In a subset of endocrine therapy-resistant prostate cancers, amino acid substitutions H874Y, T877A and T877S, which broaden ligand specificity of the ligand binding domain (LBD) of the androgen receptor (AR), have been detected. To increase our knowledge of the role of amino acid substitutions at these specific positions in prostate cancer, codons 874 and 877 were subjected to random mutagenesis. AR mutants were screened in a yeast readout system for responsiveness to 5␣-dihydrotestosterone, progesterone and dehydroepiandrosterone. At position 874, only the histidine to tyrosine substitution could broaden AR ligand specificity. At position 877, 4 ligand specificity broadening substitutions were found: T877A, T877S, T877C and T877G. The latter 2 were not found in prostate cancer. The AR mutants were tested in mammalian (Hep3B) cells for responsiveness to 13 different ligands. All mutants displayed their own ligand specificity spectrum. Importantly, AR(H874Y) and AR(T877A) could be activated by cortisol. According to the 3-dimensional structure of the AR LBD, T877 interacts directly with the 17-hydroxyl group of androgens. All amino acid substitutions identified at position 877 had smaller side chains than the threonine in the wild-type receptor, indicating that increased space in the ligand binding pocket is important in broadened ligand specificity. Because H874 does not interact directly with the ligand, its substitution by a tyrosine is expected to change the ligand binding pocket conformation indirectly. For T877C and T877G substitutions, 2-point mutations are required, and for H874Y, T877A and T877S substitutions, only a 1-point mutation is sufficient. This most likely explains that the latter 3 have been found in prostate cancer. ©
Androgens (testosterone [T] and 5␣-dihydrotestosterone[DHT]) are essential for development and maintenance of the male phenotype. They mediate their function by activation of the androgen receptor (AR), which is a member of the nuclear receptor family of transcription factors. The AR also plays a pivotal role in prostate tumor growth. Because growth of most prostate cancers depends on continuous androgenic stimulation, therapy of metastatic disease is generally based on androgen withdrawal or blockade of AR function by antiandrogens. However, after an initial regression, essentially all tumors continue to grow.Like other nuclear receptors, the AR displays a modular structure: a carboxy-terminal ligand binding domain (LBD), a central DNA binding domain (DBD), and an amino-terminal transactivation domain (TAD). Upon ligand binding, the AR regulates transcription by binding to specific androgen response elements in regulatory regions of target genes. Together with coactivators, general transcription factors and RNA polymerase II, a stable transcription initiation complex is formed (for reviews, see refs. 1-3). The size of the AR can be variable, due to variation in the length of polyglutamine and polyglycine stretches in the TAD. The amino acid numbering in our article correspon...