Hydrodynamic Characterizations of Estrogen Receptors Complexed with [3H]-4-Hydroxytamoxifen: Evidence in Support of Contrasting Receptor Transitions Mediated by Different Ligands†

Size-exclusion high-performance liquid chromatography was used to characterize the hydrodynamic molecular properties of estrogen receptors complexed with estradiol and the antiestrogen 4-hydroxytamoxifen. Cytoplasmic estrogen receptors complexed with [3H]-4-hydroxytamoxifen did not undergo reductions in hydrodynamic size after exposure to KC1 or urea. Nuclear receptors complexed with 4-hydroxytamoxifen eluted as hydrodynamically larger molecules than nuclear receptors complexed with estradiol. Because identical hydrodynamic characterizations were obtained with the covalent ligand [3H]tamoxifen aziridine, these differences in chromatographic behavior are due to differences in ligand-mediated receptor properties and are not the result of ligand dissociation. When estrogen receptors, complexed with either [3H] estradiol or [3H]-4-hydroxytamoxifen, were exposed to trypsin, the receptors complexed with 4-hydroxytamoxifen eluted as larger hydrodynamic forms than receptors complexed with estradiol. These observations are interpreted to indicate that estradiol and 4-hydroxytamoxifen mediate contrasting transitions in the molecular orientation of estrogen receptors. The consequences of the transitions mediated by 4-hydroxytamoxifen appear to be that intermolecular associations become difficult to disrupt with KC1 or urea and that the accessibility of trypsin-sensitive proteolytic sites becomes altered. Chromatin fractionation using DNase I and hypotonic Mg2+ solubilization identified a chromatin region that was less readily penetrated by receptors complexed with 4-hydroxytamoxifen than receptors complexed with estradiol. This observation supports the hypothesis that one consequence of different ligand-mediated receptor transitions is that receptors become positioned distinctively in chromatin by agonistic and antagonistic ligands. We suggest that these transitions may be related to mechanisms that separate the actions of estrogen agonists and antagonists.