Differential inhibition of estrogen and antiestrogen binding to the estrogen receptor by diethylpyrocarbonate.
Diethylpyrocarbonate differentially inhibited the specific binding, in lamb uterine cytosol, of estradiol (inhibition approximately 90% with 4 mM reagent) and 4-hydroxytamoxifen (inhibition approximately less than 50% with 4-16 mM reagent), a potent triphenylethylene antiestrogen. Saturation analysis experiments indicated that the effects of diethylpyrocarbonate were due to progressive but differing decreases in the concentration of binding sites for the two ligands, with no apparent change in the affinity constants. However, competitive binding and dissociation experiments evidenced that steroidal and nonsteroidal estrogens still bound, but with very low affinities, to diethylpyrocarbonate-modified receptor (greater than 1000-fold decrease in affinity) whereas the affinities of triphenylethylene antiestrogens were much less affected (less than 10-fold decrease). Both ligands prevented the inactivation of the estrogen receptor by diethylpyrocarbonate, estradiol being more efficient than 4-hydroxytamoxifen. These data indicate that the action of diethylpyrocarbonate results in the formation of two populations of estrogen receptor that are quantitatively nearly equivalent: the first does not bind estrogens or antiestrogens; the second does not bind estrogens significantly but still interacts with antiestrogens at a high affinity. The simplest interpretation is that these two populations arise from mutually exclusive modifications by diethylpyrocarbonate of at least two aminoacid residues located at or close to the ligand binding site; modification of one residue totally prevents the binding of estrogens and antiestrogens; the modification of the second impairs only the binding of estrogens. Considering that (i) hydroxylamine, which specifically reverses the diethylpyrocarbonate-induced modification of histidine and tyrosine residues, restored a large part (greater than 80%) of the estradiol- and 4-hydroxytamoxifen-binding capacity of diethylpyrocarbonate-inactivated cytosol, and that (ii) similar differential inhibition of estrogen and antiestrogen binding was observed following the action of tetranitromethane, it is likely that these residues are histidine(s) and/or tyrosine(s). These results evince a marked difference in the interaction of estrogens and triphenylethylene antiestrogens with the estrogen receptor, which could account for the altered activation of the receptor by triphenylethylene antiestrogens. Consequently, the screening of ligands with modified steroid receptors could be a useful method for distinguishing between potential hormone agonists and antagonists.