Molecular and Cellular Biochemistry

, Volume 389, Issue 1–2, pp 141–150 | Cite as

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

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


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.


DNA-binding domain Isoform Thyroid hormone receptor β TRβ2Δ 



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).

Supplementary material

11010_2013_1935_MOESM1_ESM.tif (900 kb)
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 (560 kb)
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 (4.6 mb)
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 (1.2 mb)
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)


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Copyright information

© Springer Science+Business Media New York 2014

Authors and Affiliations

  • Rong-Lan Zhao
    • 1
    • 2
  • Bei Sun
    • 1
  • Ying Liu
    • 1
  • Jing-Hua Li
    • 1
  • Wei-Li Xiong
    • 1
  • Dong-Chun Liang
    • 1
  • Gang Guo
    • 1
  • Ai-Jun Zuo
    • 1
  • Jing-Yu Zhang
    • 1
  1. 1.Institute of Endocrinology, Metabolic Disease Hospital, Tianjin Medical University, Key Laboratory of Hormone and DevelopmentNational Health Ministry of ChinaTianjinPeople’s Republic of China
  2. 2.Shandong Provincial Key Laboratory of Clinical Laboratory Diagnostics, Department of Medical LaboratoryWeifang Medical UniversityShandongPeople’s Republic of China

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