Steroid receptors are conditional transcription factors that, upon binding to their response elements, regulate the expression of target genes via direct protein interactions with transcriptional coactivators. We have analyzed the functional interactions between the androgen receptor (AR) and 160-kDa nuclear receptor coactivators. Upon overexpression in mammalian cells, these coactivators enhance the transcriptional activity of both the amino-terminal domain (NTD) and the ligand-binding domain (LBD) of the AR. The coactivator activity for the LBD is strictly ligand-controlled and depends on the nature of the DNA-binding domain to which it is fused. We demonstrate that the NTD physically interacts with coactivators and with the LBD and that this interaction, like the functional interaction between the LBD and p160 coactivators, relies on the activation function 2 (AF2) core domain. The mutation of a highly conserved lysine residue in the predicted helix 3 of the LBD (K720A), however, blunts the functional interaction with coactivators but not with the NTD. Moreover, this mutation does not affect the transcriptional activity of the full-size AR. A mutation in the NTD of activation function AF1a (I182A/L183A), which dramatically impairs the activity of the AR, has no effect on the intrinsic transcriptional activity of the NTD but interferes with the cooperation between the NTD and the LBD. Finally, p160 proteins in which the three LXXLL motifs are mutated retain most of their coactivator activity for the full-size AR, although they are no longer functional for the isolated LBD. Together, these data suggest that in the native AR the efficient recruitment of coactivators requires a functional association of the NTD with the LBD and that the binding of coactivators occurs primarily through the NTD.

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The nuclear receptors constitute a large family of transcription factors characterized by a well conserved DNA-binding domain. The receptors for glucocorticoids, progestins, mineralocorticoids, and androgens constitute a subgroup because they bind in vitro with high affinity to DNA elements containing a partial palindrome of the core sequence 5-TGTTCT-3. In vivo, however, the corresponding steroids differentially regulate the expression of their target genes, even when more than one receptor type is present in a particular cell.The DNA-binding domains of the androgen and of the glucocorticoid receptors bind most androgen response elements with similar relative affinities. In contrast, one element (5-GGTTCTTGGAGTACT-3) which was recently described in the promoter region of the probasin gene selectively interacts with the DNA-binding domain of the androgen receptor and not with that of the glucocorticoid receptor. From studies with chimeric elements, it can be deduced that it is the left subsequence 5-GGTTCT-3 which excludes the glucocorticoid receptor domain from binding.In co-transfection experiments where the ARE of the C3(1) gene is responsive to both androgens and glucocorticoids, the probasin element is induced only by androgens and not by glucocorticoids. The existence of response elements which are recognized preferentially by the androgen receptor provides yet another possible mechanism to explain the differences of the in vivo effects between androgens and other steroids of the subgroup.Nuclear receptors are transcription factors which mediate signals of a variety of hormones. Upon ligand binding, the receptors activate transcription by interacting with specific DNA sequences located within or near gene promoters.All members of the nuclear receptor superfamily bind with high affinity to directly or inversely repeated DNA sequences (1) by the DNA-binding domain (DBD) 1 which contains two zinc-finger motifs. Mader et al. (2) have demonstrated that differences between the glucocorticoid receptor (GR) and the estrogen receptor (ER) involving three amino acids located in the so-called P-box, are responsible for the difference in sequence recognition. The GR recognizes the sequence 5Ј-TGT-TCT-3Ј, while the ER interacts with the

The androgen and glucocorticoid hormones evoke specific in vivo responses by activating different sets of responsive genes. Although the consensus sequences of the glucocorticoid and androgen response elements are very similar, this in vivo specificity can in some cases be explained by differences in DNA recognition between both receptors. This has clearly been demonstrated for the androgen response element PB-ARE-2 described in the promoter of the rat probasin gene. Swapping of different fragments between the androgen- and glucocorticoid-receptor DNA-binding domains demonstrates that (i) the first Zn-finger module is not involved in this sequence selectivity and (ii) that residues in the second Zn-finger as well as a C-terminal extension of the DNA-binding domain from the androgen receptor are required. For specific and high-affinity binding to response elements, the DNA-binding domains of the androgen and glucocorticoid receptors need a different C-terminal extension. The glucocorticoid receptor requires 12 C-terminal amino acids for high affinity DNA binding, while the androgen receptor only involves four residues. However, for specific recognition of the PB-ARE-2, the androgen receptor also requires 12 C-terminal residues. Our data demonstrate that the mechanism by which the androgen receptor binds selectively to the PB-ARE-2 is different from that used by the glucocorticoid receptor to bind a consensus response element. We would like to suggest that the androgen receptor recognizes response elements as a direct repeat rather than the classical inverted repeat.

The basis for specificity of gene regulation by steroid hormone receptors remains an important problem in the study of steroid hormone action. One possible mechanism for steroid specificity is the difference in DNA binding characteristics of the receptors, although they share a high homology in their DNA-binding domains. Indeed, the androgen-specific expression of, for example, the probasin (PB) gene can be explained by the presence of an androgen response element (ARE) in its promoter (PB-ARE-2), specifically recognized by the androgen and not by the glucocorticoid receptor. Three residues in the DNA-binding domain of the AR were identified as main determinants for its high affinity for the PB-ARE-2. In addition, the direct repeat nature of this ARE seems to prohibit high affinity binding by the glucocorticoid receptor. This is confirmed by the fact that several imperfect direct repeats of the 5-TGT-TCT-3 core recognition sequence are recognized by the androgen receptor and not by the glucocorticoid receptor. Up to now, only differences between the androgen and glucocorticoid receptor in the transcription activation functions were invoked to explain the specificity of their genomic actions. In the present study, we describe the influence of the DNA-binding domain on the specificity of androgen action. The novelty of our working hypothesis resides in the demonstration of the capacity of the AR-DNA-binding domain to recognize elements with a direct repeat structure.Steroid hormones are important endocrine messengers that activate their receptors, which translocate to the cell nucleus and regulate gene expression mainly after interaction with DNA sequences, called response elements (1, 2). The steroid receptors are a subfamily of the nuclear receptor superfamily, a large group of structurally homologous transcription factors. A problem with the explanation of the specificity of these hormone responses arose when several studies pointed out that the class I receptors (androgen receptor (AR), 1 glucocorticoid receptor (GR), progesterone receptor, and mineralocorticoid receptor) have identical consensus response elements (3, 4) and that their DNA-binding domains were highly conserved (5). This contrasts with the fact that the in vivo expression of several genes is specifically controlled by only one steroid hormone (6). Several possible mechanisms have been described to explain the steroid specificity of transcriptional control, e.g. steroid metabolism, tissue-specific receptor presence (7), influence of coactivator complexes (8), and chromatin structure (9, 10). In addition, more recent reports indicate that the AR on the one hand and the GR, progesterone receptor, and mineralocorticoid receptor on the other exhibit different DNA binding characteristics (11-15). One AR-specific response element was found in the promoter of the rat probasin gene (PB-ARE-2) (12,16,17). Probasin is an androgen-regulated protein exclusively expressed in the dorsolateral epithelium of the prostate (18). Two cis-acting androgen response e...