Cushing C L, Bambara R A, Hilf R
Endocrinology. 1985 Jun;116(6):2419-29. doi: 10.1210/endo-116-6-2419.
Interactions of the estrogen-receptor complex (ER) with nuclei in vitro were shown to be dose, time, temperature, and tissue dependent. Specificity was demonstrated by the ability of ER charged with unlabeled 17 beta-estradiol to compete with [3H]ER for the nuclear acceptor sites. The ionic environment affected [3H]ER nuclear interactions; [3H]ER binding varied inversely with ionic strength, and apparent nuclear saturation was observed in the presence of 0.1 M KCl. Nuclear interactions of estrogen receptor charged with 17 beta-estradiol (ER) or monohydroxytamoxifen (AER) were compared. While both ER and AER (nonradiolabeled) were efficient competitors for [3H]ER nuclear binding, differences were observed when 3H-labeled ligands were used for saturation analysis of the nuclear acceptor sites. Scatchard analysis of the data revealed similar apparent Kd values for [3H]ER and [3H]AER binding to the nuclear sites (mean +/- SD, 1.2 +/- 0.5 X 10(-9) and 2.6 +/- 0.2 X 10(-9) M, respectively). However, the relative number of nuclear binding events consistently differed, with 28,000 +/- 7,300 sites/nucleus (mean +/- SD) for ER vs. 17,800 +/- 6,300 sites/nucleus for AER. Treatment of nuclei with 0-300 unit-min/ml DNAase I before incubation with receptor complexes resulted in a parallel percent decrease in the number of ER and AER nuclear binding sites. Saturation analysis performed with nuclei previously digested with 0-30 unit-min/ml DNAase I demonstrated that the apparent affinities of the receptor complexes for nuclear sites remained unchanged. Therefore, we suggest that both AER and ER bind to acceptor sites in the small portion of the chromatin hypersensitive to DNase I, but that fewer AER than ER can bind at least some of the ER nuclear acceptor sites. The lower binding capacity for AER may result in a pattern of gene expression that produces the agonist/antagonist effects observed with antiestrogens.
雌激素受体复合物（ER）与细胞核在体外的相互作用显示出剂量、时间、温度和组织依赖性。未标记的17β-雌二醇负载的ER与[3H]ER竞争核受体位点的能力证明了其特异性。离子环境影响[3H]ER与核的相互作用；[3H]ER结合与离子强度呈反比，在0.1M KCl存在下观察到明显的核饱和。比较了负载17β-雌二醇（ER）或单羟基他莫昔芬（AER）的雌激素受体的核相互作用。虽然ER和AER（未放射性标记）都是[3H]ER核结合的有效竞争者，但当使用3H标记的配体进行核受体位点的饱和分析时，观察到了差异。对数据进行Scatchard分析显示，[3H]ER和[3H]AER与核位点结合的表观Kd值相似（分别为平均±标准差，1.2±0.5×10-9和2.6±0.2×10-9M）。然而，核结合事件的相对数量始终不同，ER为28000±7300个位点/核（平均±标准差），而AER为17800±6300个位点/核。在用受体复合物孵育之前，用0-300单位-分钟/毫升DNA酶I处理细胞核，导致ER和AER核结合位点的数量平行减少。用0-30单位-分钟/毫升DNA酶I预先消化的细胞核进行的饱和分析表明，受体复合物对核位点的表观亲和力保持不变。因此，我们认为AER和ER都与对DNA酶I敏感的染色质小部分中的受体位点结合，但与ER相比，能结合至少一些ER核受体位点的AER较少。AER较低的结合能力可能导致一种基因表达模式，产生抗雌激素所观察到的激动剂/拮抗剂效应。
