Human estrogen receptor beta-specific monoclonal antibodies: characterization and use in studies of estrogen receptor beta protein expression in reproductive tissues

investigation of the role of the second, more recently described estrogen r eceptor, denoted ERP, will be critical in understanding the Molecular mecha nisms underlying tissue-specific gene regulation by estrogens. Expression o f ERP in a variety of tissues has been examined predominantly at the mRNA l evel, and there is little information regarding the cellular localization a nd size of the endogenous ER beta protein, due, in part, to the limited ava ilability of human ER beta -specific antibodies. Thus, our aim was to gener ate specific antibodies to human ER beta and use them to determine the tiss ue-specific distribution and size(s) of the ER beta protein, To this end, w e have cloned three different hybridoma cell lines that produce monoclonal antibodies specific for the hormone-binding domain of human ER beta. The an tibodies, made in mice against human ER beta amino acids 256-505 (hormone b inding 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 coul d be used to detect in vitro translated, baculovirus expressed, and cell tr ansfected and expressed ER beta protein by Western blot analyses, and all f ailed to detect ER alpha. A9 and F12 were able to immunoprecipitate efficie ntly the native form of ER beta protein in the presence and absence of estr adiol. Epitope mapping studies indicate that the E12 and F12 antibodies rec ognize overlapping peptide sequences in the N-terminal region of the hormon e-binding domain, a region that is highly conserved among species. Immunocy tochemical studies with these antibodies reveal nuclear-specific localizati on of the ER beta protein in granulosa cells of the rat ovary. Nuclear ER b eta 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 a nd an additional ca. 62-64 kDa band in these species. These results indicat e the presence of two predominant molecular size forms of the ER beta prote in in ovarian granulosa cells and demonstrate the utility of these antibodi es for detection of ER beta in the human and in several other mammalian spe cies. (C) 2001 Elsevier Science Ireland Ltd. All rights reserved.