An alternative splice variant of the mouse TRH receptor mRNA is the major form expressed in the mouse pituitary gland.

The sequences of the mouse and rat TRH receptors (TRH-Rs) show 94% similarity at the protein level. However, they differ significantly at their carboxy terminals, i.e. the mouse TRH-R ends with an asparagine at position 393 while, in the rat, residue 393 is lysine and an additional 19 amino acids are added before the first stop codon. In the mouse cDNA, the sequence encoding these additional amino acids is located 224 bp downstream in the 3' untranslated region (3'UT). As the mouse TRH-R was cloned from thyrotrope-derived TtT97 tissue and the rat TRH-R from lactotrope-derived GH cell lines, we have investigated whether this difference at the carboxy terminus represents a species-specific or cell type-specific pattern of TRH-R expression. Total RNA was isolated from mouse pituitary and TtT97 tissue, and rat pituitary and GH3 cells. Reverse transcription PCR analysis was performed using primers that would generate DNA fragments including the stop codon in either the mouse or the rat TRH-R and, in the mouse form, the extra 224 bp of 3'UT. This would generate a product of 234 bp from the rat sequence and 441 bp from the mouse sequence. In rat pituitary and GH3 cDNA, PCR generated the expected 234 bp product but not a band representing the mouse sequence. In both mouse pituitary and TtT97 cDNA, neither the expected 441 bp nor the 234 bp fragments were amplified; instead a larger, 829 bp, product was generated. Sequence analysis revealed a 388 bp insertion at position 1663 in the 3'UT compared with the published mouse TRH-R sequence. Ribonuclease protection analysis using this 829 bp fragment as a probe showed that this sequence represented the major TRH-R mRNA species in mouse pituitary and TtT97 RNA. A genomic clone containing this region of the mouse TRH-R gene was isolated and analysis of the sequence in this region revealed that this longer form of the mouse TRH-R could be generated by alternative splicing. In summary, we have shown that the carboxyterminal differences between the mouse and rat TRH-Rs are species-specific rather than cell type-specific, and that the major TRH-R mRNA expressed in mouse pituitary contains an additional 388 bp of 3'UT compared with the published sequence. As a region in the 3'UT of the published mTRH-R sequence has been shown to be important for stability of this mRNA, this additional 3'UT sequence could have major effects on the regulation and stability of the mouse TRH-R mRNA.

Half-site arrangement of hybrid glucocorticoid and thyroid hormone response elements specifies thyroid hormone receptor complex binding to DNA and transcriptional activity.

Thyroid hormone receptors bind to thyroid hormone response elements (TREs) as heterodimers with 3,5,3'-L-triiodothyronine (T3) receptor auxiliary protein (TRAP) and retinoid X receptors (RXRs). Currently, it is not known whether TR/TRAP or TR/RXR heterodimers need to bind to both TRE half-sites and whether there is a preferred orientation for TR/RXR heterodimer binding to TREs or transcriptional activation. Accordingly, we created a mutant TR alpha (TR-P box) by changing 3 amino acids in the P box region of the first zinc finger of the DNA-binding domain to that of the glucocorticoid receptor (GR), and we examined wild-type TR alpha and TR-P box complex binding to hybrid response elements containing TRE and glucocorticoid receptor element (GRE) half-sites arranged as a direct repeat with a four-nucleotide gap. TR-P box/RXR heterodimers selectively bound to the hybrid response elements in which GRE half-site was the downstream half-site, whereas TR alpha/RXR bound to hybrid response elements in which GREs were in either position. Additionally, TR/TRAP or TR/RXR heterodimer required two half-sites for binding to DNA, with strong binding to at least one of the half-sites. Last, co-transfection assays and methylation interference studies using the hybrid response elements suggest that the sequential arrangement of strong and weak half-sites in the TRE may be a critical determinant of TR/RXR heterodimer binding and transcriptional activation.

Roles of 3,5,3'-triiodothyronine and deoxyribonucleic acid binding on thyroid hormone receptor complex formation.

Thyroid hormone receptors (TRs) bind to thyroid hormone response elements (TREs) in the promoter region of target genes as monomers, homodimers, and heterodimers with nuclear proteins such as retinoid-X receptors (RXRs). Recently, we observed that T3 decreased TR homodimer, but not TR/RXR heterodimer, binding to TREs, suggesting that the latter complexes may be involved in transcriptional activation of target genes. However, little is known about TR complexes that form in solution. Thus far, there have been only limited studies comparing TR complex formation in solution and on DNA as well as examining the effects of T3 and the putative ligand for RXRs, 9-cis retinoic acid (9-cis RA), on TR complex formation. In this paper, we used a coimmunoprecipitation assay with anti-TR beta 1 antibody and the electrophoretic mobility shift assay under similar buffer and incubation conditions to demonstrate that in the absence of T3, TR beta 1 is present as a monomer in solution and binds primarily as a homodimer to the chicken lysozyme TRE, F2. In the presence of T3, TR beta 1 cannot form a homodimer on F2, but, instead, exists as a liganded monomer in solution. Kinetic studies demonstrated that T3 markedly increased the dissociation rate of TR homodimer from F2. Using similar methods, we observed TR beta 1/RXR alpha heterodimer formation in solution and 10-fold greater formation on F2. Neither T3 nor 9-cis RA significantly affected TR beta 1/RXR alpha heterodimer formation. Taken together, these results suggest that both T3 and TRE binding are important determinants of the formation of specific TR complexes in solution and on DNA.

Evidence that phosphorylation events participate in thyroid hormone action.

Using both a protein phosphatase inhibitor, okadaic acid (OA), and a protein kinase inhibitor, H7, to modify phosphorylation events in the cell, we investigated the effects of these agents on transcriptional activation via exogenous rat thyroid hormone receptor (TR) isoforms in transiently transfected cells and endogenous TRs. CV-1 cells were transiently cotransfected with expression plasmids encoding either the rat TR alpha 1 or TR beta 1, and luciferase reporter plasmids containing either the synthetic DR4 or the chick lysozyme F2 thyroid hormone response elements (TREs). For both receptor isoforms, there was an enhancement of transcriptional activity after incubation with 5 nM T3 for 24 h compared to hypothyroid levels. There was little change in transcriptional activation in the presence of 25 nM OA alone; however, for both TR isoforms and both TREs studied, OA augmented the stimulatory effects of T3. For the F2 TRE, transcriptional activation via TR alpha 1 increased from 19- to 35-fold, and that via TR beta 1 increased from 6- to 10-fold in the presence of T3 and OA compared to that with T3 alone. Similar results were found for the DR4 TRE. OA enhanced transcriptional activation by T3 in a dose-dependent manner. Increasing concentrations of OA (0, 5, 25, and 50 nM) further increased relative luciferase activity from 11-fold in the absence of OA to 45-fold in the presence of 50 nM OA. The protein kinase inhibitor, H7, caused no change in the transcriptional activity of the reporter plasmids via TR alpha 1 in the absence of T3, but completely blocked transcriptional activation by T3 for both the DR4 and the F2 TREs. H7 also blocked stimulation of endogenous GH and inhibition of endogenous TR beta 2 mRNAs by T3 in GH3 cells. These results indicate that phosphorylation events in the cell play an important role in transcriptional activation via both TR isoforms.

Roles of v-erbA homodimers and heterodimers in mediating dominant negative activity by v-erbA.

v-erbA, a viral oncogenic homolog of thyroid hormone receptor (TR), blocks the effect of T3 in TR-mediated transcription. The mechanism(s) for this dominant negative effect by v-erbA on TRs is unknown but may involve competition between v-erbA and TR-containing complexes for binding to thyroid hormone response elements (TREs) and/or protein-protein interactions between v-erbA and TR. To investigate these potential mechanisms, we used the electrophoretic mobility shift assay to compare in vitro translated v-erbA and TR alpha binding to two TREs-chick lysozyme TRE (F2) and direct repeat TRE (DR4). v-erbA bound as a homodimer to these TREs, whereas TR alpha bound as a homodimer and monomer. T3 decreased TR alpha homodimer binding to the TREs as we reported previously; however, surprisingly, high concentrations of T3 (10(-6) M) also decreased v-erbA homodimer binding to the TREs. Additionally, v-erbA formed heterodimers with nuclear proteins such as retinoid X receptor and T3 receptor auxiliary protein as well as with TR alpha. These dimers remained bound to DNA in the presence of T3. Finally, v-erbA could not mediate ligand-dependent transcriptional activation even at 10(-6) M T3 but could block ligand-dependent TR-mediated transactivation in co-transfection experiments. v-erbA also exhibited differential dominant negative activity on F2 and DR4 suggesting that half-site sequence and/or orientation may influence v-erbA-dominant negative activity. In sum, there are multiple v-erbA complexes that bind to TREs in the presence of T3, which all may contribute to v-erbA's dominant negative effect on TR-mediated transcription by competing with TR-containing complexes for binding to TREs.

Spectral shift of fluorescein and carboxyfluorescein in the anterior chamber of the rabbit eye following systemic administration.

Systemically administered fluorescein is converted to fluorescein glucuronide. The conjugate is fluorescent and interferes with the measurement of fluorescein in the anterior chamber. Carboxyfluorescein is a hydrophilic derivative of fluorescein. In the rabbit, carboxyfluorescein is not converted as readily to a fluorescent metabolite. Thus, carboxyfluorescein has potential advantages as a quantitative fluorophore for studies of aqueous humor dynamics.