Androgens, like progestins, are 3-ketosteroids with structural differences restricted to the 17␤ substituent in the steroid D-ring. To better understand the specific recognition of ligands by the human androgen receptor (hAR), a homology model of the ligand-binding domain (LBD) was constructed based on the progesterone receptor LBD crystal structure. Several mutants of residues potentially involved in the specific recognition of ligands in the hAR were constructed and tested for their ability to bind agonists. Their transactivation capacity in response to agonist (R1881) and antagonists (cyproterone acetate, hydroxyflutamide, and ICI 176344) was also measured. Substitution of His 874 by alanine, only marginally impairs the ligand-binding and transactivation capacity of the hAR receptor. In contrast, mutations of Thr 877 and, to a greater extent, Asn 705 perturb ligand recognition, alter transactivation efficiency, and broaden receptor specificity. Interestingly, the N705A mutant acquires progesterone receptor (PR) properties for agonist ligands but, unlike wild type AR and PR, loses the capacity to repress transactivation with nonsteroidal antagonists. Models of the hAR⅐LBD complexes with several ligands are presented, which suggests new directions for drug design. The androgen receptor (AR)1 is a transcription factor that requires high affinity androgen binding to initiate a series of molecular events leading to the specific gene activation required for male sex development. This crucial role is demonstrated by the abundance of AR gene mutations identified in patients presenting with androgen insensitivity syndrome (1). This syndrome encompasses a wide spectrum of male pseudohermaphroditisms ranging from complete androgen insensitivity syndrome in subjects with female phenotype to partial androgen insensitivity syndrome in men with infertility and/or stigmata of undervirilization (2). Mutations have also been described in prostate cancer, and some of these alter the ligand-binding specificity, thereby inducing a putative AR activation by adrenal androgens (3) or the antiandrogens used during treatment (4).AR is a member of the nuclear receptor (NR) family that includes receptors for steroid and thyroid hormones, vitamin D3 and retinoic acids, and numerous orphan receptors for which no ligands are known (5, 6). NRs are modular proteins that can be divided into separable domains with specific functions, such as ligand binding, dimerization, DNA binding, and transactivation. In the absence of ligand, the androgen receptor resides in the cytoplasm (7,8). Hormone binding induces a transconformation of the receptor and allows its translocation into the nucleus where it initiates transcription through specific interactions with the transcription machinery (for review see Ref. 9). Recently, the crystal structures of unliganded and liganded NR ligand-binding domains (LBD) have been solved (10 -17). These crystal structures reveal a triple-layered antiparallel ␣-helical sandwich fold, with the major difference between th...

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A short C-terminal end is present at the end of the human androgen receptor (hAR) similar to that of other steroid receptors. It is located directly after helix 12 of the ligand binding domain and has never been described as being part of the hydrophobic binding pocket. Although some fragmentary data have indicated the involvement of this region in ligand binding, its precise function still remains unclear. To gain deeper insight into the role of the hAR extreme C-terminal end, an extensive mutational analysis was carried out by using site-directed mutagenesis and alanine scanning over the 13-residue C-terminal end region. Both ligand binding and transcriptional activity were tested with each mutant. Our study demonstrates the participation of almost all of the amino acids in this region for the ligand binding function and consequently for the transcriptional activity. A conformational study by limited proteolysis was performed with the mutants that most affected the affinity of the receptor. It was remarkable that the mutants with a low binding affinity adopted an inactive conformation and were either less or not able to undergo a following conformational change to provide the active form of the receptor. Our results demonstrate the importance of hydrophobicity for the function of the C-terminal end with residues located at very precise positions. Especially, both hydrophobicity and aromaticity on position 916 are critical for providing the correct ligand binding conformation of the receptor. Furthermore, this study highlights essential criteria regarding the C-terminal amino acids which could be applied to other steroid receptors.