FUNCTIONAL IN-VIVO INTERACTION BETWEEN THE AMINO-TERMINAL, TRANSACTIVATION DOMAIN AND THE LIGAND-BINDING DOMAIN OF THE ANDROGEN RECEPTOR

The ligand binding domain (LED) and the amino-terminal, transactivatio n domain (TAD) of the androgen receptor (AR) were separately linked to the GAL4 DNA binding domain (DBD) and to the Resulting constructs wer e tested in the yeast two-hybrid system for protein-protein GALA(TAD). In the presence of androgen [methyltrienolone (R1881) or dihydrotesto sterone (DHT)I a transcriptionally active complex was formed, reflecti ng an association between the AR(LBD) and the interactions. AR(TAD). N o interactions were found in the presence of low-affinity ligands like estradiol (E2), promegestone (R5020), or progesterone (Pg), Use of th e Thr-868-Ala mutated AR(LBD) in the assay resulted not only in a clea r 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 ARC(TAD) also gave rise to the formation of a transcriptionally active complex. No interactions w ere found between two AR LBDs at the low-expression level of the two c omponents. However, LED-LED interaction was detectable by application of a high-expression vector for GAL4(TAD)AR(LED), albeit at high ligan d concentrations. To substantiate the observation of the AR LBD-TAD in teraction, CHO cells were cotransfected with expression plasmids for a truncated AR, which lacks the TAD [AR(DBD)(LBD)I, and for the separat e 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 sy stem, a maximal AR TAD-LBD interaction was obtained at approximately 1 0-fold higher ligand concentrations than required for full-length AR a ctivation. In the presence of low-affinity ligands, the AR TAD-LBD int eraction as measured by transcriptional activation was considerably we aker than the activity of the full-length AR. From the present results a concept of hormone-dependent AR activation is proposed, which requi res a functional, direct or indirect intramolecular interaction betwee n the TAD and the LED.