Selective Mutations in Estrogen Receptor αD-domain Alters Nuclear Translocation and Non-estrogen Response Element Gene Regulatory Mechanisms
Selective Mutations in Estrogen Receptor αD-domain Alters Nuclear Translocation and Non-estrogen Response Element Gene Regulatory Mechanisms
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DOI：
10.1074/jbc.m110.187773
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发表时间：
2011-04-08
影响因子：
4.8
通讯作者：
Korach, Kenneth S.
中科院分区：
文献类型：
--
作者：
Burns, Katherine A.;Li, Yin;Korach, Kenneth S.
ERα作用的三种主要机制是：1)核、基因组、直接DNA结合，2)核、基因组、“拴系”介导的蛋白质-蛋白质相互作用，以及3)非核、非基因组、快速反应。研究表明，ERα的D结构域或铰链区在上述机制1和2中起重要作用。证明ERα铰链区功能的研究已经切除了完整的D-结构域；因此，进行了定点突变，以将精确的序列功能归因于该结构域。本研究重点研究了三个新的ERα-D结构域定点定向突变体的特性和性质。共聚焦显微镜、报告分析、内源性基因表达和免疫共沉淀法表明，铰链1(H1)ERα突变体破坏了核定位，不能再执行拴系介导的反应，并失去了与c-jun的相互作用，但保留了雌激素反应元件(ERE)介导的功能。H2 ERα突变体是非核的，但在雌二醇(E-2)处理下移位到核，并维持ERE介导的功能。H2+NES ERα突变体不再与激素结合维持核易位，不再激活ERE靶基因，在ERE或拴系介导的荧光素酶分析中发挥功能，但保留了非基因组、非核、快速反应。这些研究揭示了ERα铰链区的序列(S)，它参与了拴系介导的作用以及核定位，并将重要的功能归因于该受体的这一区域。此外，这些ERα突变体的特性将使未来的研究进一步剖析和表征ERα的三个主要作用机制，并确定每个作用在雌激素激素调节中的机制作用。
The three main mechanisms of ER alpha 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 ER alpha plays an important role in mechanisms 1 and 2 above. Studies demonstrating the functionality of the ER alpha 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 ER alpha-D-domain mutants. The Hinge 1 (H1) ER alpha 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 ER alpha mutant is non-nuclear, but translocates to the nucleus with estradiol (E-2) treatment and maintains ERE-mediated functionality. The H2 + NES ER alpha 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 ER alpha 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 ER alpha mutants will allow future studies to further dissect and characterize the three main ER alpha mechanisms of action and determine the mechanistic role each action has in estrogen hormone regulation.