Journal: The Journal of Clinical Endocrinology and Metabolism

Article Title: Insight Into Molecular Determinants of T3 vs T4 Recognition From Mutations in Thyroid Hormone Receptor α and β

doi: 10.1210/jc.2018-02794

Figure Lengend Snippet: (a) Pedigree chart demonstrating that only the index patient (II.1) has the clinical phenotype of RTH α . (b) Sequence analysis of exon 8 of the THRA gene shows a de novo heterozygous missense mutation (c.767T>C) in the index patient, resulting in a Met to Thr substitution at codon 256 (p.M256T).

Article Snippet: FLAG-TR α 1 WT, M256T, and M256A receptor proteins were synthesized using the TnT® T7 Quick Coupled Transcription/Translation System (Promega, Leiden, Netherlands).

Techniques: Sequencing, Mutagenesis

Journal: The Journal of Clinical Endocrinology and Metabolism

Article Title: Insight Into Molecular Determinants of T3 vs T4 Recognition From Mutations in Thyroid Hormone Receptor α and β

doi: 10.1210/jc.2018-02794

Figure Lengend Snippet: Comparison of the architecture of the TR α 1 ligand binding pocket in the presence of T3 and T4. (a and b) Close-up view of the ligand-binding pocket of the TR α 1 crystal structure in complex with (a) T3 (PDB ID: 2h77) and (b) T4. The residue side-chains lining the niche that accommodates the outer ring of T3 and T4 are highlighted, and their molecular surface is shown except for Phe405 for clarity. The 5′-iodine group of T4 is represented by the green ball in the T4-bound TR α 1 model. The hydrophobic contacts between Met256 and the phenolic outer ring are depicted as dashed lines. (c and d) Structural models of the TR α 1-M256T mutant in complex with (c) T3 and (d) T4. (e and f) Structural models of the TR α 1-M256A mutant in complex with (e) T3 and (f) T4. (g and h) Overlay of the structural orientation of the residue side-chains that face the (g) T3 and (h) T4 ligands at the 5′ position in WT (gray), M256T (blue), and M256A (red) mutant TR α 1 models. All figures were created in YASARA Structure using PovRay imaging software.

Article Snippet: FLAG-TR α 1 WT, M256T, and M256A receptor proteins were synthesized using the TnT® T7 Quick Coupled Transcription/Translation System (Promega, Leiden, Netherlands).

Techniques: Ligand Binding Assay, Mutagenesis, Imaging, Software

Journal: The Journal of Clinical Endocrinology and Metabolism

Article Title: Insight Into Molecular Determinants of T3 vs T4 Recognition From Mutations in Thyroid Hormone Receptor α and β

doi: 10.1210/jc.2018-02794

Figure Lengend Snippet: (a, c, e) [ 125 I]T3 dissociation curves showing that compared with (a) WT, (c) the TR α 1-M256T mutation, and (e) TR α 1-M256A mutation reduces the affinity for T3 (solid line) more than for T4 (dashed line) (mean ± SEM of three experiments for WT and M256T and two experiments for M256A performed in duplicate). (b, d, f) The TR α 1-M256T and TR α 1-M256A mutations also had a larger effect on T3- than on T4-dependent transcriptional activation (mean ±SEM of three experiments performed in triplicate). The effect of the Ala substitution on the ligand binding affinity and the transcriptional activity of TR α 1 was less than the effect of the Thr substitution. The insert in (b) shows immunoblots confirming an equal expression of WT, M256T, and M256A FLAG-tagged TR α 1 and Histone 3 as a loading control in the nuclear fraction of JEG-3 cells.

Article Snippet: FLAG-TR α 1 WT, M256T, and M256A receptor proteins were synthesized using the TnT® T7 Quick Coupled Transcription/Translation System (Promega, Leiden, Netherlands).

Techniques: Mutagenesis, Activation Assay, Ligand Binding Assay, Activity Assay, Western Blot, Expressing

Journal: The Journal of Clinical Endocrinology and Metabolism

Article Title: Insight Into Molecular Determinants of T3 vs T4 Recognition From Mutations in Thyroid Hormone Receptor α and β

doi: 10.1210/jc.2018-02794

Figure Lengend Snippet: Summary of the Results of Competitive Binding, Transcriptional Activity, and Protein-Protein Interaction Assays of WT, TR α 1-M256T, and TR α 1-M256A Mutants

Article Snippet: FLAG-TR α 1 WT, M256T, and M256A receptor proteins were synthesized using the TnT® T7 Quick Coupled Transcription/Translation System (Promega, Leiden, Netherlands).

Techniques: Binding Assay, Activity Assay

Journal: The Journal of Clinical Endocrinology and Metabolism

Article Title: Insight Into Molecular Determinants of T3 vs T4 Recognition From Mutations in Thyroid Hormone Receptor α and β

doi: 10.1210/jc.2018-02794

Figure Lengend Snippet: The TR α 1-M256T mutation had a larger effect on T3- than on T4-dependent (a and b) GAL4-NCoR1 dissociation and (c and d) GAL4-SRC1 association (mean ± SEM of at least three experiments performed in triplicate). The insert in (a) shows immunoblots confirming an equal expression of WT and M256T VP16 TR α 1 fusion proteins and Histone 3 as loading control in the nuclear fraction of JEG-3 cells.

Article Snippet: FLAG-TR α 1 WT, M256T, and M256A receptor proteins were synthesized using the TnT® T7 Quick Coupled Transcription/Translation System (Promega, Leiden, Netherlands).

Techniques: Mutagenesis, Western Blot, Expressing

Journal: The Journal of Clinical Endocrinology and Metabolism

Article Title: Insight Into Molecular Determinants of T3 vs T4 Recognition From Mutations in Thyroid Hormone Receptor α and β

doi: 10.1210/jc.2018-02794

Figure Lengend Snippet: (a–c) The T4-induced transcriptional activity of three TR α 1 mutations identified in RTH α patients is lower than that is induced by T3, which is similar to WT (mean ± SEM of three experiments performed in triplicate). (d) The EC50 of T4 is ∼30- to 50-fold higher than the EC50 of T3, except for TR α 1-M256T. *** P < 0.001 (one-way ANOVA with Tukey post test).

Article Snippet: FLAG-TR α 1 WT, M256T, and M256A receptor proteins were synthesized using the TnT® T7 Quick Coupled Transcription/Translation System (Promega, Leiden, Netherlands).

Techniques: Activity Assay

Journal: The Journal of Clinical Endocrinology and Metabolism

Article Title: Insight Into Molecular Determinants of T3 vs T4 Recognition From Mutations in Thyroid Hormone Receptor α and β

doi: 10.1210/jc.2018-02794

Figure Lengend Snippet: The T3- and T4-induced transcriptional activity of (a) WT and (b) TR β 1-M310T in JEG-3 cells shows that the TR β 1-M310T mutation affects T3- more than T4-dependent transcriptional activation (mean ± SEM of four experiments performed in triplicate), which is in line with the results of TR α 1-M256T .

Article Snippet: FLAG-TR α 1 WT, M256T, and M256A receptor proteins were synthesized using the TnT® T7 Quick Coupled Transcription/Translation System (Promega, Leiden, Netherlands).

Techniques: Activity Assay, Mutagenesis, Activation Assay