Functional in Vivo Interaction between the Amino-Terminal, Transactivation Domain and the Ligand Binding Domain of the Androgen Receptor

Abstract

The ligand binding domain (LBD) and the amino-terminal, transactivation domain (TAD) of the androgen receptor (AR) were separately linked to the GAL4 DNA binding domain (DBD) and to the GAL4(TAD). Resulting constructs were tested in the yeast two-hybrid system for protein-protein interactions. In the presence of androgen [methyltrienolone (R1881) or dihydrotestosterone (DHT)] a transcriptionally active complex was formed, reflecting an association between the AR(LBD) and the AR(TAD). No interactions were found in the presence of low-affinity ligands like estradiol (E2), promegestone (R5020), or progesterone (Pg). Use of the Thr-868-Ala mutated AR(LBD) in the assay resulted not only in a clear AR TAD-LBD interaction in the presence of R1881 and DHT but also in the presence of E2, Pg, and R5020, corresponding to the alteration in ligand specificity induced by the mutation. Coexpression of the fusion protein Gal4(DBD)AR(LBD) and the separate AR(TAD) also gave rise to the formation of a transcriptionally active complex. No interactions were found between two AR LBDs at the low-expression level of the two components. However, LBD-LBD interaction was detectable by application of a high-expression vector for GAL4(TAD)AR(LBD), albeit at high ligand concentrations. To substantiate the observation of the AR LBD-TAD interaction, CHO cells were cotransfected with expression plasmids for a truncated AR, which lacks the TAD [AR(DBD)(LBD)], and for the separate AR(TAD). This resulted in stimulation of a MMTV-LUC reporter gene in the presence of R1881 but not in the absence of hormone. This finding indicates that, like in the yeast system, in mammalian cells, TAD-LBD interactions are of importance for AR activation. In the mammalian system, a maximal AR TAD-LBD interaction was obtained at approximately 10-fold higher ligand concentrations than required for full-length AR activation. In the presence of low-affinity ligands, the AR TAD-LBD interaction as measured by transcriptional activation was considerably weaker than the activity of the full-length AR. From the present results a concept of hormone-dependent AR activation is proposed, which requires a functional, direct or indirect intramolecular interaction between the TAD and the LBD.

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

“…5B). As expected, in the yeast protein interaction system, ligand-dependent interaction with AR LBD could easily be detected for GalAD-AR [17][18][19][20][21][22][23][24][25][26][27][28][29][30][31][32] . However, the interaction was weak for GalAD-AR [24][25][26][27][28][29][30][31][32][33][34][35][36][37][38][39] (Fig.…”

“…Western blot analysis for detection of GalAD fusion proteins was performed as previously described, utilizing a GAL4AD monoclonal antibody (Clontech) [18].…”

“…Previously, a ligand-dependent functional interaction between the AR subdomains NTD and LBD, has been described [17][18][19]. This N/C interaction might be intra-or intermolecular [15,[17][18][19].…”

“…This N/C interaction might be intra-or intermolecular [15,[17][18][19]. In vitro pull-down experiments indicated that the AR N/C interaction is direct [11].…”

Microarray coupled to quantitative RT–PCR analysis of androgen-regulated genes in human LNCaP prostate cancer cells

“…Earlier microarray studies for profiling androgen-regulated genes in prostate cancer cell lines have been carried out using the synthetic androgen R1881 or dihydro-testosterone (DHT), but few studies have carried out a detailed investigation of AR-regulated gene expression, in response to anti-androgens (for a review see Dehm and Tindall, 2006). In addition, AR activity can be stimulated by oestrogen (E2) and progesterone, whereas the anti-androgen cyproterone acetate (CPA) is a partial agonist for AR (Doesburg et al, 1997). Finally, some AR mutations, such as the AR-T877A mutation in LNCaP cells, increase the agonist activity of some of these weak AR agonists, as well as the antiandrogens CPA and hydroxyflutamide (OHF) (Steketee et al, 2002).…”