INVITED LECTURE Molecular biology of the androgen responses F. Claessens, G. Verrijdt, A. Haelens, L. Callewaert, U. Moehren, A. d’Alesio, T. Tanner, K. Schauwaers, S. Denayer and N. Van Tilborgh Molecular Endocrinology Laboratory, Faculty of Medicine, K.U. Leuven, Belgium 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 DNA- cellulose competition assay with partially purified andro- gen receptor from rat prostate (Claessens et al., 1990). Once the receptor cDNA was cloned, the separate DNA- binding 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 consen- sus of the first identified AREs pointed out that it is very similar to the glucocorticoid/progesterone response ele- ment (GRE/PRE) consensus 5¢-GGTACAnnnTGTTCT-3¢. Not surprisingly, these AREs also act as GRE/PRE in tran- sient 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-bind- ing domains of the androgen and glucocorticoid recep- tors 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 select- ive DNA binding. It overlaps with part of a nuclear local- ization signal and it is involved in the control of transactivation. Indeed, opposite to what is expected, deletions within this region result in a superactive andro- gen 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 contribu- ting to it. For all steroid receptors, two activation func- tions 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 recep- tor is an exception since the AF2 is weak and in most experiments difficult to demonstrate. A possible explan- ation 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 inter- acts with AF2, described as a hydrophobic cleft on the sur- face of the ligand-binding domain. This interaction seems to prevent recruitment of the known p160 co-activators to ª 2005 The Authors Journal Compilation ª 2005 Blackwell Publishing Ltd Andrologia 37 (2005) 209–210 209