by mutation analysis. When the mutated ARs are forced into the nucleus, however, the residues seem to play different roles in transactivation. Moreover, we show by FRAP analysis that during activation, the AR is distributed in the nucleus in a mobile and two immobile fractions, and that mutations in the 629RKLKK633 motif affect the distribution of the AR over these three intranuclear fractions. Taken together, the 629RKLKK633 motif is a multifunctional motif that integrates nuclear localization, receptor stability, DNA binding, transactivation potential and intranuclear mobility.

Molecular biology of the androgen responses

The androgen receptor is a ligand-inducible transcription factor with very specific target genes. This definition implies the activation by the cognate ligand through the ligand-binding domain, the recognition of the target genes by means of the DNA-binding domain and the transcriptional activation through different activation functions. When the first androgen-responsive genes were cloned, we identified receptor-binding sites by means of a DNAcellulose competition assay with partially purified androgen receptor from rat prostate (Claessens et al., 1990). Once the receptor cDNA was cloned, the separate DNAbinding domain was expressed and shown to have similar, if not identical DNA recognition properties as the full size receptor. The binding sites were proven functional in transient transfection experiments with reporter genes cloned downstream of these sites (Claessens et al., 1993). The motifs which are recognized by the receptor are called androgen response elements (ARE), and a consensus of the first identified AREs pointed out that it is very similar to the glucocorticoid/progesterone response element (GRE/PRE) consensus 5¢-GGTACAnnnTGTTCT-3¢. Not surprisingly, these AREs also act as GRE/PRE in transient transfections. The probasin promoter region also contains two AR-binding sites, but in contrast to what was observed for the earlier AREs, these are not recognized by the glucocorticoid receptor. Later on, several other selective AREs were characterized in the slp and sc enhancers (Verrijdt et al., 2000). A comparison of the DNA-binding domains of the androgen and glucocorticoid receptors revealed specific residues which are involved in the recognition of these selective AREs, but not in the recognition of the classical AREs. These residues are not situated within the first zinc-coordinated module or zinc finger, but rather in the second one, as well as in a carboxy-terminal extension of the DNA-binding motif (Schoenmakers et al., 2000). This hinted to us that the recognition of the selective AREs occurs through an alternative dimerization of the DNA-binding domain that would be specific for the androgen receptor. Indeed, when the direct repeat nature of the selective AREs was changed into inverted repeat nature, the selectivity of the AREs and of the enhancers, of which they form part, was lost (Verrijdt et al., 2000). The silico screening of human genome has led to the definition of several additional selective AREs. In collaboration with the group of Daniel Gewirth, we were able to solve a crystal structure of the DNA-binding domain of the androgen receptor complexed to a perfect direct repeat of the 5¢TGTTCT-3¢ hexamer (Shaffer et al., 2004). This revealed that the domain is folded into two zinc-coordinated modules very similar to what has been described for other nuclear receptors. The two monomers are organized in a head-to-head configuration. Specific for the androgen receptor is the increased strength of the dimerisation interface due to an enlarged contact surface as well as to three additional hydrogen bonds. A functional analysis of the carboxyterminal extension of the DBD, which is part of the hinge region, revealed that it has more functions besides contributing to selective DNA binding. It overlaps with part of a nuclear localization signal and it is involved in the control of transactivation. Indeed, opposite to what is expected, deletions within this region result in a superactive androgen receptor, even when DNA binding in band shifts becomes difficult to demonstrate. The transcription activation by the androgen receptor is complex in the sense that different domains are contributing to it. For all steroid receptors, two activation functions have been described: the activation function 1 (AF1) in the amino-terminal domain and activation function 2 (AF2) in the ligand-binding domain. The androgen receptor is an exception since the AF2 is weak and in most experiments difficult to demonstrate. A possible explanation for this was found in a strong interaction between the ligand-binding domain and the amino-terminal domain of the androgen receptor. This occurs through a motif at the amino-terminal end of the receptor that interacts with AF2, described as a hydrophobic cleft on the surface of the ligand-binding domain. This interaction seems to prevent recruitment of the known p160 co-activators to