Naturally Occurring Amino Acids in Helix 10 of the Thyroid Hormone Receptor Mediate Isoform-Specific TH Gene Regulation - PubMed (original) (raw)
Naturally Occurring Amino Acids in Helix 10 of the Thyroid Hormone Receptor Mediate Isoform-Specific TH Gene Regulation
Vitor M S Pinto et al. Endocrinology. 2017.
Abstract
Thyroid hormone (TH) action is mediated by the products of two genes, TH receptor (THR)α (THRA) and THRβ (THRB) that encode several closely related receptor isoforms with differing tissue distributions. The vast majority of THR isoform-specific effects are thought to be due to tissue-specific differences in THR isoform expression levels. We investigated the alternative hypothesis that intrinsic functional differences among THR isoforms mediate these tissue-specific effects. To achieve the same level of expression of each isoform, we created tagged THR isoforms and tested their DNA and functional properties in vitro. We found significant homodimerization and functional differences among the THR isoforms. THRA1 was unable to form homodimers on direct repeat separated by 4 bp DNA elements and was also defective in TH-dependent repression of Tshb and Rxrg in a thyrotroph cell line, TαT1.1. In contrast, THRB2 was both homodimer sufficient and fully functional on these negatively regulated genes. Using domain exchanges and individual amino acid switches between THRA1 and THRB2, we identified three amino acids in helix 10 of the THRB2 ligand-binding domain that are required for negative regulation and are absent in THRA1.
Copyright © 2017 Endocrine Society.
Figures
Figure 1.
THR isoforms have different abilities to form homodimers on DNA. (a) Schematic representation of hemagglutinin (HA)-tagged THR isoforms. AF-1, DBD, and LBD domains (N to C termini) are shown by different shading. (b) In vitro transcription/translation proteins from rabbit reticulocyte lysate were analyzed in duplicate by western blot using an anti-HA antibody. pSP72 was used as an “empty” vector negative control. (c–e) Binding of wild-type THRs to DR4, LAP, and PAL in the presence or absence of retinoic X receptor α (RXRA) were tested by electrophoretic mobility shift assay. (c) On a DR4 element, THRA1 bound exclusively as a heterodimer, THRB1 showed weak homodimer formation, and THRB2 bound as homodimer in the absence of T3. All THR isoforms heterodimerized with RXRA. (d) On an LAP element, all THRs isoforms bound as homodimers and heterodimers, but only THRA1 binding was dissociated by T3. (e) On a PAL element, THRs bound exclusively in the presence of RXRA. Samples were treated with vehicle or 100 nM T3. *Nonspecific band observed in unprogrammed lysate.
Figure 2.
THR isoforms have different functions in positive and negative regulation of gene expression. (a) Relative expression of Thrb in control (scr KD GFP) and Thrb KD cells measured by qRT-PCR and normalized to Actb mRNA (see Materials and Methods). (b) Expression level of each HA-THR isoform in Thrb KD T_α_T1.1 cells was tested by western blotting with anti-HA antibody. Relative expression of (c and d) negatively and (e and f) positively regulated genes were measured by qRT-PCR and normalized to Actb mRNA. Relative expression [_y_-axis (log2)] of (c) Tshb and (d) Rxrg decreased with increased concentrations of T3 [_x_-axis (log10); y intersects x at 0 nM]. KD of Thrb abolishes T3-dependent repression (also see percentage of repression, left panels). Expression of HA-tagged THRB2 proteins in Thrb KD T_α_T1.1 cells fully reconstituted gene repression, THRB1 had weaker ability to regulate Tshb and Rxrg, and expression of THRA1 was unable to rescue Thrb KD phenotype. Relative expression [_y_-axis (log2)] of (e) Rab27 and (f) Sema3c increased with increased concentrations of T3 [_x_-axis (log10); y intersects x at 0 nM]. KD of Thrb abolishes T3-dependent gene activation (also see percentage of activation, left panels). Expression of HA-tagged THR isoforms in Thrb KD T_α_T1.1 cells reconstituted T3-dependent gene activation. Data points are presented as mean ± standard error of the mean. Differences were considered to be significant at P < 0.05. NS, not significant.
Figure 3.
Structure of THRB LBD domain. (a) Dimerization surface of THRB LBD (Protein Data Bank code 3GWS) (9). Helix 10 is shown in blue, helix 11 is in light gray. Amino acids Tyr (Y), Asp (D), and Ser (S) that were substituted in the THRBH10 chimera are shown in red, Arg (R) affected in the R429Q mutant is shown in green. (b) Alignment of LBDs of THRA and THRB from humans and mice. Positions that show nonconserved amino acid variations between mammalian α and β isoforms are shaded. Black rectangles on the top of the sequence show location of helices (H1–H12) as described in Wagner et al. (24). The position of domain exchange in THRB2LBD THRA1LBD, and in THRB2H10-12 and THRA1H10-12 are marked with arrows. The amino acids changed in THRB2 H10 are underlined.
Figure 4.
LBD domain exchange between THRA1 and THRB2 affects receptor binding to DR4 and LAP. (a and c) Schematic structures and (b and d) protein levels of _in vitro_–translated THRA1, THRA1LBD, THRB2, and THRB2LBD are shown. pSP72 was used as an “empty” vector negative control. (e–h) EMSA of chimeric receptors in the absence and presence of RXRA. (e) On DR4, neither THRA1 nor THRA1LBD formed a homodimer. (g) THRA1LBD formed a stronger homodimer than did WT THRA1 and did not dissociate from LAP after T3 treatment. Both THRA1 and THRA1LBD formed heterodimers with RXRA on DR4 and LAP. (f) On DR4, the THRBLBD lost the ability to form a homodimer compared with that of WT THRB2. (h) In contrast to WT THRB2, THRB2LBD partially dissociated from the LAP element after T3 treatment. THRB2 and THRB2LBD showed similar abilities to form heterodimers with RXRA on both DNA elements. Samples were treated with vehicle or increasing concentrations of T3 (1 nM, 10 nM, and 100 nM). *Nonspecific band observed in unprogrammed lysate.
Figure 5.
Exchange of helices 10 to 12 in THRA1 and THRB2 affects receptor binding to DR4 and LAP. (a and c) Schematic structures and (b and d) protein levels of _in vitro_–translated THRA1, THRA1H10-12, THRB2, and THRB2H10-12 are shown. pSP72 was used as an “empty” vector negative control. (e) Similar to Fig. 4, neither THRA1 nor THRA1H10-12 formed a homodimer on DR4 in EMSA. (g) LAP THRA1H10-12 formed a homodimer that did not dissociate from LAP after T3 treatment. (f) On DR4, THRBH10-12 lost the ability to form a homodimer and (h) partially dissociated from the LAP element after T3 treatment. All tested THRs showed similar abilities to form heterodimers with RXRA on both DNA elements. Samples were treated with vehicle or increasing concentrations of T3 (1 nM, 10 nM, and 100 nM). *Nonspecific band observed in unprogrammed lysate.
Figure 6.
The substitution of YQDS amino acid to SQEA in Thrb2H10 affects receptor homodimerization on DR4 and LAP. Three-amino-acid substitution in THRB2H10, YQDS to SQEA affects receptor homodimerization on DR4 and LAP. (a) Western blot showed equal amounts of _in vitro_–translated WT THRB2 and THRB2H10. (b) EMSA of THRB2 and THRB2H10 showed that similar to THRB2LBD and THRB2H10-12, THRB2H10 was unable to form a homodimer on a DR4 element, whereas heterodimer binding with RXR was not affected. (c) Similarly, THRB2H10 dissociated to some degree from the LAP element after T3 treatment. Samples were treated with vehicle or increasing concentrations of T3. *Nonspecific band observed in unprogrammed lysate.
Figure 7.
THRB2H10 specifically affects negative regulation of gene expression. (a) Expression levels of WT THRB2 and THRB2H10 in Thrb KD T_α_T1.1 cells were tested by western blotting with an anti-HA antibody. Relative expression of (b and c) negatively and (d and e) positively regulated genes were measured by qRT-PCR and normalized to Actb mRNA (see Materials and Methods) and compared with controls (scrambled KD, GFP, and Thrb KD GFP, Fig. 2). Reexpression of HA-THRB2 in Thrb KD cells reconstituted (b) Tshb, and (c) Rxrg negative regulation, with the maximal percentage of repression (left panes) 75% to 80%. Expression of HA-THRB2H10 only partially rescued negative regulation of Tshb and Rxrg (45% to 50% of repression at 10 nM T3). WT THRB and THRB2H10 showed similar properties in activation of gene expression of (d) Rab 27 and (e) Sema3c. No significant differences (NS) were seen in percentage of activation (right panels) at 1 and 10 nM T3. Left panels show relative gene expression [_y_-axis (log2)] with increased concentration of T3 [_x_-axis (log10); y intersects x at 0 nM). Expression levels were normalized to those in scramled KD, GFP no T3. Data points are presented as mean ± standard error of the mean. Differences were considered to be significant at P < 0.05.
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