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• W2077014402 abstract "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 β1 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 β1 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. 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 β1 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 β1 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. Protein transport into and out of the nucleus occurs through nuclear pore complexes embedded in the nuclear envelope. Import and export of proteins through the nuclear pore complexes are mediated by karyopherins, which bind to a nuclear localization signal (NLS) 3The abbreviations used are: NLSnuclear localization signalTRthyroid hormone receptorTRα1thyroid hormone receptor α1TRβ1thyroid hormone receptor β1NESnuclear export signalLMBleptomycin BT3thyroid hormoneDBDDNA-binding domainLBDligand-binding domainGRglucocorticoid receptorARandrogen receptorG3GFP-GST-GFP vectorCRM1chromosome region maintenance 1RTHresistance to thyroid hormoneNnuclearCcytosolicTREthyroid hormone-responsive element. or nuclear export signal (NES) present in the cargo protein. The most well studied NLS sequences are the classical monopartite and bipartite NLS motifs, exemplified by the simian virus 40 (SV40) large T-antigen NLS (PKKKRKV) and nucleoplasmin (KRPAATKKAGQAKKKK), respectively (1McLane L.M. Corbett A.H. Nuclear localization signals and human disease.IUBMB Life. 2009; 61: 697-706Crossref PubMed Scopus (80) Google Scholar). Other nonclassical NLS motifs have been defined, and it is likely that many more remain uncharacterized. NES motifs have remained difficult to predict, with only the prototypical chromosome region maintenance 1 (CRM1)-dependent leucine-rich NES sequence being well characterized (2Kutay U. Güttinger S. Leucine-rich nuclear export signals. Born to be weak.Trends Cell Biol. 2005; 15: 121-124Abstract Full Text Full Text PDF PubMed Scopus (277) Google Scholar). In recent years the importance of regulated nucleocytoplasmic transport in gene regulation has become apparent, and there is strong interest in identifying novel NLS and NES motifs (3Umemoto T. Fujiki Y. Ligand-dependent nucleo-cytoplasmic shuttling of peroxisome proliferator-activated receptors, PPARα and PPARγ.Genes Cells. 2012; 17: 576-596Crossref PubMed Scopus (60) Google Scholar, 4Dopie J. Skarp K.P. Rajakylä E.K. Tanhuanpää K. Vartiainen M.K. Active maintenance of nuclear actin by importin 9 supports transcription.Proc. Natl. Acad. Sci. U.S.A. 2012; 109: E544-E552Crossref PubMed Scopus (162) Google Scholar, 5Vandevyver S. Dejager L. Libert C. On the trail of the glucocorticoid receptor. Into the nucleus and back.Traffic. 2012; 13: 364-374Crossref PubMed Scopus (146) Google Scholar). nuclear localization signal thyroid hormone receptor thyroid hormone receptor α1 thyroid hormone receptor β1 nuclear export signal leptomycin B thyroid hormone DNA-binding domain ligand-binding domain glucocorticoid receptor androgen receptor GFP-GST-GFP vector chromosome region maintenance 1 resistance to thyroid hormone nuclear cytosolic thyroid hormone-responsive element. Our interest for many years has been in the mechanisms regulating the nuclear import and export of thyroid hormone receptor α1 (TRα1 and NR1A1a), a member of the nuclear receptor superfamily. Thyroid hormone receptors (TRs) are encoded by two genes, one for TRα and another for TRβ, which encode the major isoforms of TR, including TRα1, TRα2, TRβ1, and TRβ2. Both TRα1 and TRβ1 shuttle between the nucleus and cytoplasm; however, they localize primarily to the nucleus at steady state (6Bunn C.F. Neidig J.A. Freidinger K.E. Stankiewicz T.A. Weaver B.S. McGrew J. Allison L.A. Nucleocytoplasmic shuttling of the thyroid hormone receptor α.Mol. Endocrinol. 2001; 15: 512-533Crossref PubMed Scopus (66) Google Scholar), where they either activate or repress target gene expression in response to thyroid hormone (7Zhang J. Lazar M.A. The mechanism of action of thyroid hormones.Annu. Rev. Physiol. 2000; 62: 439-466Crossref PubMed Scopus (575) Google Scholar, 8Lazar M.A. Thyroid hormone action. A binding contract.J. Clin. Invest. 2003; 112: 497-499Crossref PubMed Scopus (116) Google Scholar). Nuclear localization is critical for the gene regulatory function of TR. For example, mislocalization of TRα1 to the cytoplasm by its oncogenic homolog v-ErbA may contribute to oncogenic conversion of cells (9Bonamy G.M. Allison L.A. Oncogenic conversion of the thyroid hormone receptor by altered nuclear transport.Nucl. Recept. Signal. 2006; 4: e008Crossref PubMed Google Scholar, 10Bonamy G.M. Guiochon-Mantel A. Allison L.A. Cancer promoted by the oncoprotein v-ErbA may be due to subcellular mislocalization of nuclear receptors.Mol. Endocrinol. 2005; 19: 1213-1230Crossref PubMed Scopus (19) Google Scholar). It is clear that a comprehensive understanding of TR trafficking must take into account both nuclear import and export pathways. Nuclear receptors share significant similarity in the DNA-binding domain (DBD), hinge region, and ligand-binding domain (LBD); however, they show variability in their N-terminal A/B domain and in the region C-terminal to the LBD (11Laudet V. Evolution of the nuclear receptor superfamily. Early diversification from an ancestral orphan receptor.J. Mol. Endocrinol. 1997; 19: 207-226Crossref PubMed Scopus (425) Google Scholar). Although an NLS in the hinge domain has been partially characterized for TRs (12Baumann C.T. Maruvada P. Hager G.L. Yen P.M. Nuclear cytoplasmic shuttling by thyroid hormone receptors.J. Biol. Chem. 2001; 276: 11237-11245Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar, 13Casas F. Busson M. Grandemange S. Seyer P. Carazo A. Pessemesse L. Wrutniak-Cabello C. Cabello G. Characterization of a novel thyroid hormone receptor α variant involved in the regulation of myoblast differentiation.Mol. Endocrinol. 2006; 20: 749-763Crossref PubMed Scopus (20) Google Scholar, 14Lee Y. Mahdavi V. The D domain of the thyroid hormone receptor α1 specifies positive and negative transcriptional regulation functions.J. Biol. Chem. 1993; 268: 2021-2028Abstract Full Text PDF PubMed Google Scholar, 15Maruvada P. Baumann C.T. Hager G.L. Yen P.M. Dynamic shuttling and intranuclear mobility of nuclear hormone receptors.J. Biol. Chem. 2003; 278: 12425-12432Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar, 16Zhu X.G. Hanover J.A. Hager G.L. Cheng S.Y. Hormone-induced translocation of thyroid hormone receptors in living cells visualized using a receptor green fluorescent protein chimera.J. Biol. Chem. 1998; 273: 27058-27063Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar), the corresponding complete NLS in the hinge domain of TRα1 had not been fully defined. However, a wealth of information regarding NLS motifs is available from other nuclear receptors. For example, the rat constitutive androstane receptor has a ligand-independent NLS in its hinge region, as well as a ligand-dependent NLS in its LBD (17Kanno Y. Suzuki M. Miyazaki Y. Matsuzaki M. Nakahama T. Kurose K. Sawada J. Inouye Y. Difference in nucleocytoplasmic shuttling sequences of rat and human constitutive active/androstane receptor.Biochim. Biophys. Acta. 2007; 1773: 934-944Crossref PubMed Scopus (28) Google Scholar, 18Kanno Y. Suzuki M. Nakahama T. Inouye Y. Characterization of nuclear localization signals and cytoplasmic retention region in the nuclear receptor CAR.Biochim. Biophys. Acta. 2005; 1745: 215-222Crossref PubMed Scopus (35) Google Scholar), whereas nuclear import of peroxisome proliferator-activated receptor α and γ is mediated by an NLS in the A/B domain and a second NLS that spans the DBD and hinge region (3Umemoto T. Fujiki Y. Ligand-dependent nucleo-cytoplasmic shuttling of peroxisome proliferator-activated receptors, PPARα and PPARγ.Genes Cells. 2012; 17: 576-596Crossref PubMed Scopus (60) Google Scholar). Highlighting the modular nature of nuclear receptors, the glucocorticoid receptor (GR) has a bipartite NLS in its DBD and a ligand-dependent NLS in its LBD (19Picard D. Yamamoto K.R. Two signals mediate hormone-dependent nuclear localization of the glucocorticoid receptor.EMBO J. 1987; 6: 3333-3340Crossref PubMed Scopus (724) Google Scholar, 20Cadepond F. Gasc J.M. Delahaye F. Jibard N. Schweizer-Groyer G. Segard-Maurel I. Evans R. Baulieu E.E. Hormonal regulation of the nuclear localization signals of the human glucocorticosteroid receptor.Exp. Cell Res. 1992; 201: 99-108Crossref PubMed Scopus (63) Google Scholar, 21LaCasse E.C. Lochnan H.A. Walker P. Lefebvre Y.A. Identification of binding proteins for nuclear localization signals of the glucocorticoid and thyroid hormone receptors.Endocrinology. 1993; 132: 1017-1025Crossref PubMed Scopus (21) Google Scholar), and the androgen receptor (AR) has a bipartite NLS in its DBD, a ligand-dependent NLS in its LBD, and putative NLS activity at its N terminus (22Kaku N. Matsuda K. Tsujimura A. Kawata M. Characterization of nuclear import of the domain-specific androgen receptor in association with the importin α/β and Ran-guanosine 5′-triphosphate systems.Endocrinology. 2008; 149: 3960-3969Crossref PubMed Scopus (45) Google Scholar, 23Cutress M.L. Whitaker H.C. Mills I.G. Stewart M. Neal D.E. Structural basis for the nuclear import of the human androgen receptor.J. Cell Sci. 2008; 121: 957-968Crossref PubMed Scopus (175) Google Scholar). In contrast, only one NLS located in the DBD has been identified in the retinoid X receptor (24Prüfer K. Barsony J. Retinoid X receptor dominates the nuclear import and export of the unliganded vitamin D receptor.Mol. Endocrinol. 2002; 16: 1738-1751Crossref PubMed Scopus (128) Google Scholar). Many structural studies of TRα1 have been performed to correlate structural motifs with particular functions of the receptor. For example, the N-terminal A/B domain of nuclear receptors has a random coiled structure (25Kumar R. Thompson E.B. Transactivation functions of the N-terminal domains of nuclear hormone receptors. Protein folding and coactivator interactions.Mol. Endocrinol. 2003; 17: 1-10Crossref PubMed Scopus (151) Google Scholar), and the LBD of TRα1 has 12 α-helices and four β-sheets (26Wagner R.L. Apriletti J.W. McGrath M.E. West B.L. Baxter J.D. Fletterick R.J. A structural role for hormone in the thyroid hormone receptor.Nature. 1995; 378: 690-697Crossref PubMed Scopus (811) Google Scholar). Helix 12, in addition to its role in ligand binding, also has a transactivation function termed AF-2 that is necessary for coactivator binding and release of corepressors upon ligand binding (27Baniahmad A. Leng X. Burris T.P. Tsai S.Y. Tsai M.J. O'Malley B.W. The Tau 4 activation domain of the thyroid hormone receptor is required for release of a putative corepressor(s) necessary for transcriptional silencing.Mol. Cell. Biol. 1995; 15: 76-86Crossref PubMed Google Scholar, 28Barettino D. Vivanco Ruiz M.M. Stunnenberg H.G. Characterization of the ligand-dependent transactivation domain of thyroid hormone receptor.EMBO J. 1994; 13: 3039-3049Crossref PubMed Scopus (291) Google Scholar, 29Danielian P.S. White R. Lees J.A. Parker M.G. Identification of a conserved region required for hormone-dependent transcriptional activation by steroid hormone receptors.EMBO J. 1992; 11: 1025-1033Crossref PubMed Scopus (721) Google Scholar). Similarly, the A/B domain of TR has a transactivation function termed AF-1 (30Tomura H. Lazar J. Phyillaier M. Nikodem V.M. The N-terminal region (A/B) of rat thyroid hormone receptors α1, β1, but not β2 contains a strong thyroid hormone-dependent transactivation function.Proc. Natl. Acad. Sci. U.S.A. 1995; 92: 5600-5604Crossref PubMed Scopus (31) Google Scholar, 31Wilkinson J.R. Towle H.C. Identification and characterization of the AF-1 transactivation domain of thyroid hormone receptor β1.J. Biol. Chem. 1997; 272: 23824-23832Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar, 32Hollenberg A.N. Monden T. Wondisford F.E. Ligand-independent and -dependent functions of thyroid hormone receptor isoforms depend upon their distinct amino termini.J. Biol. Chem. 1995; 270: 14274-14280Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar). However, these previous studies did not address the question of whether known structural features of TRα1 include signals for nuclear import and export. Our prior studies have revealed an increasingly complex picture of TRα1 nuclear export, involving multiple pathways and export factors. We have shown that TRα1 uses both the export factor CRM1 in cooperation with calreticulin for its nuclear export and a second CRM1-independent pathway (33Grespin M.E. Bonamy G.M. Roggero V.R. Cameron N.G. Adam L.E. Atchison A.P. Fratto V.M. Allison L.A. Thyroid hormone receptor α1 follows a cooperative CRM1/calreticulin-mediated nuclear export pathway.J. Biol. Chem. 2008; 283: 25576-25588Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar). However, this study did not address the specific sequence determinants involved in nuclear export of TRα1. CRM1 mediates the nuclear export of proteins with leucine-rich NES sequences (34Cook A. Bono F. Jinek M. Conti E. Structural biology of nucleocytoplasmic transport.Annu. Rev. Biochem. 2007; 76: 647-671Crossref PubMed Scopus (413) Google Scholar). Even though some members of the nuclear receptor family have CRM1-independent NES sequences, it is not known what other karyopherins are involved in these alternative export pathways (35Pemberton L.F. Paschal B.M. Mechanisms of receptor-mediated nuclear import and nuclear export.Traffic. 2005; 6: 187-198Crossref PubMed Scopus (571) Google Scholar). There is some limited information available on the protein cargos exported by these other karyopherins (36Tran E.J. Bolger T.A. Wente S.R. SnapShot. Nuclear transport.Cell. 2007; 131: 420Abstract Full Text PDF PubMed Scopus (44) Google Scholar, 37Fried H. Kutay U. Nucleocytoplasmic transport. Taking an inventory.Cell. Mol. Life Sci. 2003; 60: 1659-1688Crossref PubMed Scopus (405) Google Scholar); however, there is limited understanding of the sequence determinants for the export of proteins that use a CRM1-independent pathway. In this report we have identified a novel NLS, designated NLS-2, in the A/B domain of TRα1 that is absent in TRβ1, and a novel, conserved CRM1-independent NES in the AF-2 region of helix 12 in the TRα1 LBD, designated NES-H12. Mutations in this NES markedly reduce both nuclear export and transactivation of thyroid hormone-mediated gene expression. We also provide evidence for additional NES activity in the region spanning helix 3 (NES-H3) and helix 6 (NES-H6) of the TRα1 LBD. The NLS and NES motifs were shown to be sufficient to target a cytosolic protein to the nucleus or a nuclear protein to the cytosol, respectively. In addition, NLS-2 and NES-H12 were shown to be necessary for efficient import or export in the context of the full-length receptor. Conservation of targeting signals among the vertebrate species suggests that nucleocytoplasmic shuttling is crucial for the normal function of TRα1. Indeed, our mutagenesis studies point to the intriguing possibility that altered shuttling of TRβ1, due to defective nuclear export, may be a contributing factor in resistance to thyroid hormone syndrome. These findings highlight the complexity of the signals that regulate the shuttling of TR and emphasize the importance of detailed molecular characterization for understanding nuclear receptor function. Rat TRα1 domains, NLS, and NES constructs, chicken TRα1 (cTRα1) A/B domain, and v-ErbA A/B domain (excluding the retroviral Gag sequence) were cloned as PCR products into a GFP-GST-GFP (G3) expression vector (designed by Ghislain Bonamy) as C-terminal tags. Oligonucleotides used for cloning were annealed, digested with the appropriate restriction endonuclease, and purified using a PCR purification kit (Qiagen, Valencia, CA). Digested oligonucleotides were ligated into the G3 vector. A/B domain mutations were made using QuikChange® mutagenesis kit according to the manufacturer's instructions (Agilent Technologies, Santa Clara, CA). Mutagenesis primers were designed using QuikChange® primer design program. GFP-TRα1 and GFP-v-ErbA expression vectors were constructed as described previously (6Bunn C.F. Neidig J.A. Freidinger K.E. Stankiewicz T.A. Weaver B.S. McGrew J. Allison L.A. Nucleocytoplasmic shuttling of the thyroid hormone receptor α.Mol. Endocrinol. 2001; 15: 512-533Crossref PubMed Scopus (66) Google Scholar). The expression vector for GFP-tagged human TRβ1 (accession number BC106929), cloned into pReceiver-M29 vector, was obtained from Capital Biosciences (Rockville, MD). PCR primers and short oligonucleotides used in cloning were synthesized by Integrated DNA Technology (Coralville, IA). A list of primer pairs along with the cloning sites and the source vector used in this study are presented in supplemental Table S1. Sequences of individual primers and oligonucleotides are listed in supplemental Table S2. HeLa cells (ATCC CCL-2) were cultured at 37 °C and 5% CO2 in minimum Eagle's medium supplemented with 10% FBS, 100 units/ml penicillin, and 100 μg/ml streptomycin (Invitrogen). For transfection, 7.5–8.0 × 105 cells were plated on 40-mm coverslips (Bioptech, Butler, PA) in 60-mm plates. The next day cells were transfected with 1.5 μg of plasmid DNA using Lipofectamine 2000, according to the manufacturer's instructions (Invitrogen). Twenty four hours post-transfection, cells were analyzed by live cell imaging. For ligand-dependent nuclear translocation experiments, cells were cultured in medium supplemented with charcoal dextran-stripped FBS. For FRAP analysis (see below), 4.5 × 105 HeLa cells were seeded in 35-mm glass bottom dishes (Mattek, Ashland, MA), followed by transfection with 0.75 μg of GFP-tagged TRα1 or TRβ1 expression plasmids and 1.5 μl of Lipofectamine 2000. In addition, cells were treated with 200 ng of cytochalasin D (Sigma) for 16 h (either pre-or post-transfection) to promote nuclear division in the absence of cytokinesis. Subsequently, cells were washed in minimum Eagle's medium to remove residual cytochalasin D for at least 23 h prior to FRAP analysis. Cells were placed in a Focht Chamber System 2 (FCS2 ®, Bioptech) and maintained at 37 °C. MEM without supplements or phenol red was preincubated overnight at 37 °C and 5% CO2, prior to use in live cell imaging experiments. Medium was replaced once per h for experiments lasting more than 1 h. Where indicated, medium contained 9.3 nm LMB (Sigma) and 100 nm T3 (Sigma) to monitor CRM1-dependent nuclear export and ligand-dependent nuclear import. Images were captured using a Nikon Plan apo-60×/1.20 water immersion objective and a CoolSnap HQ2 CCD camera (Photometrics, Tucson, AZ). GFP was visualized using Blue Excitation, B-2E/C filter block (Nikon). Image capturing and analysis were carried out using Nikon NIS-elements Version 3.1. For time lapse imaging experiments, images were captured at 3-min intervals. The intracellular distribution patterns of NES and NLS constructs were visualized by GFP fluorescence in live cells, as described above. NLS constructs were scored in replicate experiments as primarily nuclear (N), nuclear accumulation and a clearly visible cytosolic population (N > C), whole cell (N ≤ C), and primarily cytosolic (C). The efficiency of nuclear import was measured as an increase in the number of cells with a primarily nuclear distribution of the NLS construct. NES constructs were scored in the same categories, except cells scored as N > C and N ≤ C were combined into one category (C). The efficiency of nuclear export was measured as an increase in the number of cells with a cytosolic localization of the NES constructs. Mutations that inhibit nuclear export showed an increase in nuclear retention. The kinetics of shuttling of NES-H12 mutants and wild-type TRα1 and TRβ1 were carried out using FRAP analysis. All FRAP experiments were performed in an Okolab incubation system (Okolab, Italy) for a Nikon Ti-E PFS-A1 confocal system at 37 °C under 5% CO2 and 37% humidity. Imaging was carried out using an Apo LWD 40× WIS DIC N2 objective. One of the two nuclei in a single multinucleated cell was photobleached at 45% laser power for 17 s using the 488 nm line. Fluorescence recovery of the bleached nucleus was monitored every 2 min for 16 min and every 5 min for another 50 min. Protein synthesis was inhibited by 25 μg/ml cycloheximide during the imaging period. Cell plasma membranes were visualized with CellMaskTM deep red plasma membrane stain as per the manufacturer's instruction (Invitrogen). This labeling was used to confirm the presence of more than one nucleus in a single cell. For quantitative analysis of digitized images, fluorescent intensity values were generated using NIS-Elements AR (Nikon). Data were expressed as percent intensity of recovery. For TRβ1, time 0 was set as 2 min, to minimize the contribution of the small cytosolic population (≤ 15%) toward recovery rates. The recovery rate of mutant versus wild-type proteins was plotted for GFP-TRα1 and GFP-TRβ1. HeLa cells were plated at 6.0–7.0 × 105 cells in 100-mm dishes and transiently transfected for 6–7 h with 5 μg of tk-TREp-CAT reporter plasmid containing a synthetic palindromic thyroid hormone-responsive element (TRE), and 5 μg of GFP-TRα1, GFP-TRβ1, or GFP-TRα1 mutant expression plasmids (GFP-TRα1 F401A, GFP-TRα1 F401P, GFP-TRα1 R26H), or 5 μg of empty vector (pCAT®3-Basic Vector). Medium was replaced 12 h post-transfection with MEM containing 10% charcoal-dextran stripped FBS (Invitrogen) supplemented with or without 100 nm T3 (Sigma). After 12 h, cells were lysed, cell extracts prepared, and extracts were used to determine CAT expression levels by ELISA according to the manufacturer's specifications (Roche Applied Science). Protein concentration was determined by Nano Drop (ND-1000 Spectrophotometer) and adjusted to the same amount of total protein (600 μg). For each assay, a standard curve utilizing four pure protein standards was prepared, to ensure that CAT concentrations of sample extracts fell within the linear range of the assay. Replicate samples were assayed in each microplate. Prior studies suggest that the hinge region of both TRα1 and TRβ1 contains a classical NLS (13Casas F. Busson M. Grandemange S. Seyer P. Carazo A. Pessemesse L. Wrutniak-Cabello C. Cabello G. Characterization of a novel thyroid hormone receptor α variant involved in the regulation of myoblast differentiation.Mol. Endocrinol. 2006; 20: 749-763Crossref PubMed Scopus (20) Google Scholar, 14Lee Y. Mahdavi V. The D domain of the thyroid hormone receptor α1 specifies positive and negative transcriptional regulation functions.J. Biol. Chem. 1993; 268: 2021-2028Abstract Full Text PDF PubMed Google Scholar, 16Zhu X.G. Hanover J.A. Hager G.L. Cheng S.Y. Hormone-induced translocation of thyroid hormone receptors in living cells visualized using a receptor green fluorescent protein chimera.J. Biol. Chem. 1998; 273: 27058-27063Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar). Although partially characterized for TRβ1 (14Lee Y. Mahdavi V. The D domain of the thyroid hormone receptor α1 specifies positive and negative transcriptional regulation functions.J. Biol. Chem. 1993; 268: 2021-2028Abstract Full Text PDF PubMed Google Scholar, 16Zhu X.G. Hanover J.A. Hager G.L. Cheng S.Y. Hormone-induced translocation of thyroid hormone receptors in living cells visualized using a receptor green fluorescent protein chimera.J. Biol. Chem. 1998; 273: 27058-27063Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar), the NLS in the hinge domain of TRα1 has not been fully defined. To begin to map the hinge NLS in rat TRα1, we cloned the hinge region into a G3 vector for expression as C-terminal fusion protein with the G3 tag (Fig. 1A). The G3-Hinge fusion protein has a number of important properties; it can be tracked in real time in transiently transfected cells; it is too large for diffusion through the nuclear pore complex, and it serves the purpose of demonstrating that a putative NLS is sufficient to target G3, a cytosolic protein, to the nucleus. The expression vector for G3-Hinge was transfected into HeLa cells, and the fusion protein was analyzed for its ability to localize to the nucleus or cytosol, in comparison with GFP-tagged full-length TRα1 and G3 alone (Fig. 1B). GFP-TRα1 has a predominantly nuclear localization, although G3 is entirely cytosolic. As expected, the hinge domain was sufficient to localize G3 to the nucleus. Based on earlier predictions for the hinge region NLS of TRα1 (38Robbins J. Dilworth S.M. Laskey R.A. Dingwall C. Two interdependent basic domains in nucleoplasmin nuclear targeting sequence. Identification of a class of bipartite nuclear targeting sequence.Cell. 1991; 64: 615-623Abstract Full Text PDF PubMed Scopus (1247) Google Scholar), a minimal sequence was cloned into the G3 vector. This minimal sequence of the hinge region, designated NLS-1 (KRVAKRKLIEQNRERRRK), was sufficient to target the G3 fusion protein to the nucleus (Fig. 1B). This bipartite NLS is well conserved in both the oncoprotein v-ErbA and TRβ1 (Fig. 1C). Earlier studies had shown that the hinge region of TRα1 only partially targeted a cytosolic protein to the nucleus (14Lee Y. Mahdavi V. The D domain of the thyroid hormone receptor α1 specifies positive and negative transcriptional regulation functions.J. Biol. Chem. 1993; 268: 2021-2028Abstract Full Text PDF PubMed Google Scholar, 39Dang C.V. Lee W.M. Nuclear and nucleolar targeting sequences of c-Erb-A, c-Myb, N-Myc, p53, HSP70, and HIV Tat proteins.J. Biol. Chem. 1989; 264: 18019-18023Abstract Full Text PDF PubMed Google Scholar), thus pointing to the possibility of a second NLS within TRα1. When we used the program PSORT II (40Nakai K. Horton P. PSORT. A program for detecting sorting signals in proteins and predicting their subcellular localization.Trends Biochem. Sci. 1999; 24: 34-36Abstract Full Text Full Text PDF PubMed Scopus (1828) Google Scholar) to search for NLS motifs, a potential NLS was identified in the TRα1 A/B domain. To determine whether TRα1 does indeed have a second NLS apart from NLS1 in the hinge domain, a G3-tagged TRα1 A/B domain fusion protein was examined for its intracellular distribution (Fig. 2A). In support of our predictions, the A/B domain construct localized to the nucleus, indicating that the A/B domain has an NLS sufficient to direct G3, a cytosolic protein, to the nucleus (Fig. 2B). To further characterize the novel NLS in the A/B domain of TRα1, we sought to determine the minimal sequence that confers NLS" @default.
• W2077014402 created "2016-06-24" @default.
• W2077014402 creator A5008977374 @default.
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• W2077014402 date "2012-09-01" @default.
• W2077014402 modified "2023-10-15" @default.
• W2077014402 title "Multiple Novel Signals Mediate Thyroid Hormone Receptor Nuclear Import and Export" @default.
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