Mediator subunit MED1 differentially modulates mutant thyroid hormone receptor intracellular dynamics in Resistance to Thyroid Hormone syndrome.

Thyroid hormone receptor (TR) controls the expression of thyroid hormone (T3)-responsive genes, while undergoing rapid nucleocytoplasmic shuttling. In Resistance to Thyroid Hormone syndrome (RTH), mutant TR fails to activate T3-dependent transcription. Previously, we showed that Mediator subunit 1 (MED1) plays a role in TR nuclear retention. Here, we investigated MED1's effect on RTH mutants using nucleocytoplasmic scoring and fluorescence recovery after photobleaching in transfected cells. MED1 overexpression and knockout did not change the nucleocytoplasmic distribution or intranuclear mobility of C392X and P398R TRα1 at physiological T3 levels. At elevated T3 levels, however, overexpression increased P398R's nuclear retention and MED1 knockout decreased P398R's and A263V's intranuclear mobility, while not impacting C392X. Although A263V TRα1-transfected cells had a high percentage of aggregates, MED1 rescued A263V's impaired intranuclear mobility, suggesting that MED1 ameliorates nonfunctional aggregates. Results correlate with clinical severity, suggesting that altered interaction between MED1 and TRα1 mutants contributes to RTH pathology.

Mediator subunit MED1 modulates intranuclear dynamics of the thyroid hormone receptor.

The thyroid hormone receptors (TRs) mediate thyroid hormone (T3 )-dependent gene expression. The nuclear import and export signals that direct TR shuttling are well characterized, but little is known about factors modulating nuclear retention. We used fluorescence-based nucleocytoplasmic scoring and fluorescence recovery after photobleaching in transfected cells to investigate whether Mediator subunits MED1 and MED13 play a role in nuclear retention of TR. When MED1 was overexpressed, there was a striking shift towards a greater nuclear localization of TRß1 and the oncoprotein v-ErbA, subtypes with cytosolic populations at steady-state, and TRß1 intranuclear mobility was reduced. For TRα1, there was no observable change in its predominantly nuclear distribution pattern or mobility. Consistent with a role for MED1 in nuclear retention, the cytosolic TRα1 and TRß1 population were significantly greater in MED1-/- cells, compared with MED1+/+ cells. Exposure to T3 and epidermal growth factor, which induces MED1 phosphorylation, also altered TR intranuclear dynamics. Overexpression of miR-208a, which downregulates MED13, led to a more cytosolic distribution of nuclear-localized TRα1; however, overexpression of MED13 had no effect on TRß1 localization. The known binding site of MED1 overlaps with a transactivation domain and nuclear export signal in helix 12 of TR's ligand-binding domain (LBD). Coimmunoprecipitation assays demonstrated that TR's LBD interacts directly with exportins 5 and 7, suggesting that binding of exportins and MED1 to TR may be mutually exclusive. Collectively, our data provide evidence that MED1 promotes nuclear retention of TR, and highlight the dual functionality of helix 12 in TR transactivation and nuclear export.

Mislocalization of Cancer-associated Thyroid Hormone Receptor Mutants.

The thyroid hormone receptor (TR) is essential for the proper regulation of metabolism and development, as it regulates gene expression in response to thyroid hormone. Nuclear localization signals (NLSs) and nuclear export signals (NESs) allow for TR transport into and out of the nucleus, respectively. Previous research suggests that nuclear import, nuclear retention, and nuclear export of TR are associated with modulation of gene expression, the alteration of which can contribute to various diseases. Here, we examined the impact of cancer-associated mutations on TR localization patterns as a way of analyzing key structural components of TR and to further explore the correlation between TR trafficking, misfolding, and disease. Through mammalian cell transfection of expression plasmids for green fluorescent protein (GFP) and mCherry-tagged TRα1 and quantitative fluorescence microscopy, we examined particular groups of TRα1 mutations that were observed in patients with hepatocellular carcinoma, renal cell carcinoma, and thyroid cancer, and are associated with NLSs and NESs of TRα1. We also investigated structural alterations of the mutants by in silico modeling. Our results show striking shifts towards a more cytoplasmic localization for many of the mutants and an increased tendency to form cytosolic and nuclear aggregates.

Acetylation modulates thyroid hormone receptor intracellular localization and intranuclear mobility.

The thyroid hormone receptor (TR) undergoes nucleocytoplasmic shuttling, but is primarily nuclear-localized and mediates expression of genes involved in development and homeostasis. Given the proximity of TR acetylation and sumoylation sites to nuclear localization (NLS) and nuclear export signals, we investigated their role in regulating intracellular localization. The nuclear/cytosolic fluorescence ratio (N/C) of fluorescent protein-tagged acetylation mimic, nonacetylation mimic, and sumoylation-deficient TR was quantified in transfected mammalian cells. While nonacetylation mimic and sumoylation-deficient TRs displayed wild-type N/C, the acetylation mimic's N/C was significantly lower. Importins that interact with wild-type TR also interact with acetylation and nonacetylation mimics, suggesting factors other than reduced importin binding alter nuclear localization. FRAP analysis showed wild-type intranuclear dynamics of acetylation mimic and sumoylation-deficient TRs, whereas the nonacetylation mimic had significantly reduced mobility and transcriptional activity. Acetyltransferase CBP/p300 inhibition enhanced TR's nuclear localization, further suggesting that nonacetylation correlates with nuclear retention, while acetylation promotes cytosolic localization.

Thyroid hormone receptor localization in target tissues.

The thyroid hormone receptors, TRα1, TRß1 and other subtypes, are members of the nuclear receptor superfamily that mediate the action of thyroid hormone signaling in numerous tissues to regulate important physiological and developmental processes. Their most well-characterized role is as ligand-dependent transcription factors; TRs bind thyroid hormone response elements in the presence or absence of thyroid hormone to facilitate the expression of target genes. Although primarily residing in the nucleus, TRα1 and TRß1 shuttle rapidly between the nucleus and cytoplasm. We have identified multiple nuclear localization signals and nuclear export signals within TRα1 and TRß1 that interact with importins and exportins, respectively, to mediate translocation across the nuclear envelope. More recently, enigmatic cytoplasmic functions have been ascribed to other TR subtypes, expanding the diversity of the cellular response to thyroid hormone. By integrating data on localization signal motifs, this review provides an overview of the complex interplay between TR's dynamic transport pathways and thyroid hormone signaling activities. We examine the variation in TR subtype response to thyroid hormone signaling, and what is currently known about regulation of the variety of tissue-specific localization patterns, including targeting to the nucleus, the mitochondria and the inner surface of the plasma membrane.

Nuclear Import and Export of the Thyroid Hormone Receptor.

The thyroid hormone receptors, TRα1 and TRß1, are members of the nuclear receptor superfamily that forms one of the most abundant classes of transcription factors in multicellular organisms. Although primarily localized to the nucleus, TRα1 and TRß1 shuttle rapidly between the nucleus and cytoplasm. The fine balance between nuclear import and export of TRs has emerged as a critical control point for modulating thyroid hormone-responsive gene expression. Mutagenesis studies have defined two nuclear localization signal (NLS) motifs that direct nuclear import of TRα1: NLS-1 in the hinge domain and NLS-2 in the N-terminal A/B domain. Three nuclear export signal (NES) motifs reside in the ligand-binding domain. A combined approach of shRNA-mediated knockdown and coimmunoprecipitation assays revealed that nuclear entry of TRα1 is facilitated by importin 7, likely through interactions with NLS-2, and importin ß1 and the adapter importin α1 interacting with both NLS-1 and NLS-2. Interestingly, TRß1 lacks NLS-2 and nuclear import depends solely on the importin α1/ß1 heterodimer. Heterokaryon and fluorescence recovery after photobleaching shuttling assays identified multiple exportins that play a role in nuclear export of TRα1, including CRM1 (exportin 1), and exportins 4, 5, and 7. Even single amino acid changes in TRs dramatically alter their intracellular distribution patterns. We conclude that mutations within NLS and NES motifs affect nuclear shuttling activity, and propose that TR mislocalization contributes to the development of some types of cancer and Resistance to Thyroid Hormone syndrome.

Nuclear import of the thyroid hormone receptor α1 is mediated by importin 7, importin ß1, and adaptor importin α1.

The thyroid hormone receptor α1 (TRα1) is a nuclear receptor for thyroid hormone that shuttles rapidly between the nucleus and cytoplasm. Our prior studies showed that nuclear import of TRα1 is directed by two nuclear localization signals, one in the N-terminal A/B domain and the other in the hinge domain. Here, we showed using in vitro nuclear import assays that TRα1 nuclear localization is temperature and energy-dependent and can be reconstituted by the addition of cytosol. In HeLa cells expressing green fluorescent protein (GFP)-tagged TRα1, knockdown of importin 7, importin ß1 and importin α1 by RNA interference, or treatment with an importin ß1-specific inhibitor, significantly reduced nuclear localization of TRα1, while knockdown of other importins had no effect. Coimmunoprecipitation assays confirmed that TRα1 interacts with importin 7, as well as importin ß1 and the adapter importin α1, suggesting that TRα1 trafficking into the nucleus is mediated by two distinct pathways.

Multiple exportins influence thyroid hormone receptor localization.

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 toward 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.

Multiple novel signals mediate thyroid hormone receptor nuclear import and export.

Thyroid hormone receptor (TR) is a member of the nuclear receptor superfamily that shuttles between the cytosol and nucleus. The fine balance between nuclear import and export of TR has emerged as a critical control point for modulating thyroid hormone-responsive gene expression; however, sequence motifs of TR that mediate shuttling are not fully defined. Here, we characterized multiple signals that direct TR shuttling. Along with the known nuclear localization signal in the hinge domain, we identified a novel nuclear localization signal in the A/B domain of thyroid hormone receptor α1 that is absent in thyroid hormone receptor ß1 and inactive in the oncoprotein v-ErbA. Our prior studies showed that thyroid hormone receptor α1 exits the nucleus through two pathways, one dependent on the export factor CRM1 and the other CRM1-independent. Here, we identified three novel CRM1-independent nuclear export signal (NES) motifs in the ligand-binding domain as follows: a highly conserved NES in helix 12 (NES-H12) and two additional NES sequences spanning helix 3 and helix 6, respectively. Mutations predicted to disrupt the α-helical structure resulted in a significant decrease in NES-H12 activity. The high degree of conservation of helix 12 suggests that this region may function as a key NES in other nuclear receptors. Furthermore, our mutagenesis studies on NES-H12 suggest that altered shuttling of thyroid hormone receptor ß1 may be a contributing factor in resistance to thyroid hormone syndrome. Taken together, our findings provide a detailed mechanistic understanding of the multiple signals that work together to regulate TR shuttling and transcriptional activity, and they provide important insights into nuclear receptor function in general.

Recruitment of the oncoprotein v-ErbA to aggresomes.

Aggresome formation, a cellular response to misfolded protein aggregates, is linked to cancer and neurodegenerative disorders. Previously we showed that Gag-v-ErbA (v-ErbA), a retroviral variant of the thyroid hormone receptor (TRα1), accumulates in and sequesters TRα1 into cytoplasmic foci. Here, we show that foci represent v-ErbA targeting to aggresomes. v-ErbA colocalizes with aggresomal markers, proteasomes, hsp70, HDAC6, and mitochondria. Foci have hallmark characteristics of aggresomes: formation is microtubule-dependent, accelerated by proteasome inhibitors, and they disrupt intermediate filaments. Proteasome-mediated degradation is critical for clearance of v-ErbA and T(3)-dependent TRα1 clearance. Our studies highlight v-ErbA's complex mode of action: the oncoprotein is highly mobile and trafficks between the nucleus, cytoplasm, and aggresome, carrying out distinct activities within each compartment. Dynamic trafficking to aggresomes contributes to the dominant negative activity of v-ErbA and may be enhanced by the viral Gag sequence. These studies provide insight into novel modes of oncogenesis across multiple cellular compartments.

The pseudophosphatase MK-STYX interacts with G3BP and decreases stress granule formation.

MK-STYX [MAPK (mitogen-activated protein kinase) phospho-serine/threonine/tyrosine-binding protein] is a pseudophosphatase member of the dual-specificity phosphatase subfamily of the PTPs (protein tyrosine phosphatases). MK-STYX is catalytically inactive due to the absence of two amino acids from the signature motif that are essential for phosphatase activity. The nucleophilic cysteine residue and the adjacent histidine residue, which are conserved in all active dual-specificity phosphatases, are replaced by serine and phenylalanine residues respectively in MK-STYX. Mutations to introduce histidine and cysteine residues into the active site of MK-STYX generated an active phosphatase. Using MS, we identified G3BP1 [Ras-GAP (GTPase-activating protein) SH3 (Src homology 3) domain-binding protein-1], a regulator of Ras signalling, as a binding partner of MK-STYX. We observed that G3BP1 bound to native MK-STYX; however, binding to the mutant catalytically active form of MK-STYX was dramatically reduced. G3BP1 is also an RNA-binding protein with endoribonuclease activity that is recruited to 'stress granules' after stress stimuli. Stress granules are large subcellular structures that serve as sites of mRNA sorting, in which untranslated mRNAs accumulate. We have shown that expression of MK-STYX inhibited stress granule formation induced either by aresenite or expression of G3BP itself; however, the catalytically active mutant MK-STYX was impaired in its ability to inhibit G3BP-induced stress granule assembly. These results reveal a novel facet of the function of a member of the PTP family, illustrating a role for MK-STYX in regulating the ability of G3BP1 to integrate changes in growth-factor stimulation and environmental stress with the regulation of protein synthesis.

Thyroid hormone receptor alpha1 follows a cooperative CRM1/calreticulin-mediated nuclear export pathway.

The thyroid hormone receptor alpha1 (TRalpha) exhibits a dual role as an activator or repressor of its target genes in response to thyroid hormone (T(3)). Previously, we have shown that TRalpha, formerly thought to reside solely in the nucleus bound to DNA, actually shuttles rapidly between the nucleus and cytoplasm. An important aspect of the shuttling activity of TRalpha is its ability to exit the nucleus through the nuclear pore complex. TRalpha export is not sensitive to treatment with the CRM1-specific inhibitor leptomycin B (LMB) in heterokaryon assays, suggesting a role for an export receptor other than CRM1. Here, we have used a combined approach of in vivo fluorescence recovery after photobleaching experiments, in vitro permeabilized cell nuclear export assays, and glutathione S-transferase pull-down assays to investigate the export pathway used by TRalpha. We show that, in addition to shuttling in heterokaryons, TRalpha shuttles rapidly in an unfused monokaryon system as well. Furthermore, our data show that TRalpha directly interacts with calreticulin, and point to the intriguing possibility that TRalpha follows a cooperative export pathway in which both calreticulin and CRM1 play a role in facilitating efficient translocation of TRalpha from the nucleus to cytoplasm.