Selective mutations in estrogen receptor alpha D-domain alters nuclear translocation and non-estrogen response element gene regulatory mechanisms.
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ABSTRACT: The three main mechanisms of ER? 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? plays an important role in mechanisms 1 and 2 above. Studies demonstrating the functionality of the ER? 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?- D-domain mutants. The Hinge 1 (H1) ER? 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? mutant is non-nuclear, but translocates to the nucleus with estradiol (E2) treatment and maintains ERE-mediated functionality. The H2+NES ER? 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? 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? mutants will allow future studies to further dissect and characterize the three main ER? mechanisms of action and determine the mechanistic role each action has in estrogen hormone regulation.
SUBMITTER: Burns KA
PROVIDER: S-EPMC3069464 | biostudies-literature | 2011 Apr
REPOSITORIES: biostudies-literature
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