Among nuclear receptors, the androgen receptor (AR) is unique in that its ligand-binding domain (LBD) interacts with the FXXLF motif in the N-terminal domain, resembling coactivator LXXLL motifs. We compared AR- and estrogen receptor alpha-LBD interactions of the wild-type AR FXXLF motif and coactivator transcriptional intermediary factor 2 LXXLL motifs and variants of these motifs. Random mutagenesis revealed a key role for the F residues in FXXLF motifs in high-affinity and selective AR LBD interaction. The FXXLF motif in full-length AR and transcriptional intermediary factor 2 LXXLL motifs competed for an overlapping binding site. A computer model of the AR LBD/AR FXXLF complex showed that the bulky F residues are buried in a deep coactivator-binding groove. The corresponding groove in estrogen receptor alpha LBD is considerably shallower, explaining lack of binding of any of the FXXLF motifs tested. FXXLF and LXXLL motif interaction depended on different charged amino acid residues in the AR LBD present at opposite ends of the coactivator groove. In conclusion, our data demonstrate the importance of a deep hydrophobic groove and alternative usage of charged amino acids in specifying peptide binding to the AR LBD.

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...

BACKGROUND: The phosphatidylinositol 3-kinase (PI3K) -AKT pathway is activated in many cancers. Mutational hotspots in AKT1 and in the regulatory and catalytic subunits of PI3K have been detected in multiple tumour types. In AKT1, the E17K substitution leads to a PI3K-independent activation of AKT1. METHODS: A mutational profiling of AKT1 and of the mutational hotspots in PIK3CA and PIK3R1 was carried out in samples from primary and recurrent prostate tumours. RESULTS: We show that, in prostate cancer, AKT1(E17K) had a prevalence of 1.4%. The mutation seemed to be associated with a favourable clinical course but it was not associated with a specific tumour growth pattern. Activating mutations in PIK3CA or PIK3R1 were not found in prostate cancer. CONCLUSION: The E17K substitution in AKT1 is rare in prostate cancer. It seems associated with a favourable clinical outcome but not with a specific histology of the tumour.

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We characterized the specifically androgen-regulated gene (SARG), which is expressed in the androgen receptor (AR) and glucocorticoid receptor (GR) positive cell line lymph node carcinoma of the prostate-1F5 (LNCaP-1F5). SARG mRNA expression can be up-regulated by androgens, but not by glucocorticoids. SARG mRNA expression is high in prostate tissue. SARG is composed of four exons and spans a region of 14·5 kbp on chromosome 1q32·2. Transcripts of 5·5, 3·3 and 2·3 kb are the result of alternative polyadenylation. SARG mRNA splice variants lack exon 2 and vary in length of exon 1. The SARG protein has a length of 601 amino acids and is located in the cytoplasm. By screening the 18 kbp genomic sequence flanking the transcription start site we identified the imperfect direct repeat 58-TGTGCTaacTGTTCT-38 in intron 1 as an active androgen response element (ARE-SARG+4·6). A 569 bp genomic DNA fragment containing this element functioned as an androgen-specific enhancer in transiently transfected LNCaP-1F5 cells. ARE-SARG+4·6 cooperated with flanking sequences for optimal activity. Inactivation of ARE-SARG+4·6 completely abolished the androgen response of the enhancer. Chromatin immunoprecipitation (ChIP) experiments showed chromatin structural changes of the enhancer in the presence of R1881. ARE-SARG+4·6 was able to bind to the androgen receptor, but not to the glucocorticoid receptor, correlating with its androgen-specific activity in transfections.

The results indicate that PTEN dependent gene expression is important in cell-cycle regulation and is mediated by both Akt-dependent and -independent mechanisms.