Molecular mechanisms of androgen receptor functions

The androgens testosterone (T) and dihydrotestosterone (DHT) are steroid hormones, which are necessary for development and maintenance of the functions of the male sex organs, including the prostate. Androgens also play an important role in benign abnormalities of the prostate and in the growth of prostate cancer. Prostate tumors, which are not yet metastatic, are treated with radiotherapy or by surgical removal of the complete prostate. Therapy of metastasized prostate cancer aims on inhibition of androgen action, by inhibtion of the production of T in the testis (chemical castration) and by administration of anti-androgens. T and DHT exert their function by specific binding as a ligand to the androgen receptor (AR). The AR is a member of the family of nuclear receptors. It is expressed in androgen target cells, and functions as a ligand induced transcription factor. In this thesis described research project focusses on several molecular mechanisms of AR functions. Chapter 1 gives an overview of the current knowledge on nuclear receptors in general and different aspects of AR functions in particular. Among the latter are: receptor structure, interaction with other proteins involved in transcription, the ligand-dependent interaction between the N-terminal domain (NTD) and the ligand binding C-terminal domain (LBD) of the AR (N/C interaction), expression of androgen-specific regulated genes, and the role of AR mutations in prostate cancer

Functional interactions of the AF-2 activation domain core region of the human androgen receptor with the amino-terminal domain and with the transcriptional coactivator TIF2 (transcriptional intermediary factor2)

Previous studies in yeast and mammalian cells showed a functional interaction between the amino-terminal domain and the carboxy-terminal, ligand-binding domain (LBD) of the human androgen receptor (AR). In the present study, the AR subdomains involved in this in vivo interaction were determined in more detail. Cotransfection experiments in Chinese hamster ovary (CHO) cells and two-hybrid experiments in yeast revealed that two regions in the NH2-terminal domain are involved in the functional interaction with the LBD: an interacting domain at the very NH2 terminus, located between amino acid residues 3 and 36, and a second domain, essential for transactivation, located between residues 370 and 494. Substitution of glutamic acid by glutamine at position 888 (E888Q) in the AF-2 activation domain (AD) core region in the LBD, markedly decreased the interaction with the NH2-terminal domain. This mutation neither influenced hormone binding nor LBD homodimerization, suggesting a role of the AF-2 AD core region in the functional interaction between the NH2-terminal domain and the LBD. The AF-2 AD core region was also involved in the interaction with the coactivator TIF2 (transcriptional intermediary factor 2), as the E888Q mutation decreased the stimulatory effect of TIF2 on AR AF-2 activity. Cotransfection of TIF2 and the AR NH2-terminal domain expression vectors did not result in synergy between both factors in the induction of AR AF-2 activity. TIF2 highly induced AR AF-2 activity on a complex promoter [mouse mammary tumor virus (MMTV)], but it was hardly active on a minimal promoter (GRE-TATA). In contrast, the AR NH2-terminal domain induced AR AF-2 activity on both promoter constructs. These data indicate that both the AR NH2-terminal domain and the coactivator TIF2 functionally interact, either directly or indirectly, with the AF-2 AD core region in the AR-LBD, but the level of transcriptional response induced by TIF2 depends on the promoter context

Both androgen receptor and glucocorticoid receptor are able to induce prostate-specific antigen expression, but differ in their growth-stimulating properties of LNCaP cells

Androgen receptor-positive LNCaP cells were stably transfected with a rat glucocorticoid receptor (GR) expression plasmid. Ligand-binding studies in the generated cell lines revealed high-affinity binding of the cognate ligands to their receptors. Transfection experiments with the newly derived cell lines showed that, like androgen receptor, GR can induce activity of a prostate-specific antigen promoter fragment linked to the luciferase gene. Similarly, dexamethasone can stimulate expression of endogenous prostate-specific antigen messenger RNA. Cell proliferation could be induced by R1881. In contrast, dexamethasone treatment of the GR-positive sublines had no stimulatory effect on cell growth. Using the differential display technique, a so far unknown complementary DNA fragment, designated 21.1, specifically induced by androgens and not by glucocorticoids, has been identified. In conclusion, the newly generated cell lines, together with the parental LNCaP cell line, form an attractive system with which to study the mechanism of specificity of steroid hormone regulation of gene expression

Amino acids 3-13 and amino acids in and flanking the 23FxxLF27 motif modulate the interaction between the N-terminal and ligand-binding domain of the androgen receptor

The N-terminal domain (NTD) and the ligand-binding domain (LBD) of the androgen receptor (AR) exhibit a ligand-dependent interaction (N/C interaction). Amino acids 3-36 in the NTD (AR3-36) play a dominant role in this interaction. Previously, it has been shown that a PhixxPhiPhi motif in AR3-36, 23FxxLF27, is essential for LBD interaction. We demonstrate in the current study that AR3-36 can be subdivided into two functionally distinct fragments: AR3-13 and AR16-36. AR3-13 does not directly interact with the AR LBD, but rather contributes to the transactivation function of the AR.NTD-AR.LBD complex. AR16-36, encompassing the 23FxxLF27 motif, is predicted to fold into a long amphipathic alpha-helix. A second PhixxPhiPhi candidate protein interaction motif within the helical structure, 30VREVI34, shows no affinity to the LBD. Within AR16-36, amino acid residues in and flanking the 23FxxLF27 motif are demonstrated to modulate N/C interaction. Substitution of Q24 and N25 by alanine residues enhances N/C interaction. Substitution of amino acids flanking the 23FxxLF27 motif by alanines are inhibitory to LBD interaction