Receptor Biology, Laboratory of Reproductive and Developmental Toxicology, Protein Expression Core Facility, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC 27709, USA.
J Biol Chem. 2011 Apr 8;286(14):12640-9. doi: 10.1074/jbc.M110.187773. Epub 2011 Feb 1.
The three main mechanisms of ERα action are: 1) nuclear, genomic, direct DNA binding, 2) nuclear, genomic, "tethered"-mediated, protein-protein interactions, and 3) non-nuclear, non-genomic, rapid action responses. Reports suggest the D-domain or hinge region of ERα plays an important role in mechanisms 1 and 2 above. Studies demonstrating the functionality of the ERα hinge region have resected the full D-domain; therefore, site directed mutations were made to attribute precise sequence functionality to this domain. This study focuses on the characterization and properties of three novel site directed ERα- D-domain mutants. The Hinge 1 (H1) ERα mutant has disrupted nuclear localization, can no longer perform tethered mediated responses and has lost interaction with c-Jun, but retains estrogen response element (ERE)-mediated functions as demonstrated by confocal microscopy, reporter assays, endogenous gene expression and co-immunoprecipitation. The H2 ERα mutant is non-nuclear, but translocates to the nucleus with estradiol (E2) treatment and maintains ERE-mediated functionality. The H2+NES ERα mutant does not maintain nuclear translocation with hormone binding, no longer activates ERE-target genes, functions in ERE- or tethered-mediated luciferase assays, but does retain the non-genomic, non-nuclear, rapid action response. These studies reveal the sequence(s) in the ERα hinge region that are involved in tethered-mediated actions as well as nuclear localization and attribute important functionality to this region of the receptor. In addition, the properties of these ERα mutants will allow future studies to further dissect and characterize the three main ERα mechanisms of action and determine the mechanistic role each action has in estrogen hormone regulation.
雌激素受体 α 的三种主要作用机制为：1）核内，基因组，直接 DNA 结合；2）核内，基因组，“连接”介导的蛋白-蛋白相互作用；3）非核内，非基因组，快速作用反应。有报道称，雌激素受体 α 的 D 结构域或铰链区在上述机制 1 和 2 中发挥重要作用。证明雌激素受体 α 铰链区功能的研究切除了完整的 D 结构域；因此，对该区域进行了定点突变，以确定其精确的序列功能。本研究集中于三种新型定点雌激素受体 α-D 结构域突变体的特性和性质。铰链 1（H1）雌激素受体 α 突变体核定位被破坏，不能再进行连接介导的反应，与 c-Jun 的相互作用丧失，但保留了雌激素反应元件（ERE）介导的功能，如共聚焦显微镜、报告基因检测、内源性基因表达和共免疫沉淀所示。H2 雌激素受体 α 突变体是非核内的，但在雌二醇（E2）处理时可转移到核内，并保持 ERE 介导的功能。H2+NES 雌激素受体 α 突变体与激素结合时不保持核易位，不再激活 ERE 靶基因，在 ERE 或连接介导的荧光素酶检测中不起作用，但仍保留非基因组、非核内、快速作用反应。这些研究揭示了雌激素受体 α 铰链区中涉及连接介导作用以及核定位的序列，并为该受体区域赋予了重要功能。此外，这些雌激素受体 α 突变体的特性将允许未来的研究进一步剖析和表征雌激素受体 α 的三种主要作用机制，并确定每种作用在雌激素激素调节中的机制作用。
