Multiple forms of nuclear estrogen receptor in the immature rat uterus after in vitro exchange with [3H]estradiol or [3H] antiestrogens

We have compared the properties of salt-extracted uterine nuclear estrogen receptors (ER) labeled with [³H]estradiol (E₂) or [³H]antiestrogens after in vivo exposure of immature rats to these compounds or after in vitro exchange. As expected, nuclear ER labeled during a l h in vivo treatment with [³H]E₂ migrated predominantly at 5S, although this peak was skewed towards the lighter side. [³H]4-Hydroxytamoxifen ([³H]OH tamoxifen, the active metabolite of tamoxifen)-nuclear ER complexes showed comparable sedimentation rates (4.6 ± 0.05S) after injection of the [³H]antiestrogen. On the other hand, when nuclear extracts from rats treated with unlabeled E₂ in vivo were subjected to exchange with [³H]E₂ or [³H]antiestrogens for 16 h at 23°C, a marked difference. in sedimentation profiles was observed. While the [³H]E₂ -nuclear ER complex sedimented primarily as a 3S species with variable amounts of a heavier (4.0–4.5S) shoulder, complexes between nuclear ER and [³H]tamoxifen, [³H]OH tamoxifen or [³H]CI628M formed sharp, symmetrical peaks at 4S. Both the 3S and 4S components represented forms of ER as they were eliminated in the presence of excess unlabeled DES, and they were displaced to 7–8S after reaction with specific anti-ER antibodies. The 3S peak was also abolished by addition of excess nonradioactive nafoxidine or OH tamoxifen during exchange with [³H]E₂. These results suggest that more rapidly sedimenting forms of uterine nuclear ER (4–5S) may be converted to a 3S species by the action of endogenous proteases and that association of these large forms with antiestrogens may stabilize them in a conformation less or differentially susceptible to cleavage.