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...

In an effort to better define the molecular mechanisms of the functional specificity of human estrogen receptor alpha, we have carried out equilibrium binding assays to study the interaction of the receptor with a palindromic estrogen response element derived from the vitellogenin ERE. These assays are based on the observation of the fluorescence anisotropy of a fluorescein moiety covalently bound to the target oligonucleotide. The low anisotropy value due to the fast tumbling of the free oligonucleotide in solution increases substantially upon binding the receptor to the labeled ERE. The quality of our data are sufficient to ascertain that the binding is clearly cooperative in nature, ruling out a simple monomer interaction and implicating a dimerization energetically coupled to DNA binding in the nanomolar range. The salt concentration dependence of the affinity reveals formation of high stoichiometry, low specificity complexes at low salt concentration. Increasing the KCl concentration above 200 mM leads to specific binding of ER dimer. We interpret the lack of temperature dependence of the apparent affinity as indicative of an entropy driven interaction. Finally, binding assays using fluorescent target EREs bearing mutations of each of the base pairs in the palindromic ERE half-site indicate that the energy of interaction between ER and its target is relatively evenly distributed throughout the site.

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