A novel multifunctional motif in the amino-terminal A/B domain of T3Ralpha modulates DNA binding and receptor dimerization.

We reported previously that deletion of the 50-amino acid NH2-terminal A/B domain of the chicken (c) or rat thyroid hormone (T3) receptor-alpha (T3Ralpha) decreased the T3-dependent stimulation of genes regulated by native thyroid hormone response elements (TREs). This requirement of the NH2-terminal A/B domain for transcriptional activation was mapped to amino acids 21-30 of cT3Ralpha. Expression of transcription factor IIB (TFIIB) in cells was shown to enhance T3-dependent transcriptional activation by cT3Ralpha, and this enhancement by TFIIB was dependent on the same 10-amino acid sequence. In vitro binding studies indicated that cT3Ralpha interacts efficiently with TFIIB, and this interaction requires amino acids 23KRKRK27 in the A/B domain. In this study we document the functional importance of these five basic residues in transcriptional activation by cT3Ralpha, further supporting the biological significance of these residues and their interaction with TFIIB. Interestingly, we also find that the same amino acids also affect DNA binding and dimerization of cT3Ralpha. Gel mobility shift assays reveal that a cT3Ralpha mutant that has all five basic amino acids changed from 23KRKRK27 to 23TITIT27 binds to a palindromic TRE predominantly as a homodimer, whereas cT3Ralpha with the wild-type 23KRKRK27 sequence binds predominantly as a monomer. This results from both a marked decrease in the ability of the cT3Ralpha mutant to bind as a monomer and from an enhanced ability to dimerize as reflected by an increase in DNA-bound T3R-retinoic X receptor heterodimers. These effects of 23KRKRK27 on DNA binding, dimerization, transcriptional activation, and the association of T3Ralpha with TFIIB support the notion that this basic amino acid motif may influence the overall structure and function of T3Ralpha and, thus, play a role in determining the distinct context-dependent transactivation potentials of the individual T3R isoforms.

A 10-amino-acid sequence in the N-terminal A/B domain of thyroid hormone receptor alpha is essential for transcriptional activation and interaction with the general transcription factor TFIIB.

The effects of the thyroid hormone (3,5,3'-triiodo-L-thyronine [T3]) on gene transcription are mediated by nuclear T3 receptors (T3Rs). alpha- and beta-isoform T3Rs (T3R alpha and -beta) are expressed from different genes and are members of a superfamily of ligand-dependent transcription factors that also includes the receptors for steroid hormones, vitamin D, and retinoids. Although T3 activates transcription by mediating a conformational change in the C-terminal approximately 220-amino-acid ligand-binding domain (LBD), the fundamental mechanisms of T3R-mediated transcriptional activation remain to be determined. We found that deletion of the 50-amino-acid N-terminal A/B domain of chicken T3R alpha (cT3R alpha) decreases T3-dependent stimulation of genes regulated by native thyroid hormone response elements about 10- to 20-fold. The requirement of the A/B region for transcriptional activation was mapped to amino acids 21 to 30, which contain a cluster of five basic amino acids. The A/B region of cT3R alpha is not required for T3 binding or for DNA binding of the receptor as a heterodimer with retinoid X receptor. In vitro binding studies indicate that the N-terminal region of cT3R alpha interacts efficiently with TFIIB and that this interaction requires amino acids 21 to 30 of the A/B region. In contrast, the LBD interacts poorly with TFIIB. The region of TFIIB primarily involved in the binding of cT3R alpha includes an amphipathic alpha helix contained within residues 178 to 201. Analysis using a fusion protein containing the DNA-binding domain of GAL4 and the entire A/B region of cT3R alpha suggests that this region does not contain an intrinsic activation domain. These and other studies indicate that cT3R alpha mediates at least some of its effects through TFIIB in vivo and that the N-terminal region of DNA-bound cT3R alpha acts to recruit and/or stabilize the binding of TFIIB to the transcription complex. T3 stimulation could then result from ligand-mediated changes in the LBD which may lead to the interaction of other factors with cT3R alpha, TFIIB, and/or other components involved in the initiation of transcription.

Interactions of thyroid hormone receptor with the human immunodeficiency virus type 1 (HIV-1) long terminal repeat and the HIV-1 Tat transactivator.

Thyroid hormone (T3) receptor (T3R) regulates the human immunodeficiency virus type 1 (HIV-1) long terminal repeat (LTR) by binding to and activating thyroid hormone response elements (TREs) embedded within the viral NF-kappa B and Sp1 motifs. The TREs within the NF-kappa B sites are necessary for activation by T3 in the absence of Tat, while those in the Sp1 motifs function as TREs only when Tat is expressed, suggesting that Tat and T3R interact in the cell. Transactivation of the HIV-1 LTR by T3R alpha and several receptor mutants revealed that the 50-amino-acid N-terminal A/B region of T3R alpha, known to interact with the basal transcription factor TFIIB, is critical for activation of both Tat-dependent and Tat-independent responsive sequences of the LTR. A single amino acid change in the highly conserved tau 1 region in the ligand-binding domain of T3R alpha eliminates Tat-independent but not Tat-dependent activation of the HIV-1 LTR by T3. Ro 5-3335 [7-chloro-5-(2-pyrryl)-3H-1,4-benzodiazepin-2(H)-one], which inhibits Tat-mediated transactivation of HIV-1, also inhibits the functional interaction between Tat and T3R alpha. Binding studies with glutathione-S-transferase fusion proteins and Western (immunoblot) analysis indicate that T3R alpha interacts with Tat through amino acids within the DNA-binding domain of T3R alpha. Mutational analysis revealed that amino acid residues in the basic and C-terminal regions of Tat are required for the binding of Tat to T3R alpha, while the N terminus of Tat is not required. These studies provide functional and physical evidence that stimulation of the HIV-1 LTR by T3 involves an interaction between T3R alpha and Tat. Our results also suggest a model in which multiple domains of T3R alpha interact with Tat and other factors to form transcriptionally important complexes.

Cyclic alternating chemotherapy for small cell carcinoma of the lung.

Eighty-two previously untreated patients with small cell cancer of the lung were treated with six cycles of two alternating drug regimens: a new combination of mitomycin, methotrexate, and etoposide; and cyclophosphamide, doxorubicin, and vincristine. No maintenance chemotherapy was used. Consolidative thoracic irradiation and prophylactic cranial irradiation were employed. The median survival time for 32 limited-disease patients was 59 weeks, and for 50 extensive-disease patients was 35 weeks. Four-year survival was 12% for limited-disease patients and 2% for extensive-disease patients. These results were not superior to conventional combination chemotherapy regimens.