Scholl S, Lippman M E
Endocrinology. 1984 Oct;115(4):1295-301. doi: 10.1210/endo-115-4-1295.
Upon treatment of MCF-7 cells with estradiol at 37 C, practically all of the estrogen receptor is found in the nuclear compartment. This nuclear estrogen receptor form sediments close to 5S on high salt sucrose density gradients, similar to receptor activated in vitro by gentle heating. Upon extraction of the nuclear receptor, this 5S form is not stable at 0-4 C under our buffer conditions and is converted to a 4S form in 10-14 h. Incubation of cells in the presence of dense amino acids for various amounts of time in the absence of estradiol, followed by a 20- to 30-min labeling period with 3 nM [3H]estradiol at 37 C, permits separation of the newly synthesized estrogen receptor on sucrose gradients by means of its different sedimentation properties. With the fresh nuclear extract, we observe a progressive shift to a heavier form on sucrose gradients in proportion to the time of incubation in dense amino acids. The 5S nuclear receptor gradually becomes denser as the time of incubation of cells in dense medium increases and the peak moves from light to heavy without resolving into discrete heavy and light forms at intermediary time points. In contrast, when the nuclear extract, prepared after varying periods of incubation of the cells with dense amino acids and brief treatment with [3H]estradiol, is left standing at 0-4 C for a period of 10-14 h, we observe discrete peaks of heavy and light receptors. The resolution into discrete peaks of heavy and light forms at each time point permits a calculation of the turnover rate for this receptor (t 1/2 = approximately 2.25 h). These findings are consistent with a model in which the 5S nuclear receptor is an oligomer of randomly associated heavy and light monomers, whereas the aged 4S nuclear receptor is a monomer. We conclude from these data that estrogen receptor synthesis is extremely rapid in log growth phase MCF-7 cells; and the activated nuclear receptor appears to be an oligomer of at least two monomers closely related in size and metabolized at a similar rate. The random association between different monomers (heavy and light) results in a broad peak of nuclear receptor and is at least consistent with a dimerization reaction occurring as part of receptor activation.
在37℃用雌二醇处理MCF - 7细胞后，几乎所有的雌激素受体都存在于细胞核区室中。这种核雌激素受体形式在高盐蔗糖密度梯度上沉降接近5S，类似于通过温和加热在体外激活的受体。提取核受体后，在我们的缓冲条件下，这种5S形式在0 - 4℃不稳定，10 - 14小时内会转化为4S形式。在无雌二醇的情况下，将细胞在含有重氨基酸的条件下孵育不同时间，然后在37℃用3 nM [3H]雌二醇标记20 - 30分钟，通过其不同的沉降特性可在蔗糖梯度上分离新合成的雌激素受体。对于新鲜的核提取物，我们观察到在蔗糖梯度上逐渐向较重形式转变，转变程度与在重氨基酸中孵育的时间成正比。随着细胞在重培养基中孵育时间的增加，5S核受体逐渐变得更致密，峰从较轻位置移向较重位置，在中间时间点不会分离成离散的重、轻形式。相反，当用重氨基酸孵育细胞不同时间并经[3H]雌二醇短暂处理后制备的核提取物在0 - 4℃放置10 - 14小时，我们观察到重、轻受体的离散峰。每个时间点分离成重、轻形式的离散峰使得能够计算该受体的周转率（半衰期约为2.25小时）。这些发现与一个模型一致，该模型认为5S核受体是随机结合的重、轻单体的寡聚体，而老化的4S核受体是单体。从这些数据我们得出结论，在对数生长期的MCF - 7细胞中雌激素受体合成极快；并且活化的核受体似乎是至少两个大小相近且代谢速率相似的单体的寡聚体。不同单体（重、轻）之间的随机结合导致核受体出现一个宽峰，这至少与作为受体激活一部分发生的二聚化反应一致。
