Participation of critical residues from the extreme C-terminal end of the human androgen receptor in the ligand binding function.

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.

Specific recognition of androgens by their nuclear receptor. A structure-function study.

Androgens, like progestins, are 3-ketosteroids with structural differences restricted to the 17beta 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 hAR.LBD complexes with several ligands are presented, which suggests new directions for drug design.

A stable prostatic bioluminescent cell line to investigate androgen and antiandrogen effects.

We developed a new stable prostatic cell line expressing the human androgen receptor (AR) and the AR-responsive reporter gene to generate a powerful tool for investigating androgen action and for rapid screening of agonists and antagonists. The AR-deficient PC-3 cells were stably transfected with pSG(5)-puro-hAR and pMMTV-neo-Luc. After selection with puromycin and neomycin, one highly inducible clone was isolated and named PALM, for PC-3-Androgen receptor-Luciferase-MMTV. The expression of hAR was confirmed by western blot and steroid-binding assays on the whole cells. The transcriptional activity of the clone was measured after incubation of cells with increasing concentrations of synthetic R1881 or natural androgens (DHT and testosterone). The three agonists had the same maximal activity at 0.1 microM and the fold induction was equal to 20. The agonist and antagonist activities of the steroidal antiandrogens (cyproterone acetate and RU2956) and the non-steroidal antiandrogens (nilutamide, bicalutamide, inocoterone and hydroxyflutamide) measured with the PALM cells were in good correlation with the results obtained with transiently transfected cells. The selectivity in steroid transactivation was demonstrated with estradiol, progesterone, cortisol, dexamethasone and aldosterone. Spironolactone and RU486 showed partial agonist and antagonist activities, whereas R5020 presented only a partial antagonist activity. We here demonstrate that this stable transfectant provides an accurate tool for studying wild-type human AR activation and its regulation by androgens and antiandrogens in a human prostatic epithelial cell, which is routinely available and remains androgen-responsive in vitro.

[Androgen-independent prostate carcinoma and androgen-receptor: recent progress in molecular genetics]. / Cancer de la prostate hormono-indépendant et récepteurs des androgènes: les avancées en génétique moléculaire.

Prostate cancer is an androgen-dependent tumor which presents an androgen-independent regrowth after clinical regression in response to antiandrogen treatment. Four hypotheses have been developed to understand how androgen signal transduction pathway mediate androgen-independent tumor progression: over expression of the wild-type androgen-receptor gene, androgen-receptor gene mutation, excessive recruitment of transcriptional co-activator ARA-70 and a cross-talk between the androgen-receptor and the growth factor receptor pathways. In this work, C. Sawyers's group elegantly demonstrates, in LAPC-4 androgen-independent prostate cancer sublines, that forced hyperexpression of HER-2/Neu receptor tyrosine kinase allowed androgen-independent growth, that HER-2/Neu activated the androgen-receptor pathway in the absence of androgens and synergized with low levels of androgen to superactivate the pathway. These important data could have therapeutic implications for the management of androgen-independent prostate cancer.