Human estrogen receptor beta-specific monoclonal antibodies: characterization and use in studies of estrogen receptor beta protein expression in reproductive tissues
Human estrogen receptor beta-specific monoclonal antibodies: characterization and use in studies of estrogen receptor beta protein expression in reproductive tissues
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DOI：
10.1016/s0303-7207(01)00492-0
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发表时间：
2001-07-05
影响因子：
4.1
通讯作者：
Katzenellenbogen, BS
中科院分区：
文献类型：
--
作者：
Choi, I;Ko, C;Katzenellenbogen, BS
研究第二种最近被描述的雌激素受体（表示为ERP）的作用对于理解雌激素对组织特异性基因调控的分子机制是至关重要的。ERP在多种组织中的表达主要在mRNA水平上进行检测，并且关于内源性ER β蛋白的细胞定位和大小的信息很少，部分原因是人ER β特异性抗体的可用性有限。因此，我们的目的是产生针对人ER β的特异性抗体，并使用它们来确定ER β蛋白的组织特异性分布和大小。为此，我们克隆了三种不同的杂交瘤细胞系，其产生针对人ER β的结合结构域的特异性单克隆抗体。在小鼠中制备的抗人ER β氨基酸256-505（缺乏F结构域的激素结合结构域）的抗体命名为CFK-E12（E12）、CMK-A9（A9）和CWK-F12（1712），并确定E12为IgG γ 1同种型，A9和F12为IgG γ 2b同种型。Western blot分析表明，三种单克隆抗体均能检测到体外翻译、杆状病毒表达和细胞转染表达的ER β蛋白，但均不能检测到ER α。A9和F12能够在雌二醇存在和不存在的情况下有效地免疫沉淀天然形式的ER β蛋白。表位作图研究表明，E12和F12抗体识别重叠的肽序列的N-末端区域的蛋白结合域，该区域是高度保守的物种。这些抗体的免疫细胞化学研究揭示了大鼠卵巢颗粒细胞中ER β蛋白的核特异性定位。核ER β也特异性地定位于小鼠和大鼠附睾的上皮细胞和一些基质细胞中。从人、大鼠、小鼠和猪的卵巢颗粒细胞中提取蛋白质，进行蛋白质印迹分析，结果表明，52 kDa和一个额外的ca。62-64 kDa条带。这些结果表明，在卵巢颗粒细胞中存在两种主要分子大小形式的ER β蛋白，并证明了这些抗体用于检测人类和其他几种哺乳动物物种中的ER β的实用性。(C)2001爱思唯尔科学爱尔兰有限公司保留所有权利。
investigation of the role of the second, more recently described estrogen receptor, denoted ERP, will be critical in understanding the Molecular mechanisms underlying tissue-specific gene regulation by estrogens. Expression of ERP in a variety of tissues has been examined predominantly at the mRNA level, and there is little information regarding the cellular localization and size of the endogenous ER beta protein, due, in part, to the limited availability of human ER beta -specific antibodies. Thus, our aim was to generate specific antibodies to human ER beta and use them to determine the tissue-specific distribution and size(s) of the ER beta protein, To this end, we have cloned three different hybridoma cell lines that produce monoclonal antibodies specific for the hormone-binding domain of human ER beta. The antibodies, made in mice against human ER beta amino acids 256-505 (hormone binding domain lacking the F domain), are designated CFK-E12 (E12), CMK-A9 (A9) and CWK-F12 (1712) and were determined to be the IgG gamma1 isotype for E12, and IgG gamma 2b for A9 and F12. All three monoclonal antibodies could be used to detect in vitro translated, baculovirus expressed, and cell transfected and expressed ER beta protein by Western blot analyses, and all failed to detect ER alpha. A9 and F12 were able to immunoprecipitate efficiently the native form of ER beta protein in the presence and absence of estradiol. Epitope mapping studies indicate that the E12 and F12 antibodies recognize overlapping peptide sequences in the N-terminal region of the hormone-binding domain, a region that is highly conserved among species. Immunocytochemical studies with these antibodies reveal nuclear-specific localization of the ER beta protein in granulosa cells of the rat ovary. Nuclear ER beta is also specifically localized in epithelial and some stromal cells of mouse and rat epididymis. Western blot analysis with protein extracts from ovarian granulosa cells of human, rat, mouse, and pig showed a ca. 52 kDa and an additional ca. 62-64 kDa band in these species. These results indicate the presence of two predominant molecular size forms of the ER beta protein in ovarian granulosa cells and demonstrate the utility of these antibodies for detection of ER beta in the human and in several other mammalian species. (C) 2001 Elsevier Science Ireland Ltd. All rights reserved.