Skip to main content
SpringerLink
Log in

Cloning and identification of a novel thyroid hormone receptor β isoform expressed in the pituitary gland

  • Published:
Molecular and Cellular Biochemistry Aims and scope Submit manuscript

Abstract

We have previously identified a novel Trβ isoform (TrβΔ) in the rat, in which a novel exon N (108 bps) was found between exon 3 and exon 4 of TrβΔ, which represents the only difference between TrβΔ and Trβ1. In this study, we searched for an elongated Trβ2-like subtype with one additional exon N. We successfully isolated the entire mRNA/cDNA of a novel elongated Trβ2 isoform via PCR in the rat pituitary gland. The mRNA/cDNA was only 108 bps (exon N) longer than that Trβ2, and the extension of the sequence was between exon 3 and 4 of Trβ. The whole sequence of this novel Trβ isoform has been published in NCBI GenBank (HM043807.1); it is named TRbeta2Delta (Trβ2Δ). In adult rat pituitary tissue, quantitative real-time RT-PCR analysis showed that the mRNA levels of Trβ2Δ and Trβ2 were roughly equal (P > 0.05). We cloned, expressed, and purified the His-Trβ2Δ protein [recombinant TRβ2Δ (rTRβ2Δ)]. SDS-PAGE and western blotting revealed that the molecular weight of rTRβ2Δ was 58.2 kDa. Using a radioligand binding assay and an electrophoretic mobility shift assay, rTRβ2Δ-bound T3 with high affinity and recognized thyroid hormone response element (TRE) binding sites. Finally, in vitro transfection experiments further confirmed that rTRβ2Δ binding T3 significantly promotes the transcription of target genes via the TRE. Here, we have provided evidence suggesting that rTRβ2Δ is a novel functional TR isoform.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6

Similar content being viewed by others

References

  1. Cordas EA, Ng L, Hernandez A, Kaneshige M, Cheng SY, Forrest D (2012) Thyroid hormone receptors control developmental maturation of the middle ear and the size of the ossicular bones. Endocrinology 153:1548–1560

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  2. Dong H, Paquette M, Williams A, Zoeller RT, Wade M, Yauk C (2010) Thyroid hormone may regulate mRNA abundance in liver by acting on microRNAs. PLoS One 5:e12136

    Article  PubMed Central  PubMed  Google Scholar 

  3. Silvestri E, Schiavo L, Lombardi A, Goglia F (2005) Thyroid hormones as molecular determinants of thermogenesis. Acta Physiol Scand 184:265–283

    Article  CAS  PubMed  Google Scholar 

  4. Cheng SY, Leonard JL, Davis PJ (2010) Molecular aspects of thyroid hormone actions. Endocr Rev 31:139–170

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  5. Gauthier K, Billon C, Bissler M, Beylot M, Lobaccaro JM, Vanacker JM, Samarut J (2010) Thyroid hormone receptor beta (TRbeta) and liver X receptor (LXR) regulate carbohydrate-response element-binding protein (ChREBP) expression in a tissue-selective manner. J Biol Chem 285:28156–28163

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  6. Lin JZ, Sieglaff DH, Yuan C, Su J, Arumanayagam AS, Firouzbakht S, Cantu Pompa JJ, Reynolds FD, Zhou X, Cvoro A, Webb P (2013) Gene specific actions of thyroid hormone receptor subtypes. PLoS One 8:e52407

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  7. Rebaï M, Kallel I, Rebaï A (2012) Genetic features of thyroid hormone receptors. J Genet 91:367–374

    Article  PubMed  Google Scholar 

  8. Yen PM, Ando S, Feng X, Liu Y, Maruvada P, Xia X (2006) Thyroid hormone action at the cellular, genomic and target gene levels. Mol Cell Endocrinol 246:121–127

    Article  CAS  PubMed  Google Scholar 

  9. Chan IH, Privalsky ML (2009) Isoform-specific transcriptional activity of overlapping target genes that respond to thyroid hormone receptors alpha1 and beta1. Mol Endocrinol 23:1758–1775

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  10. Mavinakere MS, Powers JM, Subramanian KS, Roggero VR, Allison LA (2012) Multiple novel signals mediate thyroid hormone receptor nuclear import and export. J Biol Chem 287:31280–31297

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  11. Williams GR (2000) Cloning and characterization of two novel thyroid hormone receptor beta isoforms. Mol Cell Biol 20:8329–8342

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  12. Shupnik MA (2000) Thyroid hormone suppression of pituitary hormone gene expression. Rev Endocr Metab Disord 1:35–42

    Article  CAS  PubMed  Google Scholar 

  13. Tian H, Mahajan MA, Wong CT, Habeos I, Samuels HH (2006) The N-terminal A/B domain of the thyroid hormone receptor-beta2 isoform influences ligand-dependent recruitment of coactivators to the ligand-binding domain. Mol Endocrinol 20:2036–2051

    Article  CAS  PubMed  Google Scholar 

  14. Chen Y, Young MA (2010) Structure of a thyroid hormone receptor DNA-binding domain homodimer bound to an inverted palindrome DNA response element. Mol Endocrinol 24:1650–1664

    Article  CAS  PubMed  Google Scholar 

  15. Figueira AC, Lima LM, Lima LH, Ranzani AT, Mule Gdos S, Polikarpov I (2010) Recognition by the thyroid hormone receptor of canonical DNA response elements. Biochemistry 49:893–904

    Article  CAS  PubMed  Google Scholar 

  16. Harvey CB, Bassett JH, Maruvada P, Yen PM, Williams GR (2007) The rat thyroid hormone receptor (TR) Deltabeta3 displays cell-, TR isoform-, and thyroid hormone response element-specific actions. Endocrinology 148:1764–1773

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  17. Yen PM, Sunday ME, Darling DS, Chin WW (1992) Isoform-specific thyroid hormone receptor antibodies detect multiple thyroid hormone receptors in rat and human pituitaries. Endocrinology 130:1539–1546

    CAS  PubMed  Google Scholar 

  18. Hahm JB, Privalsky ML (2013) Research resource: identification of novel coregulators specific for thyroid hormone receptor-β2. Mol Endocrinol 27:840–859

    Article  CAS  PubMed  Google Scholar 

  19. Lee S, Young BM, Wan W, Chan IH, Privalsky ML (2011) A mechanism for pituitary-resistance to thyroid hormone (PRTH) syndrome: a loss in cooperative coactivator contacts by thyroid hormone receptor (TR) beta2. Mol Endocrinol 25:1111–1125

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  20. Wan W, Farboud B, Privalsky ML (2005) Pituitary resistance to thyroid hormone syndrome is associated with T3 receptor mutants that selectively impair beta2 isoform function. Mol Endocrinol 19:1529–1542

    Article  CAS  PubMed  Google Scholar 

  21. Mengeling BJ, Pan F, Privalsky ML (2005) Novel mode of deoxyribonucleic acid recognition by thyroid hormone receptors: thyroid hormone receptor beta-isoforms can bind as trimers to natural response elements comprised of reiterated half-sites. Mol Endocrinol 19:35–51

    Article  CAS  PubMed  Google Scholar 

  22. Jones I, Srinivas M, Ng L, Forrest D (2003) The thyroid hormone receptor beta gene: structure and functions in the brain and sensory systems. Thyroid 13:1057–1068

    Article  CAS  PubMed  Google Scholar 

  23. Hodin RA, Lazar MA, Wintman BI, Darling DS, Koenig RJ, Larsen PR, Moore DD, Chin WW (1989) Identification of a thyroid hormone receptor that is pituitary-specific. Science 244:76–79

    Article  CAS  PubMed  Google Scholar 

  24. Ringold GM (1985) Steroid hormone regulation of gene expression. Annu Rev Pharmacol Toxicol 25:529–566

    Article  CAS  PubMed  Google Scholar 

  25. Williams GR, Bland R, Sheppard MC (1994) Characterization of thyroid hormone (T3) receptors in three osteosarcoma cell lines of distinct osteoblast phenotype: interactions among T3, vitamin D3, and retinoid signaling. Endocrinology 135:2375–2385

    CAS  PubMed  Google Scholar 

  26. Jeyakumar M, Katzenellenbogen JA (2009) A dual-acceptor time-resolved Föster resonance energy transfer assay for simultaneous determination of thyroid hormone regulation of corepressor and coactivator binding to the thyroid hormone receptor: mimicking the cellular context of thyroid hormone action. Anal Biochem 386:73–78

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  27. Lévy-Bimbot M, Major G, Courilleau D, Blondeau JP, Lévi Y (2012) Tetrabromobisphenol-A disrupts thyroid hormone receptor alpha function in vitro: use of fluorescence polarization to assay corepressor and coactivator peptide binding. Chemosphere 87:782–788

    Article  PubMed  Google Scholar 

  28. Mochizuki K, Ishihara A, Goda T, Yamauchi K (2012) RNA polymerase II phosphorylation at serine 2 and histone H3 tri-methylation at lysine 36 are key steps for thyroid hormone receptor β gene activation by thyroid hormone in Rana catesbeiana tadpole liver. Biochem Biophys Res Commun 417:1069–1073

    Article  CAS  PubMed  Google Scholar 

  29. Richter CP, Münscher A, Machado DS, Wondisford FE, Ortiga-Carvalho TM (2011) Complete activation of thyroid hormone receptor β by T3 is essential for normal cochlear function and morphology in mice. Cell Physiol Biochem 28:997–1008

    Article  CAS  PubMed Central  PubMed  Google Scholar 

Download references

Acknowledgments

This work was supported by the National Natural Science Foundation of China (Grant nos. 30800955 and Grants 81301737), Natural Science Foundation of Tianjin, China (Grant no. 09JCZDJC21000), and Technology Innovation and Research Foundation of Weifang Medical University, China (Grant no. K11QC1013).

Author information

Authors and Affiliations

  1. Institute of Endocrinology, Metabolic Disease Hospital, Tianjin Medical University, Key Laboratory of Hormone and Development, National Health Ministry of China, Tianjin, 300070, People’s Republic of China

    Rong-Lan Zhao, Bei Sun, Ying Liu, Jing-Hua Li, Wei-Li Xiong, Dong-Chun Liang, Gang Guo, Ai-Jun Zuo & Jing-Yu Zhang

  2. Shandong Provincial Key Laboratory of Clinical Laboratory Diagnostics, Department of Medical Laboratory, Weifang Medical University, Shandong, 261053, People’s Republic of China

    Rong-Lan Zhao

Authors
  1. Rong-Lan Zhao
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Bei Sun
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Ying Liu
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Jing-Hua Li
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Wei-Li Xiong
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Dong-Chun Liang
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Gang Guo
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Ai-Jun Zuo
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Jing-Yu Zhang
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding authors

Correspondence to Ai-Jun Zuo or Jing-Yu Zhang.

Additional information

Rong-Lan Zhao and Bei Sun have contributed equally to this study.

Electronic supplementary material

Below is the link to the electronic supplementary material.

11010_2013_1935_MOESM1_ESM.tif

Supplement Fig. 1. The schematic picture of TRβ gene deduced from published picture [11] and data obtained in our study (TRβΔ, TRβ2Δ). Solid lines and shaded boxes are, respectively, used to represent exons in the upper and beneath part of the diagram; dotted lines and thin continuous lines are, respectively, used to represent intron in the upper and beneath part of the diagram. The novel exon N (108 bps) is located between exon 3 and exon 4 (TIFF 899 kb)

11010_2013_1935_MOESM2_ESM.tif

Supplement Fig. 2. Electrophoresis of the PCR products. TRβ2Δ was detected in the adult rat liver, heart, lung, kidney, skeletal muscle, and pituitary. Trβ2Δ is only detected in the pituitary (TIFF 560 kb)

11010_2013_1935_MOESM3_ESM.tif

Supplement Fig. 3. Comparison of the ligand binding between TRβ2Δ and TRβ2. a The saturation curve of T3 binding TRβ2Δ or TRβ2. b Scatchard plot. Binding studies of 125I-labeled T3 to TRβ2Δ or TRβ2 were used for Scatchard analysis to determine the affinity constant (K a). 125I-T3 (4.5 pM) was incubated with various concentrations of cold T3 (0, 10, 50, 100, 300, 600, 1,200, and 2,400 pM). Each point represents the mean of quintuple reactions (TIFF 4748 kb)

11010_2013_1935_MOESM4_ESM.tif

Supplement Fig. 4. Luciferase activity in cell lysates. COS-7 cells seeded in 6-well plates were transiently transfected with 5.0 μg of Trβ2+pGL3/TRE or Trβ2Δ+pGL3/TRE DNA; non-transfected cells served as the control group. After transfection for 6 h, serum-free DMEM replaced the transfection solution, a final concentration of 10 nM of T3 was added to each T3 intervention group, the cells were lysed 48 h later, and a luciferase assay was performed. Using a BCA protein assay kit to measure the total protein concentrations of each group, luciferase activity is reported in RLUs/mg protein and represents the mean ± standard deviation of three independent experiments (TIFF 1203 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Zhao, RL., Sun, B., Liu, Y. et al. Cloning and identification of a novel thyroid hormone receptor β isoform expressed in the pituitary gland. Mol Cell Biochem 389, 141–150 (2014). https://doi.org/10.1007/s11010-013-1935-9

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s11010-013-1935-9

Keywords

Search

Navigation