Proteinprotein interaction of the AR

The AR is able to perform interactions with proteins. The AR monomer can build an intermolecular interaction bridge and makes contact with a variety of other proteins. These interactions are vital for the AR-mediated transcriptional transac-tivation of target genes as they modulate the activity of the receptor.

Direct contact between the amino-terminal and the carboxyl-terminal regions (N/C-interaction) of the AR was found using a two-hybrid system and glutathione-S-transferase fusion protein studies. Hereby, distinct amino-terminal sequence motifs in the receptor mediates interaction with the AF2 region of the LBD (He etal. 2000). Because androgens are able to bind the LBD located at the C-terminus of the AR, the intermolecular interaction between the receptor sides is controlled by the hormone itself but independent of the binding to DNA (Langley et al. 1995). The N/C-interaction of AR is required for potent agonists to be effective at low concentrations but is not required for weak agonist activity at higher ligand concentrations. Therefore, the formation of the N/C-interaction contributes to the stability of AR at low ligand concentrations (Kemppainen et al. 1999). As found recently, there is also strong correlation between the strength of the N/C-terminal interactions and the corresponding clinical phenotype in androgen insensitive syndrome (AIS), indicating the importance of the structural conformation of the receptor (Ghali et al. 2003). Furthermore, comodulators such as TIF2 can augment the N/C-terminal interaction of the AR and the nuclear orphan receptor DAX-1, which has low expression level in benign prostate hyperplasia (BPH) tissue, is able to disrupt the N/C-terminal interaction of the AR but does not influence the interaction between AR and SRC-1 (Agoulnik et al. 2003; Ghali et al. 2003). Therefore some orphan receptors and comodulators are important mediators of hormonal signals in human diseases.

As it became clear that the transcriptional activities of AR and other members of the NR superfamily are modulated by coregulatory proteins, many groups tried to identify interacting partners. To search for possible binding proteins for the AR the yeast two-hybrid system, established over ten years ago (Fields and Song 1989), anddirect cDNA expression library screening using affinity chromatography, were performed (e.g. Hsiao and Chang 1999). Mostly, the cDNA libraries used to identify AR-interacting proteins are deriving from prostate and testis and only rarely from other tissues, such as brain or hepatic cell lines. Up to now, over 70 proteins described as able to interact with the AR, were compiled in different reviews (Heinlein and Chang 2002; Hermanson etal. 2002; McKenna etal. 1999; Robyr etal. 2000) and also elsewhere (ww2.mcgill.ca/androgendb). When the properties of these proteins were analysed in detail, it becomes clear that most were not exclusive for the AR as they interact with other NRs as well. In addition, some putative AR interacting proteins may be located either in a tissue where the AR is not present, or even localised differently inside a single cell. This promiscuity makes the search for AR-specific comodulators difficult especially when it is not clear what defines comodulator selectivity. Nevertheless, some comodulators specific for the AR were described (e.g. FHL2). The transcriptional effect of FHL2 was measured using transient transfection experiments in vitro that examine the ability of the comodulator to alter the AR-mediated transcriptional activity on an artificial reporter construct whereas the transactivation of other nuclear receptors (GR, PR, and MR) were not mediated by FHL2 (Muller etal. 2000).

Coregulatory proteins are able to interact with the hormone receptor either direct or indirect via secondary proteins to enhance (coactivator) or reduce (corepressor) the receptor mediated transactivation of target genes. The modulators may stabilise the receptor/ligand binding and influence the translocation of the receptor into the nucleus. ARA70, one of the first comodulator for AR described, enhances AR-dependent transcription in prostate cells. Additionally, the interaction of ARA70 with hormone bound AR enhances stability of the receptor (Yeh and Chang 1996). E2 usually binds AR with a 100-fold lower affinity than DHT and does not mediate AR-dependent transactivation. However in the presence of ARA70, E2 activates AR-mediated transcription in PC3 (Han etal. 2001) and DU145 (Yeh etal. 1998). It maybe possible that AR-dependent genes are induced by E2 in tissues where ARA70 expression is elevated causing androgen effects mediated by E2. Additionally, it was shown that ARA70 enables the AR antagonists hydroxyflutamide and casodex to behave as agonists (Miyamoto et al. 1998; Yeh et al. 1997). This is especially relevant for prostate cancer patients treated with androgen antagonists as part of androgen ablation therapy. When these compounds were used in the clinic, it must be considered if comodulators are present in the tissue, as they lead to enhancement of gene activation.

Comodulators determine the tissue specificity of SERMs with respect to agonistic and antagonistic activities (Shang and Brown 2002). Recently, it was shown that the coactivator/corepressor ratio also modulate PR-mediated transcription by a selective receptor modulator (RU38486) acting as an agonist in T47D cells and as an antagonist in HeLa cells (Liu et al. 2002). It may be possible that similar mechanisms are valid for AR-mediated transactivation in different cells where the relative amount of comodulators play a vital role.

The mechanism how comodulators act on transcription in detail is only partially understood. They may work as bridging factors between the DNA-bound nuclear receptor and the basal transcriptional machinery, as chromatin modelling factors, as protein modifying enzymes or even as DNA binding factors to regulate their corresponding genes. Importantly, the tissue distribution of comodulators differs in the organism and so far not much information is available concerning the regulation and activation of AR interacting proteins.

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