Metzger D A, Korach K S
Department of Obstetrics and Gynecology, Duke University Medical Center, Durham, North Carolina 27710.
Endocrinology. 1990 Apr;126(4):2190-5. doi: 10.1210/endo-126-4-2190.
An integral part of the mechanism of estrogen action is the interaction of estrogen receptor (ER) complexes with specific nuclear acceptor sites to effect alterations in genomic expression. The localization of nuclear acceptor sites has been in question, but an increasing body of indirect evidence implicates the nuclear matrix. To assess the binding characteristics of [3H]estradiol-receptor complexes (3HER) to nuclear matrix, ER from ovariectomized mice was partially purified by ammonium sulfate precipitation and incubated under cell-free conditions with mouse uterine nuclear matrix at 4 C. The binding capacity of the nuclear matrix was determined to be 36.4 +/- 5.7 fmol/100 micrograms DNA, with a Kd of 0.23 +/- 0.03 nM. Binding to nuclear matrix sites was specific, as determined by the ability of increasing concentrations of unlabeled ER complexes to inhibit binding of 3HER. Spleen, used as a nontarget tissue, contained fewer binding sites (n = 4.07 fmol/100 micrograms DNA) than matrix from liver (n = 14.2). The binding affinity was the same in all three tissues. Injection of animals with estradiol before death was associated with loss of assayable nuclear matrix binding sites, implying occupancy of sites by ER in vivo. Unbound receptor (R) also demonstrated the ability to bind to uterine matrix (n = 40.2 +/- 2.7 fmol/100 micrograms DNA; Kd = 0.26 +/- 0.05 nM) as well as to competitively inhibit the binding of 3HER complexes. However, heat-inactivated receptor displayed no binding or competing activity, nor did the progesterone receptor. The two forms of the receptor can be functionally distinguished by extraction with 0.6 M KCl; 43% of ER, but no R, were resistant to KCl extraction. These results indicate that nuclear acceptor sites are associated with the nuclear matrix. Furthermore, these sites demonstrate the criteria expected of specific binding sites, i.e. high affinity, limited capacity, hormone receptor, and relative tissue specificity. The apparent association of uncomplexed receptor to nuclear acceptor sites may explain the uterine tissue nuclear localization of ER in the absence of hormone.
雌激素作用机制的一个重要组成部分是雌激素受体（ER）复合物与特定核受体位点相互作用，从而影响基因组表达的改变。核受体位点的定位一直存在疑问，但越来越多的间接证据表明与核基质有关。为了评估[3H]雌二醇 - 受体复合物（3HER）与核基质的结合特性，通过硫酸铵沉淀法对去卵巢小鼠的ER进行部分纯化，并在无细胞条件下于4℃与小鼠子宫核基质孵育。测定核基质的结合能力为36.4±5.7 fmol/100μg DNA，Kd为0.23±0.03 nM。通过增加未标记的ER复合物浓度抑制3HER结合的能力确定，与核基质位点的结合是特异性的。用作非靶组织的脾脏含有的结合位点（n = 4.07 fmol/100μg DNA）比肝脏基质（n = 14.2）少。所有三种组织中的结合亲和力相同。在处死前给动物注射雌二醇与可检测的核基质结合位点的丧失有关，这意味着体内ER占据了这些位点。未结合的受体（R）也显示出与子宫基质结合的能力（n = 40.2±2.7 fmol/100μg DNA；Kd = 0.26±0.05 nM），并能竞争性抑制3HER复合物的结合。然而，热灭活的受体没有显示出结合或竞争活性，孕酮受体也没有。受体的两种形式可以通过用0.6 M KCl提取在功能上加以区分；43%的ER对KCl提取有抗性，而R没有。这些结果表明核受体位点与核基质相关。此外，这些位点显示出特异性结合位点所预期的标准，即高亲和力、有限容量、激素受体和相对组织特异性。未复合的受体与核受体位点的明显关联可能解释了在没有激素的情况下ER在子宫组织中的核定位。
