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 interaction between the amino-terminal domain and the carboxy-terminal, ligand-binding domain (LBD) of the human andro- gen receptor (AR). In the present study, the AR subdomains involved in this in vivo interaction were determined in more detail. Cotransfection ex- periments in Chinese hamster ovary (CHO) cells and two-hybrid experiments in yeast revealed that two regions in the NH2-terminal domain are in- volved 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, lo- cated 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 inter- action with the NH2-terminal domain. This muta- tion neither influenced hormone binding nor LBD homodimerization, suggesting a role of the AF-2 AD core region in the functional interaction be- tween the NH2-terminal domain and the LBD. The AF-2 AD core region was also involved in the inter- action with the coactivator TIF2 (transcriptional in- termediary factor 2), as the E888Q mutation de- creased 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 ac- tivity 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 coacti- vator TIF2 functionally interact, either directly or indirectly, with the AF-2 AD core region in the AR- LBD, but the level of transcriptional response in- duced by TIF2 depends on the promoter context. (Molecular Endocrinology 12: 1172-1183, 1998)