Selective Mutations in Estrogen Receptor αD-domain Alters Nuclear Translocation and Non-estrogen Response Element Gene Regulatory Mechanisms

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.