IDENTIFICATION OF CHARGED RESIDUES IN AN N-TERMINAL PORTION OF THE HORMONE-BINDING DOMAIN OF THE HUMAN ESTROGEN-RECEPTOR IMPORTANT IN TRANSCRIPTIONAL ACTIVITY OF THE RECEPTOR

We have shown that charged amino acids near C530 of the human estrogen receptor (ER) are involved in receptor discrimination between estroge n and antiestrogen. We now examine the role of charged residues, adjac ent to the three other cysteines (381, 417, and 447) in the hormone-bi nding domain of the human ER, in the hormone-binding, DNA-binding, and transcription activation abilities of the receptor. Mutation of the o ne charged amino acid nearest to C381 gave a mutant receptor (E380Q) r equiring two to three times less estradiol (E(2)) than wild type (WT) ER to achieve maximal activity and having activity in the absence of a dded estrogen that was 6-fold higher than that of WT receptor. The enh anced ability of this mutant to bind to estrogen response element DNA in the absence and presence of estrogen may, at least in part, explain its elevated, seemingly constitutive trans-activation activity and it s increased sensitivity to estrogen. While more sensitive to E(2), thi s E380Q mutant was less sensitive than WT ER to antiestrogen for suppr ession of transcriptional activity. Mutation of all three charged resi dues nearest to C381 (the triple mutant D374N, E380Q, and E385Q) resul ted in a greatly reduced potency of the receptor in trans-activation w ith no change in estrogen-binding affinity. When K449 (near C447), hig hly conserved among steroid receptors, was mutated to Q, 400-fold more E(2) was required for maximal reporter gene trans-activation due to a n unstable, temperature-sensitive hormone-receptor complex. In contras t, the mutant K416Q (near C417) was unaltered in E(2)-binding or recep tor transcriptional activity. These studies reveal a region in the N-t erminal portion of the hormone binding domain (ca. amino acids 374-385 ) where alterations in charged residues result in either increases or decreases in receptor transcriptional activity with no change in recep tor affinity for hormone. Our findings suggest that this region may be important in DNA binding and protein-protein interactions that modula te transcriptional activity of the ER. In addition, the region near C4 47, which is well conserved among steroid receptors, appears to be imp ortant in maintaining the receptor in a conformation that is stable at physiological (37 C) temperatures. To our knowledge, this is the firs t report of an ER (E380Q) with a sensitivity to E(2) for trans-activat ion greater than that of WT receptor and having high trans-activation activity in the absence of added hormone.