Iwabuchi Erina, Miki Yasuhiro, Ono Katsuhiko, Onodera Yoshiaki, Sasano Hironobu
Department of Pathology, Tohoku University Graduate School of Medicine, Sendai, Japan.
Department of Disaster Obstetrics and Gynecology, International Research Institute of Disaster Science (IRIDes), Tohoku University, Sendai, Japan.
Acta Histochem Cytochem. 2017 Apr 27;50(2):85-93. doi: 10.1267/ahc.17011. Epub 2017 Apr 22.
The estrogen receptor (ER) functions as a dimer and is involved in several different biological functions. However ER dimeric proteins have not been identified by methodologies. Structured illumination microscopy (SIM) has been recently developed, which enabled the localization of protein and protein interaction. Therefore, in this study, we firstly demonstrated that ERs formed both homodimers and heterodimers in breast carcinoma cell lines using Nikon's SIM (N-SIM). ERα/α homodimers were detected in the nuclei of both ERα-positive MCF-7 and T-47D cells; 23.0% and 13.4% of ERα proteins formed ERα/α homodimers, respectively. ERα/β heterodimers were also detected in MCF-7 and T-47D. Approximately 6.6% of both ERα and ERβ1 proteins formed ERα/β1 heterodimers in MCF-7. In addition, 18.1% and 22.4% of ERα and ERβ proteins formed ERα/β2 heterodimers and ERα/β5 heterodimers in MCF-7, respectively. In addition, by using proximity ligation assay (PLA) in MCF-7, estradiol-induced ERα/α homodimers and ERα/β1 heterodimers were both detected after 15 to 45 min of treatment and at 15 min, respectively. The percentage of total ER proteins could also be determined using N-SIM. By using both methods, it has become possible to evaluate precise localization and ratio of ER dimers among different cell types.
雌激素受体（ER）作为二聚体发挥作用，并参与多种不同的生物学功能。然而，尚未通过相关方法鉴定出ER二聚体蛋白。结构化照明显微镜（SIM）最近已被开发出来，它能够实现蛋白质的定位以及蛋白质相互作用的研究。因此，在本研究中，我们首先使用尼康的SIM（N-SIM）证明了ERs在乳腺癌细胞系中形成了同二聚体和异二聚体。在ERα阳性的MCF-7和T-47D细胞的细胞核中均检测到了ERα/α同二聚体；分别有23.0%和13.4%的ERα蛋白形成了ERα/α同二聚体。在MCF-7和T-47D中也检测到了ERα/β异二聚体。在MCF-7中，约6.6%的ERα和ERβ1蛋白形成了ERα/β1异二聚体。此外，在MCF-7中，分别有18.1%和22.4%的ERα和ERβ蛋白形成了ERα/β2异二聚体和ERα/β5异二聚体。此外，通过在MCF-7中使用邻近连接分析（PLA），在处理15至45分钟后以及在15分钟时分别检测到了雌二醇诱导的ERα/α同二聚体和ERα/β1异二聚体。总ER蛋白的百分比也可以使用N-SIM来确定。通过使用这两种方法，已能够评估不同细胞类型中ER二聚体的精确定位和比例。
