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

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 (E 2 ) treatment and maintains ERE-mediated functionality. The H2؉NES ER␣ mutant does not maintain nuclear translocation with hormone binding, no longer activates EREtarget 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.Many of the biological effects of estrogen are mediated through the estrogen receptors (ERs), 2 ER␣ and ER␤, which belong to the nuclear receptor superfamily (1). Focusing on ligand-dependent activation, to date, there are three main mechanisms of action for ER␣ that include 1) nuclear, genomic, direct DNA binding, 2) nuclear, genomic, "tethered"-mediated protein-protein interactions, and 3) non-nuclear, non-genomic, rapid action responses (2-8). Mechanism 1 involves liganded ER␣ bound to estrogen response elements (EREs) of target genes to mediate changes in gene expression via the "classical" ER␣ DNA binding responses (4, 9). The 2nd mechanism involves the recruitment and interaction of ER␣ which "tethers" to other transcription factors, such as c-Jun and Sp1, forming a protein-protein complex that interacts directly with the AP-1 and Sp1 DNA response elements, respectively (7, 10, 11). Lastly, the 3rd mechanism, involves a small population of non-nuclear ER␣ that mediates rapid signaling events which include cellular calcium mobilization, nitric oxide synthesis, and activation of intracellular signaling cascades such as those involving MAPK/ERK, Src, or Akt (5, 12).ER␣, as with all nuclear receptors, maintains the classical domain demarcations. Each domain ...

Estrogen Receptor β (ERβ1) Transactivation Is Differentially Modulated by the Transcriptional Coregulator Tip60 in a cis-Acting Element-dependent Manner

“…ER␣ also binds to PGC-1 at its hinge domain in a ligand-independent manner (52). Although the hinge domain of ERs is not as well characterized, it has been shown to affect protein degradation and activity of ER␤1 (53,54), ER␣ tethered-mediated AP-1 transactivation (55), and the functional synergy between AF-1 and AF-2 of ERs (56). Because AF-1 and AF-2 domains are responsible for E 2 -independent and E 2 -dependent activation of the transactivation of ERs (50), we speculate that the atypical interaction interface between ER␤1 and Tip60 at the hinge domain may contribute to the unique regulation of ER␤1 activity by Tip60.…”

IQGAP1 Binds to Estrogen Receptor-α and Modulates Its Function

“…Initially believed to serve exclusively as a flexible linker between the DBD and ligand-binding domain, evidence accumulated over the last decade has identified important functional roles for the hinge region of ER␣. For example, a recent study reveals that selective mutations in the D domain of ER␣ alter its nuclear translocation and its ability to activate its target genes activator protein-1, pS2, and specific protein-1 (43). One of the mutant ER␣ constructs generated in that study can translocate to the nucleus and bind to DNA in the presence of E 2 but is unable to interact with the necessary tethering factors to activate activator protein-1 and specific protein-1 response elements.…”

Steroid Receptors in the Uterus and Ovary

“…This form of ER did not mediate transcriptional responses but maintained estrogen-induced MAPK phosphorylation. 94 Targeting steroid receptors to the membrane involves palmitoylation, which is facilitated by HSP27. 88 The palmitoylation promotes interaction with caveolin-1, which then results in localization of the receptor in membrane caveolin rafts.…”