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Trafficking of a Resistance to Thyroid Hormone Syndrome Mutant: A Role of Calcium Signaling?
Author(s) -
O’Dwyer Jerry
Publication year - 2020
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.2020.34.s1.02974
Subject(s) - nuclear localization sequence , nuclear transport , nuclear receptor , importin , thyroid hormone receptor , wild type , biology , receptor , mutant , thyroid , nuclear export signal , transcription factor , microbiology and biotechnology , medicine , endocrinology , cytoplasm , cell nucleus , gene , genetics
Thyroid hormone receptors (TRs) are members of the nuclear receptor superfamily that serve as transcription factors for thyroid hormone‐responsive genes. TRs undergo nucleocytoplasmic shuttling mediated by nuclear localization signals (NLS) and nuclear export signals (NES) that interact with importins and exportins, respectively. Mutations in TRs offset the balance between import and export, and TR mislocalization has been hypothesized to contribute to disease pathogenesis such as in cancer and Resistance to Thyroid Hormone (RTH) syndrome (1). Severe bone malformations and delayed linear growth often characterize the phenotype of patients with RTH syndrome. A patient with a specific, atypical RTH point mutation in TRα1 (N359Y) also exhibit hypercalcemia (2). The purpose of this study was to quantify the intracellular localization pattern of RTH(N359Y) and to test the hypothesis that calcium plays a role in regulating trafficking of this RTH mutant. First, we performed in silico protein prediction modelling for both wild‐type TRα1 and RTH(N359Y). Both TRs have similar structure overall with perturbations in the DNA‐binding‐domain and Hinge domain. Transient transfection of HeLa (human) cells with GFP‐tagged wild‐type TRα1 and RTH(N359Y) were utilized to quantify the distribution patterns through the nuclear‐to‐cytoplasmic fluorescence ratio. RTH(N359Y) had similar localization patterns compared to wild‐type (p>0.05), with both receptors primarily localized to the nucleus. In contrast, when exposed to calcium‐enriched media, RTH(N359Y) showed a significantly increased nuclear localization compared to the wild‐type (p<0.05). To further test our hypothesis, we examined the distribution patterns of a membrane‐associated, N‐terminal truncated TRα1, namely p30, which has been reported to associate with Caveolin‐1 in the lipid‐microdomain with other proteins involved with the cGMP‐NO signaling cascade (3). Despite lacking the DNA‐binding‐domain, p30 reconstituted in MC3T3 lead to T3‐dependent proliferation. Therefore, if p30 localizes at the plasma membrane with Caveolin‐1, thyroid hormone stimulation could mediate import of calcium from the extracellular media and serve as an in vivo approach to test the impact of calcium on receptor trafficking for wild‐type TRα1 and RTH(N359Y). Confocal analysis, however, showed that GFP‐tagged p30 does not localize at the plasma membrane, up to 36 hours post‐transfection. Therefore, other approaches will need to be utilized to ascertain the role of calcium in the trafficking of this RTH mutant. An understanding of how calcium regulates the trafficking of RTH(N359Y) could provide insight into the connection between RTH syndrome and hypercalcemia and more generally the synergistic role of calcium signaling and nuclear trafficking. Support or Funding Information NIH Grant #2R15DK058028 to L.A.A.1 Zhang J , Roggero VR , Allison LA. ( 2018 ) Nuclear Import and Export of the Thyroid Hormone Receptor . Vitam. Horm. 106 , 45 – 66 . 2 Espiard , S. et al ( 2015 ) A Novel Mutation in THRA Gene Associated With an Atypical Phenotype of Resistance to Thyroid Hormone . J. Clin. Endocrinol. Metab. 100 , 2841 – 28483 Kayanaraman , H. et al ( 2014 ) Nongenomic Thyroid Hormone Signaling Occurs Through a Plasma Membrane-Localized Receptor . Sci. Signal. 7 ( 326 ), ra48 . DOI: 10.1126/scisignal.2004911