The analysis of chimeric human/rainhow trout estrogen receptors reveals amino acid residues outside of P- and D-boxes important for the transactivation function

The amino acid sequence of rainbow trout estrogen receptor (rtER) is highly conserved in the C domain but presents few similarities in the A/B and E d omains with human estrogen receptor alpha (hER) [NR3A1], A previous study h as shown that rtER and hER have differential functional activities in yeast Saccharomyces cerevisiae, To determine the domain(s) responsible for these differences, chimeric human/rainbow trout estrogen receptors were construc ted, The A/B, C/D or E/F regions of rtER were replaced by corresponding reg ions of hER and expressed in yeast cells, Ligand-binding and transcription activation abilities of these hybrid receptors were compared with those of wild-type rtER or hER. Surprisingly, our data revealed that the human C/D d omains play an important role in the magnitude of transactivation of ER, Tw o other chimeric ERs carrying either a C or D domain of hER showed that the C domain was responsible for this effect whereas the D domain did not affe ct hybrid receptor activities, Moreover, a chimeric hER carrying the C doma in of rtER showed maximal transcriptional activity similar to that observed with rtER, Gel shift assays showed that, whereas rtER and hER present a si milar binding affinity to an estrogen response element (ERE) element, the r tER C domain is responsible for a weaker DNA binding stability compared to those of hER, In addition, the human C domain allows approximately 2 times faster association of ER to an ERE, Utilization of reporter genes containin g one or three EREs confirms that rtER requires protein-protein interaction s for its stabilization on DNA and that the C domain is involved in this st abilization. Moreover, AF-1 may be implicated in this synergistic effect of EREs, Interestingly, although E domains of these two receptors are much le ss conserved, replacement of this domain in rtER by its human counterpart r esulted in higher estradiol sensitivity but no increase in the magnitude of transactivation, Data from the chimeric receptors, rtER(hC) and hER(rtC), demonstrated that rtER AF-1 and AF-2 activation domains activated transcrip tion in the presence of estradiol similar to both AF-1 and AF-2 hER, This i mplies that these domains, which show poor sequence homology, may interact with similar basal transcription factors.