Function of N-terminal transactivation domain of the estrogen receptor requires a potential alpha-helical structure and is negatively regulated by the A domain

Transcriptional activation by the estrogen receptor (NR3A1, or ER) requires specific ligand-inducible activation functions located in the amino (AF-1) and the carboxyl (AF-2 and AF-2a) regions of the protein. Although several detailed reports of ER structure and function describe mechanisms whereby AF-2 activates transcription, less precise data exist for AF-1. We recently reported that the rainbow trout and human estrogen receptors (rtERs and hE Rs, respectively), two evolutionary distant proteins, exhibit comparable AF -1 activities while sharing only 20% homology in their N-terminal region. T hese data suggested that the basic mechanisms whereby AF-1 and the ER N-ter minal region activate transactivation might be evolutionary conserved. Ther efore, a comparative approach between rtER and hER could provide more detai led information on AF-1 function. Transactivation analysis of truncated rec eptors and Gal4DBD (DNA binding domain of the Gal4 factor) fusion proteins in Saccharomyces cerevisiae defined a minimal region of 11 amino acids, loc ated at the beginning of the B domain, necessary for AF-1 activity in rtER. Hydrophobic cluster analysis (HCA) indicated the presence of a potential c y-helix within this minimal region that is conserved during evolution. Both rtER and hER sequences corresponding to this potential alpha -helical stru cture were able to induce transcription when fused to the Gal4DBD, indicati ng that this region can transactivate in an autonomous manner. Furthermore, point mutations in this 11-amino acid region of the receptors markedly red uced their transcriptional activity either within the context of a whole ER or a Gal4DBD fusion protein. Data were confirmed in mammalian cells and. I nterestingly, ERs with an inverted alpha -helix were as active as their cor responding wild-type proteins, indicating a conserved role in AF-1 for thes e structures. Moreover, using two naturally occurring rtER N-terminal varia nts possessing or not the A domain (rtER(L) and rtER(S), respectively), tog ether with A domain-truncated hER and chimeric rtER/hER receptors, we demon strated that the A domain of the ER plays an inhibitory role in ligand-inde pendent activity of the receptor. In vitro and in vivo protein-protein inte raction assays using both rtER and hER demonstrated that this repression is likely to be mediated by a ligand-sensitive direct interaction between the A domain and the C-terminal region of the ER.