Multiple exportins influence thyroid hormone receptor localization

Abstract

The thyroid hormone receptor (TR) undergoes nucleocytoplasmic shuttling and regulates target genes involved in metabolism and development. Previously, we showed that TR follows a CRM1/calreticulin-mediated nuclear export pathway. However, two lines of evidence suggest TR also follows another pathway: export is only partially blocked by leptomycin B (LMB), a CRM1-specific inhibitor; and we identified nuclear export signals in TR that are LMB-resistant. To determine whether other exportins are involved in TR shuttling, we used RNA interference and fluorescence recovery after photobleaching shuttling assays in transfected cells. Knockdown of exportins 4, 5, and 7 altered TR shuttling dynamics, and when exportins 5 and 7 were overexpressed, TR distribution shifted towards the cytosol. To further assess the effects of exportin overexpression, we examined transactivation of a TR-responsive reporter gene. Our data indicate that multiple exportins influence TR localization, highlighting a fine balance of nuclear import, retention, and export that modulates TR function.

Nuclear import of the thyroid hormone receptor α1 is mediated by importin 7, importin β1, and adaptor importin α1

“…No role has yet been reported for these karyopherins, or for transportins 1 and 2 (Twyffels et al, 2014), in import of nuclear receptors, so they were considered less likely candidates. In a separate study focused on TRα1 nuclear export pathways, we confirmed that transportins 1 and 2 are not involved in nuclear import (or export) of TRα1 (Subramanian et al, 2015). …”

“…Twenty six hours post-transfection, HeLa cell protein lysates were prepared and analyzed by immunoblotting as described (Subramanian et al, 2015). Antibodies were used with the following concentrations: anti-GAPDH (Santa Cruz Biotechnology Inc, Dallas, TX), 1:5000; anti-importin β1 (Santa Cruz), 1:2000; anti-importin 4 (Santa Cruz), 1:333; anti-importin 5 (Santa Cruz), 1:10,000; anti-importin 7 (Abcam, Cambridge, MA), 1:1000; anti-importin 8 (Abcam), 1:250; anti-importin 9 (Abcam), 1:250; anti-importin 11 (Abcam), 1:333; anti-importin 13 (Santa Cruz), 1:100; anti-importin α1 (Santa Cruz), 1:2000; anti-importin α3 (Thermo Scientific), 0.2 μg/ml; horseradish peroxidase (HRP)-conjugated donkey anti-rabbit IgG (GE Healthcare Life Sciences), 1:25,000; HRP-sheep anti-mouse IgG (GE Healthcare Life Sciences), 1:25,000; or HRP-mouse anti-goat IgG (Santa Cruz Biotechnology), 1:25,000.…”

Mediator subunit MED1 modulates intranuclear dynamics of the thyroid hormone receptor

“…This is particularly intriguing since our prior studies have shown that AF‐2 overlaps with one of TR's nuclear export signals, NES‐H12 (see Figure A). Previously, we used RNA interference‐mediated knockdown to show that exportin 4, 5, and 7 influence TRα1 localization, suggesting these exportins are candidates for mediating TRα1 nuclear export, in combination with the previously characterized CRM1/calreticulin pathway . To determine whether these exportins influence localization via direct protein‐protein interactions, we performed coimmunoprecipitation assays.…”

“…mCherry‐tagged TRα1, TRβ1, and v‐ErbA expression plasmids were prepared by subcloning the corresponding coding regions from GFP‐TRα1, GFP‐v‐ErbA, and GFP‐TRβ1 into pmCherry‐C1 (Clontech). GFP‐GST‐GFP‐Hinge, containing NLS‐1 of TRα1, and GFP‐GST‐GFP‐Hinge‐LBD were previously described .…”

“…[21][22][23] Transcription factors, such as TRα1 and TRβ1, cross the nuclear envelope through the nuclear pore complexes, facilitated by transport proteins called importins and exportins. [24][25][26][27][28][29][30] Although localized primarily to the nucleus at steady-state, TRα1 and TRβ1 shuttle between the nucleus and cytosol, directed by multiple nuclear export signal (NES) and nuclear localization signal (NLS) motifs [31][32][33][34] ( Figure 1A). Along with a NLS in the hinge domain (NLS-1), TRα1 houses a NLS in the A/B domain that is absent in TRβ1 and inactive in the retroviral oncoprotein v-ErbA.…”