Nardulli A M, Katzenellenbogen B S
Endocrinology. 1986 Nov;119(5):2038-46. doi: 10.1210/endo-119-5-2038.
The rate at which the estrogen receptor (ER) is synthesized and degraded (turns over) in target cells is a major factor regulating the levels of ER and cell sensitivity to estrogen. We have used the ability of tamoxifen aziridine [(TAZ)(Z)-1-[4-(2-[N-aziridinyl]ethoxy)phenyl]1,2-diphenyl-1-buten e] to affinity label the ER in intact cells to study the dynamics of ER turnover in uterine cells in vitro and in intact rat uteri in vivo. In primary cultures of rat uterine cells (from 21-day-old rats), ER is degraded with a half-life of 3-4 h, as determined by pulse-chase experiments in which ER in cells are covalently labeled with [3H]TAZ (20 nM) and then exposed to a chase of 10(-6) M estradiol. Density shift experiments, in which uterine cells are exposed for different time periods to medium containing dense (15N,13C,2H) amino acids and the shift of receptor from a normal density to a more dense species is analyzed on sucrose gradients, also confirm this rapid turnover for the uterine ER. A similar half-life is obtained for ER in rat uteri in vivo, after intralumenal installation of [3H]TAZ to label effectively ER covalently. Analysis of nuclear ER on sodium dodecyl sulfate-polyacrylamide gels during the chase period reveals the loss of the 65,000 mol wt [3H]TAZ-labeled receptor species; no lower mol wt [3H]TAZ-labeled fragments are observed. Likewise, immunoblot analyses of receptor with the ER monoclonal antibody H222Sp gamma reveal only the 65,000 mol wt receptor species, with no evidence of any smaller receptor forms in cells. Interestingly, incubation of uterine cells with 10(-5) M cycloheximide (which inhibits 98% of protein synthesis) completely arrests ER turnover, suggesting that ER turnover, which is rapid, is probably dependent upon the synthesis of other rapidly turning over proteins. Hence, we conclude that ER turnover is rapid in uterine cells, that turnover of uterine ER occurs at a rate similar to that previously reported for ER turnover in MCF-7 human breast cancer cells, and that ER turnover is similar in uterine cells under in vitro and in vivo conditions. These findings indicate that the receptor is a dynamic and rapidly turning over protein whose level could be adjusted quickly by alterations in the rate of synthesis and/or degradation.
雌激素受体（ER）在靶细胞中的合成与降解速率（周转率）是调节ER水平以及细胞对雌激素敏感性的一个主要因素。我们利用他莫昔芬氮丙啶[（TAZ）（Z）-1-[4-(2-[N-氮丙啶基]乙氧基)苯基]-1,2-二苯基-1-丁烯]对完整细胞中的ER进行亲和标记的能力，来研究体外培养的子宫细胞以及完整大鼠子宫中ER周转的动力学。在大鼠子宫细胞（来自21日龄大鼠）的原代培养物中，通过脉冲追踪实验确定ER的半衰期为3 - 4小时。在该实验中，细胞中的ER用[3H]TAZ（20 nM）进行共价标记，然后用10^(-6) M的雌二醇进行追踪。密度转移实验也证实了子宫ER的这种快速周转率，在该实验中，子宫细胞在含有重（15N、13C、2H）氨基酸的培养基中暴露不同时间段，然后在蔗糖梯度上分析受体从正常密度向更高密度种类的转移情况。在体内，通过向大鼠子宫腔内注入[3H]TAZ以有效共价标记ER后，也得到了类似的半衰期。在追踪期间，对十二烷基硫酸钠 - 聚丙烯酰胺凝胶上的核ER进行分析，结果显示65,000 mol wt [3H]TAZ标记的受体种类消失；未观察到更低mol wt的[3H]TAZ标记片段。同样，用ER单克隆抗体H222Spγ对受体进行免疫印迹分析，结果仅显示65,000 mol wt的受体种类，细胞中没有任何更小受体形式的证据。有趣的是，用10^(-5) M环己酰亚胺（抑制98%的蛋白质合成）孵育子宫细胞会完全阻止ER周转，这表明快速的ER周转可能依赖于其他快速周转蛋白质的合成。因此，我们得出结论：子宫细胞中ER周转迅速，子宫ER的周转速率与之前报道的MCF - 7人乳腺癌细胞中ER周转速率相似，并且在体外和体内条件下子宫细胞中的ER周转情况相似。这些发现表明该受体是一种动态且周转迅速的蛋白质，其水平可通过合成和/或降解速率的改变而迅速调节。
