Interaction of the antiestrogen [ 3H]H1285 with the two forms of the molybdate-stabilized calf uterine estrogen receptor

Abstract

The high affinity antiestrogen [ 3H]H1285 bound to the cytosol calf uterine estrogen receptor dissociated very slowly ( t 1 2 1,3 approx 30 h at 20°C) and did not demonstrate a change in dissociation rate in the presence of molybdate, which is characteristic of [ 3H]estradiol-receptor complexes. [ 3H]H1285-Receptor complexes sediment at approx 6S on 5–20% sucrose density gradients containing 0.3 M KC1 with or without 10 mM molybdate. This is in contrast to [ 3H]estradiol-receptor complexes which sedimented at approx 4.5S without molybdate and at approx 6S with molybdate. These results suggest a physicochemical difference in the estrogen receptor when occupied by antiestrogens versus estrogens. We recently reported that the cytoplasmic uterine estrogen receptor, when bound by estradiol and prepared in 10 mM molybdate, eluted from DEAE-Sephadex columns as Peak I (0.21 M KC1) & Peak II (0.25 M KC1). However, [ 3H]H1285 bound to the estrogen receptor eluted only as one peak at 0.21 M KC1, also suggesting that the initial interaction of antiestrogens with the estrogen receptor is different. We have extended these studies and report that H 1285 can compete with [ 3H]estradiol for binding to both forms of the estrogen receptor and [ 3H]H1285 can bind to both forms if the unoccupied receptor is first separated by DEAE-Sephadex chromatography. However, if the receptor is first bound by unlabeled H1285, eluted from the column and post-labeled by exchange with [ 3H]estradiol, only one peak is measured. Thus, it appears that H1285 binding alters the properties of the receptor such that all receptor components seem to elute as one form. These partially purified [ 3H]1285-receptor complexes obtained from DEAE-Sephadex columns sedimented as 5.5S in sucrose density gradients in contrast to the sedimentation values for the [ 3H]estradiol-receptor components eluting as Peak I (4.5S) and Peak II (6.3S). These differences in the physicochemical characteristics of the estrogen receptor when bound by estrogen versus antiestrogens may be related to some of the biological response differences induced by these ligands.