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 FACTOR-2)

Previous studies in yeast and mammalian cells showed a functional inte raction between the amino-terminal domain end the carboxy-terminal, li gand-binding domain (LBD) of the human androgen receptor (AW). In the present study, the AW 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 funct ional interaction with the LED: an interacting domain at the very NH2 terminus, located between amino acid residues 3 and 36, and a second d omain, essential for transactivation, located between residues 370 and 494. Substitution of glutamic acid by glutamine at position 888 (E888 Q) 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 LED homodimerization, suggesti ng a role of the AF-2 AD core region in the functional interaction bet ween the NH2-terminal domain and the LBD. The AF-2 AD core region was also involved in the interaction with the coactivator TIF2 (transcript ional intermediary factor 2), as the E888Q mutation decreased the stim ulatory effect of TIF2 on AR AF-2 activity. Cotransfection of TIF2 and the AR NH2-terminal domain expression vectors did not result in syner gy between both factors in the induction of AR AF-2 activity. TIF2 hig hly induced AR AF-2 activity on a complex promoter [mouse mammary tumo r virus (MMTV)], but it was hardly active on a minimal promoter (GRE-T ATA). 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, eithe r 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 t he promoter context.