A Novel Multifunctional Motif in the Amino-terminal A/B Domain of T3Ralpha Modulates DNA Binding and Receptor Dimerization*

Emir HadzicDagger §, Ioanis Habeos, Bruce M. Raaka, and Herbert H. Samuels

From the Division of Molecular Endocrinology, Departments of Medicine and Pharmacology, and the Dagger  Department of Cell Biology, New York University Medical Center, New York, New York 10016

    ABSTRACT
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Abstract
Introduction
Procedures
Results
Discussion
References

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.

    INTRODUCTION
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Abstract
Introduction
Procedures
Results
Discussion
References

Steroid, retinoid, and thyroid hormone nuclear receptors are ligand-dependent transcription factors that couple extracellular signals directly to transcriptional responses. These receptors activate or repress transcription of target genes by binding to specific DNA sequences referred to as hormone response elements (HREs)1 (1). The nuclear receptor superfamily can be divided into the steroid hormone receptor family and the thyroid hormone/retinoid receptor family (1, 2), which includes receptors that mediate the effects of thyroid hormone [L-triiodothyronine (T3) (the T3Rs), all-trans-retinoic acid (the RARs), 9-cis-retinoic acid (the RARs and RXRs), and 1,25-dihydroxyvitamin D3 as well as several orphan receptors (e.g. COUP-TF, c-erbAalpha 2) whose ligand(s), if any, are unknown (3-5).

The T3Rs are encoded by two distinct but closely related genes (alpha  and beta ) which, in humans (h), map to chromosomes 17 and 3, respectively (6). Each gene expresses several alternatively spliced isoforms. The T3Ralpha gene in the rat (r) and man expresses the T3-binding isoform T3Ralpha 1 along with c-erbAalpha 2, which does not bind T3 because of alternative splicing at the COOH terminus (3, 7, 8). The closely related chicken (c) alpha  gene expresses only cT3Ralpha , which is more than 90% similar at the amino acid level to rT3Ralpha 1 and hT3Ralpha 1 (6, 9, 10). The T3Rbeta gene expresses T3Rbeta 1 and T3Rbeta 2 that differ only in their NH2-terminal A/B regions, which are distinct from the A/B region of T3Ralpha 1 (3, 11). Except for the A/B domains, the T3Ralpha and T3Rbeta receptors are more than 90% similar at the amino acid level. Thus, three T3Rs are expressed which differ primarily in the A/B domain, suggesting that this region may play a role in mediating different effects of these receptors.

One of the central issues in understanding the actions of the T3Rs and other nuclear receptors is elucidation of the details by which target genes are recognized. The T3Rs and certain other members of thyroid hormone/retinoid receptor family bind to their HREs as monomers, homodimers (12-16), or as heterodimers with the RXRs (17-24). In particular, the T3Rs bind to and activate transcription from a wide variety of response elements organized as direct repeats (DR), inverted repeats (IR), or everted repeats (ER) of the optimized AGGTCA hexanucleotide half-site (25-27) and from native half-site motifs that diverge from the AGGTCA core binding sequence (28).

Recognition of specific base pairs within the half-site core binding motif is mediated by the highly conserved DNA binding domain (DBD), which defines the nuclear receptor superfamily. This highly conserved DBD contains 66-68 amino acids that are organized into two zinc finger structures that include 9 perfectly conserved cysteines followed by a carboxyl-terminal extension (29, 30). A helix in the carboxyl-terminal extension, with its extensive minor groove contacts, effectively extends the contact surface of the DBD beyond the consensus 6-base pair half-site (31). The ability of nuclear hormone receptors to distinguish among specific HREs is conferred by 3 amino acids at the base of the first zinc finger in the DBD (the P box) (32). This region is organized into an alpha -helix that penetrates the major groove and recognizes the specific nucleotide sequence of the HRE.

Amino acids at the base of the second zinc finger (the D box) are thought to provide a dimerization interface for protein-protein interactions on certain HREs (32). Structural studies indicate that the DBDs of certain thyroid hormone/retinoid family members form a cooperative dimerization interface, when bound to DRs but not to IRs and ERs (31). For IRs and ERs, binding of homodimers or heterodimers is thought to result from a dimerization interface located within the ligand binding domain. In addition to homodimer and heterodimer binding, the T3Rs also bind IRs, DRs, and other DNA configurations as monomers (14). In the absence of RXR, T3Ralpha binds more efficiently as monomers to these elements, and T3Rbeta isoforms bind more efficiently as homodimers (33).

We reported previously that a 10-amino acid sequence within the A/B domain of cT3Ralpha or rT3Ralpha 1 was essential for ligand-dependent activation of native HREs and for interaction of T3Ralpha with TFIIB (34). Interestingly, deletion of the 50-amino acid A/B domain of cT3Ralpha markedly reduced monomer binding and increased homodimer binding of the receptor, suggesting that the A/B domain of T3Ralpha imposes preferential monomer binding of the receptor (34). In this study we show that the same 5 basic amino acids 23KRKRK27 which are necessary for efficient binding to TFIIB are required for transcriptional activity of cT3Ralpha . These same amino acids are also responsible for imposing preferential monomer binding and influencing the efficiency of heterodimer formation with RXR. To our knowledge this is the first identification of specific NH2-terminal residues involved in the differential binding of T3R isoforms to DNA.

    EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results
Discussion
References

Plasmids-- Delta MTV-TREp-CAT (14, 25), Delta MTV-TRE-GH-CAT (25), and Delta MTV-TRE-Mal-CAT (35) have been described previously. These CAT reporter genes contain a single copy of each TRE cloned into the HindIII site at -88 of Delta MTV-CAT, a mouse mammary tumor viral LTR-CAT reporter that lacks glucocorticoid response elements (25, 32). The TREp (also known as the TRE-IR) is an inverted repeat of optimized AGGTCA half-sites (AGGTCA TGACCT). TRE-GH and TRE-Mal are from the rat growth hormone (13) and rat malic enzyme genes (36), respectively, and contain direct and inverted repeats. The TRE1/2 is the same as the TREp except that it contains a single G to C change in one of the half-sites (AGGTCA to ACGTCA).

Full-length cT3Ralpha cDNA, corresponding to amino acids 1-408, was cloned into a pEXPRESS (pEX) vector (pEX-cT3Ralpha ) (37). pEX vectors contain the Rous sarcoma viral (RSV) LTR linked to a phage T7 RNA polymerase promoter from pET8c (38), an Asp718 (KpnI) site, followed by stop codons in each reading frame and an SV40 polyadenylation signal. pEX can be used for expression in both Escherichia coli and mammalian cells and for synthesis of in vitro transcripts using purified T7 RNA polymerase (37). pRSV-T7-cT3Ralpha was made by inserting an oligonucleotide containing a T7 polymerase binding site with 5'- and 3'-HindIII cohesive ends between the RSV LTR and the cT3Ralpha cDNA in pRSV-cT3Ralpha (34). The oligonucleotide was designed so that the 5'-HindIII site is inactivated, but the 3'-HindIII site is regenerated upon insertion, allowing for future cloning at this site. pRSV-T7-cT3Ralpha (21-408) was formed by digesting pRSV-T7-cT3Ralpha (31-408) with HindIII and PflMI and inserting an oligonucleotide corresponding to amino acids 21-30. pRSV-T7-cT3Ralpha (31-408), which lacks the first 30 amino acids of cT3Ralpha , was constructed by removing the HindIII-PflMI fragment corresponding to amino acids 1-30 of cT3Ralpha from pRSV-T7-cT3Ralpha and inserting an oligonucleotide containing a polylinker (5'-HindIII-XbaI-NcoI-PflMI-3') into the digested vector. The NcoI site in this and other pRSV-T7-cT3Ralpha mutants contains the ATG initiation codon.

pEX-cT3Ralpha (51-408), lacking the entire A/B region, was constructed from pEX-cT3Ralpha (51-157) and pEX-cT3Ralpha . DNA corresponding to amino acids 119-408 was excised from pEX-cT3Ralpha with MscI and Asp718 and subcloned into pEX-cT3Ralpha (51-157) after digestion of the vector with MscI, which cleaves at codon 118, and Asp718, which cleaves just after codon 157. pEX-cT3Ralpha (51-157) was constructed by polymerase chain reaction of wild-type cT3Ralpha using appropriate primers. pRSV-T7-cT3Ralpha (21-408, 7/8) was constructed by cleaving pRSV-T7-cT3Ralpha (21-408) with HindIII and PflMI and replacing the DNA corresponding to amino acids 21-30 with an oligonucleotide that changed amino acids 23KRKRK27 to 23TITIT27. pRSV-T7-cT3Ralpha (21-408, 9/10) was constructed in the same way using an oligonucleotide that changed amino acids 23KRKRK27 to 23TITRK27. pRSV-T7-cT3Ralpha (21-408, 11/12) was constructed as pRSV-T7-cT3Ralpha (21-408, 7/8) using an oligonucleotide that changed amino acids 23KRKRK27 to 23KRTIT27. pRSV-T7-cT3Ralpha (21-408, 13/14) was constructed in the same way using an oligonucleotide that changed amino acids 23KRKRK27 to 23TIKIT27. pEX-TFIIB was made from human TFIIB in pET11d (39). The TFIIB cDNA was excised from the pET11d vector with NcoI and BamHI and cloned into the NcoI-BglII site of a pEX vector that contained a BglII site at the 3'-end instead of the Asp718 site (34). pGST-RXR contains the entire mouse RXRalpha cDNA cloned into pGEX2T (40). This plasmid was constructed and provided to us by Paul T. van der Saag.

Cell Transfections and CAT Assays-- HeLa cells were transfected by electroporation (14, 34). After transfection, cells were cultured in Dulbecco's modified Eagle's medium (Life Technologies, Inc.) supplemented with 15 mM Hepes, 0.1 mg/ml pyruvate, 50 µg/ml streptomycin sulfate, 50 µg/ml penicillin (DHAP medium) containing 10% (v/v) hormone-depleted calf serum with and without 1 µM T3 for 48 h (as indicated) before harvesting for CAT assay. CAT expression was measured by determining the extent of [14C]chloramphenicol (50 mCi/mmol; NEN Life Science Products) acetylation as described previously (41, 42). Protein concentration was determined (43), and amounts of cell protein assayed for CAT activity were varied to maintain the percentage of [14C]chloramphenicol acetylated in the linear range (<30%) during a 16-h incubation. CAT activity values were normalized to represent the percent of [14C]chloramphenicol acetylated by a specific amount of cell protein in 16 h at 37 °C. All experiments were performed in duplicate, and the results represent the means of at least three independent transfections. Within each transfection, duplicate samples varied less than 10%.

Gel Mobility Shift Assays-- cT3Ralpha , cT3Ralpha (21-408), cT3Ralpha (21-408, 7/8), cT3Ralpha (51-408), and the various cT3Ralpha NH2-terminal amino acid mutants were expressed by in vitro translation in reticulocyte lysates (24). A fraction of each translation received L-[35S]cysteine in the incubation, and the amount of each 35S-synthesized protein was analyzed by SDS-gel electrophoresis and quantitated using a Molecular Dynamics PhosphorImager with ImageQuant software. The number of fmol of each cT3Ralpha protein synthesized in the lysate was estimated by the relative amounts of the individual 35S-proteins and quantitation of reticulocyte lysate-synthesized cT3Ralpha by binding with L-[125I]T3 (24). Reticulocyte lysate-translated cT3Ralpha proteins (about 10 fmol/µl of lysate) were incubated with 5 fmol (30,000 dpm) of the 32P-labeled TREp or the TRE1/2 element. The 30-µl incubation mixture contained 25 mM Tris (pH 7.8), 0.5 mM EDTA, 75 mM KCl, 1 mM dithiothreitol, 0.2 µg of poly(dI-dC), 0.05% Triton X-100 (v/v), 30 µg of ovalbumin, 0.3 µM ZnCl2, 0.2 µg of RNase A, and 10% glycerol (v/v) and either 0.5, 1.5, 3.0, or 4.5 µl of reticulocyte lysate (24). Control reticulocyte lysate was added to the 0.5-, 1.5-, or 3.0-µl receptor preparations to adjust the final amount of lysate in each sample to 4.5 µl. Unless indicated otherwise, a comparison of the different cT3Ralpha proteins used the same number of fmol of receptor. Samples were analyzed by electrophoresis at 4 °C for 50 min in nondenaturing 6% polyacrylamide gels (acrylamide:bisacrylamide, 37.5:1) in buffer containing 10 mM Tris, 7.5 mM acetic acid, 40 µM EDTA (pH 7.8) (12, 14). The gels were then dried and autoradiographed. The assignment of receptor monomers and homodimers is based on the mobility of purified cT3Ralpha (14). Certain gel shift studies were quantitated using a PhosphorImager as indicated.

Influence of the NH2 Terminus of cT3Ralpha on the Formation of cT3Ralpha ·RXR Heterodimers in Solution in the Absence of DNA-- The binding of 35S-cT3Ralpha and mutants to GST and GST-RXR in vitro were carried out as described previously (34). L-[35S]Cysteine-labeled wild-type cT3Ralpha , cT3Ralpha (21-408), and the cT3Ralpha (21-408, 7/8) NH2-terminal mutant were prepared using TNT reticulocyte lysates (Promega). 25,000 dpm of 35S-labeled protein was incubated with 25 ng of GST-RXR or 10 ng of GST protein immobilized on glutathione-agarose beads in 300 µl of Buffer A for 1 h at 4 °C on an Orbitron rotator. Buffer A consists of 50 mM KCl, 25 mM Hepes (pH 7.9), 6% glycerol, 5 mM EDTA, 5 mM MgCl2, 1 mM dithiothreitol, and 0.05% Triton X-100 (34). The amount of GST protein used (10 ng) was equivalent to the amount of GST in the GST-RXR fusion protein (25 ng). Beads were collected by centrifugation at 4 °C for 5 min at ~500 × g and washed three times with 1 ml of Buffer A. The bound proteins were eluted with SDS-gel loading buffer and analyzed by SDS-gel electrophoresis followed by autoradiography. 35S-Labeled wild-type or mutant proteins in the binding assays were analyzed by electrophoresis and autoradiography to ensure that equal amounts of input radioactivity of the labeled protein were used.

    RESULTS
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Abstract
Introduction
Procedures
Results
Discussion
References

Residues 23KRKRK27 of the NH2-terminal A/B Domain of cT3Ralpha Are Necessary for Maximal Transcriptional Activation of Native TREs-- Our previous studies aimed at elucidating the functional role of the NH2-terminal A/B region of cT3Ralpha indicated that amino acids 21-30 of cT3Ralpha , which are also conserved in the NH2-terminal A/B domains of rT3Ralpha 1 and hT3Ralpha 1, are essential for both transcriptional activation and for efficient binding to TFIIB (34). More detailed in vitro binding studies revealed that residues 23KRKRK27 centered within amino acids 21-30 are required for efficient binding of cT3Ralpha with TFIIB (34). This suggests that the functional activity that we originally mapped to amino acids 21-30 may depend solely on these 5 basic residues. To test this possibility we compared the functional activities of cT3Ralpha (21-408) and cT3Ralpha (21-408, 7/8) in which 23KRKRK27 was changed to 23TITIT27. In transfection experiments with a reporter gene regulated by a single idealized TRE organized as an inverted repeat (TREp) of the optimized AGGTCA half-site (Delta MTV-TREp-CAT), cT3Ralpha (21-408) and cT3Ralpha (21-408, 7/8) showed similar activity (Fig. 1A). In contrast, with Delta MTV-TRE-GH-CAT or Delta MTV-TRE-Mal-CAT, which contain native TREs, cT3Ralpha (21-408) was much more active (Fig. 1, B and C). These results are similar to our previous findings that showed that the functional effect of the NH2 terminus is much more prominent on reporters containing lower affinity native TREs compared with reporters containing the idealized higher affinity TREp (34). Finally, coexpression of TFIIB, although enhancing the activity of both receptors, results in a much higher level of T3-dependent stimulation by cT3Ralpha (21-408) (Fig. 1C). The ability of TFIIB to enhance the activity of cT3Ralpha (21-408, 7/8) partially is not altogether unexpected and is similar to our previous results with cT3Ralpha (51-408). This most likely stems from the low affinity of amino acids 119-154 of the cT3Ralpha D region for TFIIB (34).


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Fig. 1.   Maximal transcriptional activation of native TREs by cT3Ralpha requires amino acids 23KRKRK27. Panel A, HeLa cells were transfected by electroporation with 5 µg of a reporter containing an idealized TREp inverted repeat (AGGTCA TGACCT) inserted at position -88 of Delta MTV-CAT (Delta MTV-TREp-CAT) (14). Cells were cotransfected with 2 µg of either cT3Ralpha (21-408) or cT3Ralpha (21-408, 7/8) expression vector and incubated with or without T3 (1 µM) for 48 h. Panel B, same as in panel A except that the reporter was Delta MTV-TRE-GH-CAT, which contains the native TRE from the rat growth hormone gene promoter. Panel C, HeLa cells were transfected with 5 µg of reporter Delta MTV-TRE-Mal-CAT containing the native TRE from the rat malic enzyme gene promoter (35). Cells were cotransfected with 0.75 µg of either cT3Ralpha (21-408) or cT3Ralpha (21-408, 7/8) expression vector with or without 2 µg of TFIIB expression vector and were incubated in the absence or presence of 1 µM T3 for 48 h. CAT activity was determined as described under "Experimental Procedures."

Residues 23KRKRK27 Affect DNA Binding of cT3Ralpha as Monomers and Homodimers-- The NH2 terminus of T3R isoforms may confer cell type and promoter specificity not just through divergent, isotype-distinct, interactions with other transcription factors (34, 44) but also through conferring distinct DNA binding properties to receptor isotypes (45, 46). For example, the DNA binding properties of v-erbA differ from those of c-erbA (cT3Ralpha ) and more closely resemble those of the RARs. The structural basis behind this difference in DNA recognition by v-erbA results from one or more changes within the v-erbA NH2-terminal domain (45, 47).

In a previous study we found that in the absence of RXR, cT3Ralpha binds to the TREp predominantly as a monomer, whereas cT3Ralpha (51-408) binds preferentially as a homodimer (34). Similar results were included in a study by Wong and Privalsky (47) but were not discussed further. This finding suggests that, in addition to the DBD, all or part of the NH2-terminal A/B domain may affect the DNA binding properties of cT3Ralpha . We also found that without RXR, cT3Ralpha (21-408) binds the native rat TRE-GH as a monomer, whereas cT3Ralpha (51-408) binds this element poorly as a monomer (34). To determine whether this different DNA binding of NH2-terminal cT3Ralpha mutants is influenced by the basic amino acid residues 23KRKRK27, we compared the binding of cT3Ralpha (1-408), cT3Ralpha (51-408), and cT3Ralpha (21-408, 7/8) to the TREp. As shown in Fig. 2A, both cT3Ralpha (21-408, 7/8) (lanes 7-9) and cT3Ralpha (51-408) (lanes 10-12) bind much more efficiently as homodimers when compared with wild-type cT3Ralpha (1-408) (lanes 4-6). That this difference results from the 23KRKRK27 sequence is shown by comparing the DNA binding of cT3Ralpha (21-408) and cT3Ralpha (21-408, 7/8) (Fig. 2B, lanes 1-3 and 4-6, respectively). Increasing amounts of cT3Ralpha (21-408) result in an increase in monomer binding, whereas cT3Ralpha (21-408, 7/8) binds as a homodimer even at very low concentrations. Therefore, the basic amino acid sequence 23KRKRK27 affects not only receptor transactivation potential and its binding to TFIIB but its DNA binding properties as well.


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Fig. 2.   Residues 23KRKRK27 affect DNA binding of cT3Ralpha . Wild-type cT3Ralpha , cT3Ralpha (21-408), cT3Ralpha (21-408, 7/8), and cT3Ralpha (51-408) were expressed by in vitro translation in reticulocyte lysates and incubated with 5 fmol (30,000 dpm) of the 32P-labeled TREp element. The conditions for gel mobility shift assays are described under "Experimental Procedures" (24, 34). The gels were then dried and autoradiographed. The assignment of receptor monomers (M) and homodimers (D) is based on the mobility of purified cT3Ralpha (14). Panel A, no binding to the TREp was detected using 0.5, 1.5, or 4.5 µl of control lysate (lanes 1-3, respectively). No homodimer binding was detected with 0.5, 1.5, or 4.5 µl of cT3Ralpha -expressing reticulocyte lysate (lanes 4-6, respectively). Monomer binding was detected using 1.5 µl of cT3Ralpha (21-408, 7/8)- and cT3Ralpha (51-408)-expressing lysates (lanes 8 and 11, respectively), and both monomer and homodimer binding were detected using 4.5 µl of the same lysates (lanes 9 and 12, respectively). Panel B, no homodimer binding was detected with 1.5, 3.0, or 4.5 µl of reticulocyte lysate expressing cT3Ralpha (21-408) (lanes 1-3, respectively). Monomer binding was detected using 1.5 µl of lysate expressing cT3Ralpha (21-408, 7/8) (lane 4), and both monomer and homodimer binding were detected using 3.0 and 4.5 µl of the same lysate (lanes 5 and 6, respectively).

T3 Inhibits Both Monomer and Homodimer DNA Binding of cT3Ralpha NH2-terminal Mutants-- Ligand binding to DNA-bound T3R is accompanied by a conformational change of the receptor (48) which may alter the T3R-DNA interaction (49). For example, T3 inhibits homodimer formation in vitro on various direct and everted, but not inverted, TREs (49, 50). In addition, T3 increases the electrophoretic mobility of T3R monomers and homodimers and of T3R-RXR heterodimers on inverted repeat TREs without changing their apparent stability (14, 24).

Given the involvement of the NH2-terminal domain and its 5 basic amino acids in cT3Ralpha -DNA binding, T3-induced conformational changes of cT3Ralpha may, depending on the physical integrity or structure of the A/B domain, result in distinct receptor DNA binding affinities. To test this possibility, gel mobility shift studies were performed with the TREp using wild-type cT3Ralpha (1-408), cT3Ralpha (51-408), cT3Ralpha (21-408), and cT3Ralpha (21-408, 7/8) in the absence or presence of T3. As shown in Fig. 3, cT3Ralpha (1-408) and cT3Ralpha (21-408) in the absence of T3 bind the TREp predominantly as monomers (lanes 1 and 3). As expected, T3 increased the electrophoretic mobility of these complexes without altering their apparent stability (lanes 2 and 4). Thus, deletion of the first 20 amino acids from the A/B domain does not affect receptor DNA binding in either the absence or presence of T3. In the absence of T3, cT3Ralpha (21-408, 7/8) and cT3Ralpha (51-408) bound as homodimers and somewhat less efficiently as monomers (lanes 5 and 7). Surprisingly (lanes 6 and 8), T3 almost completely eliminates monomer and homodimer DNA binding of cT3Ralpha (21-408, 7/8) and cT3Ralpha (51-408).


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Fig. 3.   T3 strongly inhibits DNA binding of cT3Ralpha (21-408, 7/8) and cT3Ralpha (51-408) to the TREp. Wild-type cT3Ralpha , cT3Ralpha (21-408), cT3Ralpha (21-408, 7/8), and cT3Ralpha (51-408) were expressed by in vitro translation in reticulocyte lysates and incubated with 5 fmol (30,000 dpm) of the 32P-labeled TREp element. Homodimer binding was not detected with reticulocyte lysates expressing either cT3Ralpha or cT3Ralpha (21-408) in the absence (lanes 1 and 3) or presence (lanes 2 and 4) of T3. cT3Ralpha (21-408, 7/8) and cT3Ralpha (51-408) in the absence of T3 bound as a monomer and homodimer (lanes 5 and 7). In the presence of 1 µM T3, binding of cT3Ralpha (21-408, 7/8) or cT3Ralpha (51-408) as monomer and homodimer was inhibited strongly (lanes 6 and 8).

NH2-terminal Mutants of cT3Ralpha Bind Poorly to a TRE1/2-- The preferential binding of cT3Ralpha (21-408, 7/8) or cT3Ralpha (51-408) to the TREp as a homodimer compared with the predominant monomeric binding of cT3Ralpha (21-408) or wild-type cT3Ralpha may result from an increased potential to homodimerize and/or a decreased potential to bind DNA as a monomer. This latter instance might result in an increased amount of this mutant receptor available to bind as a homodimer. To assess effects of the 23KRKRK27 sequence in the NH2 terminus on monomer binding, we compared the binding of cT3Ralpha (21-408) and cT3Ralpha (21-408, 7/8) with a TRE1/2 in the absence and presence of T3 (Fig. 4). The TRE1/2 is the same as the TREp except that it contains a single G to C change in one of the half-sites (AGGTCA to ACGTCA). Whereas cT3Ralpha (21-408) binds to the TRE1/2 in the absence or presence of T3 (lanes 1 and 2), cT3Ralpha (21-408, 7/8) does not bind to this element without or with T3 (lanes 3 and 4). This suggests that NH2-terminal amino acids 23KRKRK27 impose preferential monomer DNA binding on cT3Ralpha .


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Fig. 4.   Binding of cT3Ralpha (21-408) and cT3Ralpha (21-408, 7/8) to the TRE1/2. cT3Ralpha (21-408) and cT3Ralpha (21-408, 7/8) were expressed by in vitro translation in reticulocyte lysates and incubated with 5 fmol (30,000 dpm) of the 32P-labeled TRE1/2 element. cT3Ralpha (21-408) bound TRE1/2 in both the absence (lane 1) and presence (lane 2) of 1 µM T3. cT3Ralpha (21-408, 7/8) did not bind TRE1/2 irrespective of T3 incubation (lanes 3 and 4).

The DNA Binding Properties of cT3Ralpha Are Not Directly Related to the Number of Basic Residues Contained within 23KRKRK27-- To assess whether all or some of the basic residues within 23KRKRK27 are necessary for preferential monomer binding of wild-type cT3Ralpha , we performed gel mobility shift assays with NH2-terminal mutants containing different numbers and combinations of basic and neutral amino acid residues. As shown in Fig. 5A, wild-type cT3Ralpha and cT3Ralpha (21-408) bind the TREp predominantly as monomers (lanes 1 and 3), whereas cT3Ralpha (51-408) and cT3Ralpha (21-408, 7/8) bind predominantly as homodimers (lanes 2 and 4). Interestingly, mutant cT3Ralpha (21-408, 9/10), which has only the first 3 basic amino acid residues changed (23TITRK27), binds the TREp very poorly as a monomer and not at all as homodimer (lane 5). In contrast, mutant cT3Ralpha (21-408, 11/12, lane 6), which has the last 3 basic amino acid residues changed (23KRTIT27), binds the TREp almost as efficiently as cT3Ralpha (21-408, 7/8) or cT3Ralpha (51-408) with slightly more monomer than homodimer. Finally, mutant cT3Ralpha (21-408, 13/14) (lane 7), which has only the middle basic amino acid preserved (23TIKIT27), binds in a way similar that of cT3Ralpha (21-408, 11/12) but less efficiently. These data indicate that the affinity and mode of binding of these mutants to the TREp are not related directly to the number of the basic amino acid residues within amino acids 21-30. However, all 5 basic amino acid residues 23KRKRK27 are necessary for predominant high affinity monomer binding to TREp.


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Fig. 5.   Binding of cT3Ralpha variants to the TREp and TRE1/2. Panel A, wild-type cT3Ralpha , cT3Ralpha (51-408), cT3Ralpha (21-408), cT3Ralpha (21-408, 7/8), cT3Ralpha (21-408, 9/10), cT3Ralpha (21-408, 11/12), and cT3Ralpha (21-408, 13/14) were expressed by in vitro translation in reticulocyte lysates and incubated with either 5 fmol (30,000 dpm) of the 32P-labeled TREp (lanes 1-7, respectively) or the TRE1/2 element (lanes 8-14, respectively). Panel B, binding of the indicated proteins to the TREp in the absence or presence of 1 µM T3. Panel C, binding of the indicated proteins to the TRE1/2 in the absence or presence of T3.

To assess the effect of the changes in 23KRKRK27 on monomer binding we used the TRE1/2. As expected, wild-type cT3Ralpha and cT3Ralpha (21-408) bind the TRE1/2 (Fig. 5A, lanes 8 and 10), whereas cT3Ralpha (51-408) and cT3Ralpha (21-408, 7/8) bind very poorly (lanes 9 and 11). Mutants cT3Ralpha (21-408, 9/10) and cT3Ralpha (21-408, 13/14) (lanes 12 and 14) bind the TRE1/2 weakly. Interestingly, cT3Ralpha (21-408, 11/12) binds the TRE1/2 more efficiently than the other mutants (lane 13), which corresponds to its more efficient binding to the TREp (lane 6) compared with the other mutants.

The effect of T3 on the binding of the various NH2-terminal mutants to the TREp and the TRE1/2 is shown in Fig. 5, B and C. The expected pattern of DNA binding of wild-type cT3Ralpha , cT3Ralpha (51-408), cT3Ralpha (21-408), and cT3Ralpha (21-408, 7/8) in the absence or presence of T3 is shown in lanes 1-8 of Fig. 5, B and C. cT3Ralpha (21-408, 9/10) in the absence of T3 binds poorly as a monomer and not at all as a homodimer, and this is reduced further by T3 (lanes 9 and 10 in Fig. 5, B and C). Homodimer binding of cT3Ralpha (21-408, 11/12) and cT3Ralpha (21-408, 13/14) to the TREp is abolished by T3 (lanes 11-14 in Fig. 5B), and monomer binding is reduced markedly (lanes 11-14 in Fig. 5, B and C).

Amino acids 23KRKRK27 Affect the Efficiency of T3R·RXR Heterodimer Formation-- Although amino acids 23KRKRK27 influence monomer binding of receptor to DNA, these residues may also affect the ability of the receptor to dimerize. We examined this possibility by assessing the ability of cT3Ralpha (21-408) or cT3Ralpha (21-408, 7/8) to bind as heterodimers with RXR on the TREp (Fig. 6). Incubations were performed either in the absence or the presence of T3 and/or 9-cis-RA. As shown previously, cT3Ralpha (21-408) alone binds to the TREp predominantly as a monomer, and the electrophoretic mobility of this complex is increased by T3 (Fig. 6, lanes 1 and 2). In contrast, cT3Ralpha (21-408, 7/8) binds the TREp predominantly as a homodimer, and this complex is abolished by T3 (lanes 7 and 8). Interestingly, in the absence of T3 cT3Ralpha (21-408) forms heterodimeric complexes with RXR less efficiently than cT3Ralpha (21-408, 7/8) (lanes 3 and 9), and T3 increases the electrophoretic mobility of both complexes (lanes 4 and 10). 9-cis-RA alone does not affect either complex (lanes 5 and 11), whereas the combination of T3 and 9-cis-RA affects the mobility of both complexes as with T3 alone (lanes 6 and 12). Similar results were also found using a 32P-DR+4 containing AGGTCA half-sites instead of the TREp (data not shown). Importantly, these results indicate that amino acids 23KRKRK27 affect both monomer DNA binding and dimerization.


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Fig. 6.   Heterodimer formation between RXR and cT3Ralpha (21-408) or cT3Ralpha (21-408, 7/8). cT3Ralpha (21-408) and cT3Ralpha (21-408, 7/8) were expressed by in vitro translation in reticulocyte lysates and incubated with 5 fmol (30,000 dpm) of the 32P-labeled TREp element. Weak homodimer (D) and predominant monomer (M) binding of cT3Ralpha (21-408) in the absence of T3 (lane 1) was altered to exclusive monomer binding by 1 µM T3 (lane 2). In the presence of baculovirus-expressed mRXRbeta (5 ng), cT3Ralpha (21-408) formed monomer and heterodimer (HD) complexes (lane 3), which were not altered significantly by either T3 (lane 4) or 9-cis-RA (lane 5) or both (lane 6). Predominant homodimer binding of cT3Ralpha (21-408, 7/8) in the absence of T3 (lane 7) was abolished by T3 (lane 8). In the presence of mRXRbeta , cT3Ralpha (21-408, 7/8) formed almost exclusively heterodimers (lane 9). T3 effected the complete disappearance of the weak homodimer complex without altering the apparent stability of the heterodimer complex (lane 10). 9-cis-RA alone did not affect either heterodimer or weak homodimer complexes (lane 11). Binding in the presence of both T3 and 9-cis-RA was no different from binding in the presence of T3 alone (compare lane 12 with lane 10, respectively).

This increase in bound heterodimers of the cT3Ralpha mutant may reflect enhanced heterodimer formation and/or the binding of preformed heterodimer to DNA or a decrease in the "off rate" of prebound complexes. The stability of heterodimeric complexes between RXR and cT3Ralpha (21-408) or cT3Ralpha (21-408, 7/8) on the labeled TREp was examined by incubating preformed complexes for various times with a 1,000-fold excess of unlabeled TREp (Fig. 7A). The results shown in lanes 2-5 and 7-10 of Fig. 7A were quantitated using a PhosphorImager (Fig. 7B). Only ~20% of the total TREp-bound cT3Ralpha (21-408) is in the form of heterodimeric complexes in the presence of RXR (lane 2). In contrast, ~90% of the total TREp-bound cT3Ralpha (21-408, 7/8) is in the form of heterodimer-bound complexes in the presence of RXR (lane 7). Interestingly, the off rate of cT3Ralpha (21-408, 7/8)·RXR and cT3Ralpha (21-408)·RXR complexes prebound to the TREp does not differ significantly (Fig. 7B).


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Fig. 7.   Relative stability of various DNA-bound cT3Ralpha (21-408) and cT3Ralpha (21-408, 7/8) complexes. Panel A, cT3Ralpha (21-408) and cT3Ralpha (21-408, 7/8) were expressed by in vitro translation in reticulocyte lysates and incubated with 5 fmol (30,000 dpm) of the 32P-labeled TREp element. As a competitor, nonradioactive TREp (1,000 × molar excess) was added for different periods of time as indicated. Baculovirus-expressed mRXRbeta (5 ng) was included as indicated. Panel B, heterodimeric complexes shown in panel A were quantitated with a Molecular Dynamics PhosphorImager using ImageQuant software and are expressed as arbitrary (relative) units.

The results of Fig. 7B imply that the observed difference in the efficiency of heterodimer binding to the TREp results from an intrinsic difference in the formation rate and/or DNA binding of these complexes. To provide evidence for enhanced formation, we studied the binding of 35S-cT3Ralpha (21-408) and 35S-cT3Ralpha (21-408, 7/8) to GST and GST-RXR in the absence of DNA (Fig. 8). GST alone did not bind to either of the 35S-cT3Ralpha proteins. GST-RXR bound the NH2-terminal mutant more efficiently than 35S-cT3Ralpha (21-408), and this difference is similar to the relative increase in binding of cT3Ralpha (21-408, 7/8)·RXR heterodimers to the TREp as shown in Fig. 7.


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Fig. 8.   The NH2-terminal 23KRKRK27 motif influences the formation of cT3Ralpha /RXR heterodimers in solution. 25,000 dpm of 35S-labeled T3Ralpha (21-408) and cT3Ralpha (21-408, 7/8) were incubated with glutathione-agarose bound GST-RXR or GST in 300 µl of Buffer A (34) for 1 h at 4 °C as described under "Experimental Procedures." Beads were collected by centrifugation at 4 °C for 5 min at ~500 × g and washed three times with 1 ml of Buffer A. The bound proteins were eluted with SDS gel loading buffer and analyzed by SDS-gel electrophoresis followed by autoradiography. 35S-Labeled wild-type or mutant proteins in the binding assays were analyzed by electrophoresis and autoradiography to ensure that equal amounts of input radioactivity of the labeled proteins were used.

    DISCUSSION
Top
Abstract
Introduction
Procedures
Results
Discussion
References

Amino Acids 23KRKRK27 of the A/B Domain Are Required for Maximal Transcriptional Activation of Native TREs by cT3Ralpha -- A conserved sequence of 10 amino acids from NH2-terminal A/B domain of cT3Ralpha , rT3Ralpha 1, and hT3Ralpha 1 is essential both for transcriptional activation and for efficient binding to TFIIB (34). Within this sequence, 5 basic amino acid residues, 23KRKRK27, proved absolutely necessary for efficient receptor binding to TFIIB (34). A sequence similar to the 23KRKRK27 sequence found in T3Ralpha is not found in the A/B domains of T3Rbeta 1 or T3Rbeta 2. Indeed, we have found that removal of the A/B domain of rat T3Rbeta 1 does not alter its ability to interact with TFIIB or to activate the rat TRE-GH or TRE-Mal in Delta MTV-CAT (data not shown). However, all nuclear receptors contain a conserved basic sequence that follows their DBD, which appears to play a role in TFIIB binding (34).

In this study we show that the functional role of the A/B domain in ligand-dependent transcriptional activation by cT3Ralpha requires the 5 basic amino acid residues 23KRKRK27. This was determined by directly comparing the activities of cT3Ralpha (21-408) and cT3Ralpha (21-408, 7/8) in which all 5 basic residues (23KRKRK27) were changed to 23TITIT27. Like cT3Ralpha (51-408), which lacks the entire NH2-terminal A/B domain, cT3Ralpha (21-408, 7/8) was only slightly less active than cT3Ralpha (21-408) in transient transfection experiments with a reporter gene containing a single idealized TRE organized as an inverted repeat (Delta MTV-TREp-CAT; 34 and this study, Fig. 1A). However, in transfection experiments where native TREs from the rat growth hormone or malic enzyme gene promoters were used, cT3Ralpha (21-408, 7/8) was much less active in the presence or absence of TFIIB (Fig. 1, B and C). Indeed, the requirement for amino acids 21-30 of the A/B domain for optimal transcriptional activation of native TREs appears to depend solely on amino acids 23KRKRK27. cT3Ralpha (21-408, 13/14), where 23KRKRK27 was changed to 23TIKIT27, was somewhat more active than cT3Ralpha (21-408, 7/8) but much less active than wild-type cT3Ralpha (1-408). The other 23KRKRK27 mutants were closer in activity to cT3Ralpha (1-408) (not shown) even though their affinity for TFIIB is reduced (34). This probably reflects an integrative effect of the lower affinity of the mutants for TFIIB (34) and, as we show in this study, their increased ability to bind to response elements as homodimers and as heterodimers with RXR.

Amino Acids 23KRKRK27 Affect DNA Binding of cT3Ralpha as Monomers and Homodimers-- Previous studies from our laboratory indicated that the NH2-terminal region of cT3Ralpha might affect DNA binding of receptor to the TREp and the native TRE from the rat growth hormone gene promoter by altering the extent of monomer binding and dimerization (34). Hollenberg et al. (51) also found that the NH2 terminus of hT3Ralpha 1 reduced the ability of the receptor to dimerize. Our current study documents that the basic amino acid sequence 23KRKRK27 plays a key role in both DNA binding and dimerization of cT3Ralpha in addition to its role in transcriptional activation and TFIIB binding. First, both cT3Ralpha (21-408, 7/8), in which amino acids 23KRKRK27 were changed to 23TITIT27, and cT3Ralpha (51-408) bind to the TREp more efficiently as homodimers than cT3Ralpha (Fig. 2A). More direct evidence for the involvement of these residues in the DNA binding of receptor is provided by the finding that cT3Ralpha (21-408) binds predominantly as a monomer even at high concentrations, whereas cT3Ralpha (21-408, 7/8) binds efficiently as a homodimer even at low concentrations (Fig. 2A). We obtained results in gel shift studies using a DR+4 containing AGGTCA half-sites similar to those with the TREp (not shown). Interestingly, although the binding of cT3Ralpha and cT3Ralpha (21-408) to the TREp is not affected significantly by T3, the binding of cT3Ralpha (21-408, 7/8) and cT3Ralpha (51-408) to this element is inhibited strongly (Fig. 3). Hence, residues 23KRKRK27 may either stabilize a "proper" conformation of the DBD, or they may ensure optimal positioning of the DBD with respect to the TREp half-site. These alternative mechanisms may occur either through a direct interaction between amino acids 23KRKRK27 and the DBD or through interaction of these basic residues with some other region of the receptor such as the ligand binding domain, which may affect the structure of the DBD. Alternatively, residues 23KRKRK27 may affect both the integrity and positioning of the DBD and in addition may contact DNA directly. Irrespective of the actual mechanism by which amino acids 23KRKRK27 affect receptor DNA binding, they seem absolutely necessary in the presence of T3 for the receptor to bind to the TREp (Fig. 3).

The predominant DNA binding of cT3Ralpha (21-408, 7/8) as a homodimer may reflect decreased monomer binding potential, increased homodimerization potential, or a combination of both. DNA binding studies using the TRE1/2 to preclude homodimer binding suggest that amino acids 23KRKRK27 affect the ability of receptor to bind DNA as a monomer (Fig. 4). Although cT3Ralpha (21-408, 7/8) does not bind significantly to the TRE1/2, it does bind to the TREp as a monomer (e.g. compare Fig. 4, lane 3, Fig. 5C, lane 7 with Fig. 2A, lane 8, Fig. 2B, lane 4, Fig. 3, lane 5). One possible explanation for this finding would be that to bind to DNA cT3Ralpha (21-408, 7/8) has to make an initial contact exclusively as a homodimer. Once this homodimer-DNA contact is established one cT3Ralpha (21-408, 7/8) molecule could dissociate, leaving a relatively unstable monomer·DNA complex behind.

DNA Binding of cT3Ralpha as Monomers and Homodimers Is Influenced by Different Basic Amino Acids within the 23KRKRK27 Sequence-- The basic residues 23KRKRK27 are not equally important for preferential monomer binding of wild-type alpha -receptor. For example, cT3Ralpha (21-408, 9/10), which has amino acids 23TITRK27, binds to the TREp very poorly and only as a monomer (Fig. 5). In contrast, cT3Ralpha (21-408, 11/12), which has amino acids 23KRTIT27, binds to the TREp in a way similar to that of cT3Ralpha (51-408) with slightly more monomer than homodimer. Finally, cT3Ralpha (21-408, 13/14), which has amino acids 23TIKIT27, binds to the TREp in a way similar to that of cT3Ralpha (21-408, 11/12) but less efficiently. Hence, the affinity and the mode of binding of these mutants to the TREp are not related directly to the number of the basic amino acid residues within the sequence 23-27, and therefore these residues do not contribute equally to the receptor DNA binding. However, all 5 basic amino acid residues 23KRKRK27 are necessary for predominant high affinity monomer binding to TREp. In contrast, the affinity of cT3Ralpha for TFIIB does correlate directly with the number of these basic amino acids (data not shown).

The NH2-terminal 23KRKRK27 Sequence Influences the Extent of cT3Ralpha ·RXR Heterodimer Formation-- In addition to the effect of residues 23KRKRK27 on the binding of receptor monomers to DNA, these residues also influence the intrinsic dimerization potential of T3Ralpha . Fig. 6 indicates that cT3Ralpha (21-408, 7/8) binds as a heterodimer with RXR to the TREp element much more efficiently than cT3Ralpha (21-408). Thus, ~90% of the total TREp-bound cT3Ralpha (21-408, 7/8) is bound as a heterodimer with RXR, whereas only ~20% cT3Ralpha (21-408) participates in such complexes (Fig. 7A, lanes 2 and 7). Both complexes showed similar dissociation rates in the presence of a 1,000-fold excess of unlabeled TREp (Fig. 7A, lanes 2-5 and 7-10, and Fig. 7B), suggesting that the increased amount of heterodimers found with the cT3Ralpha mutant results from the more efficient formation and/or binding cT3Ralpha (21-408, 7/8)·RXR heterodimers to DNA. GST binding studies (Fig. 8) suggest that this increase results from the more efficient formation of heterodimers in solution, which then bind to DNA. The increased dimerization potential of the cT3Ralpha (21-408, 7/8) on the TREp does not result in enhanced activation of the Delta MTV-TREp-CAT reporter compared with cT3Ralpha (1-408) or cT3Ralpha (21-408) (Fig. 1A). This discrepancy may reflect the weaker interaction of cT3Ralpha (21-408, 7/8) with TFIIB, which could result in a number of transcriptionally active cT3Ralpha (21-408, 7/8)·RXR·TFIIB complexes on the optimized element similar to those with cT3Ralpha (21-408) or wild-type cT3Ralpha (1-408).

Influence of the NH2-terminal A/B Domain on DNA Binding of Other Members of the Thyroid Hormone/Retinoid Receptor Subfamily-- Several additional reports have provided evidence for involvement of the NH2 termini of related members of the nuclear hormone receptor family in DNA binding. Two NH2-terminal amino acids of v-erbA, His12 and Cys32 (which correspond to Arg24 and Tyr44 of cT3Ralpha ) have been shown, in conjunction with amino acid changes in the zinc finger domain, to contribute to a restricted half-site DNA binding specificity (45, 47, 52). RXRalpha and RXRgamma , but not RXRbeta , have been suggested to activate transcription by forming tetrameric complexes on DNA elements consisting of four reiterated weak half-sites (53). These isoform-specific DNA binding properties mapped to the NH2-terminal A/B domains. Replacing the RXRbeta A/B domain with that of RXRgamma resulted in both tetramer binding to DNA and transcriptional activation by the chimeric protein. That the NH2-terminal domain and the zinc finger region of nuclear hormone receptors may functionally cooperate was also shown by a study of the DNA binding properties of the RORs (46, 54). Thus, the differential DNA binding activities of RORalpha 1, RORalpha 2, and RORalpha 3 depend on their distinct NH2 termini, which, when fused to heterologous nuclear hormone receptors (e.g. hT3Rbeta 1), may impose novel DNA binding specificities. Finally, T3Ralpha and T3Rbeta bind differently to the same DNA element (33). Whereas T3Ralpha binds predominantly as a monomer to the TREp (14), T3Rbeta 1 binds predominantly as a homodimer, which is thought to result in part from its increased ability to dimerize (33, 51, 55). Importantly, T3Rbeta 1 and T3Ralpha show no homology in their NH2-terminal A/B domains. In particular, T3Rbeta 1 does not contain a sequence in its A/B domain similar to the T3Ralpha -specific KRKRK, which may account for the finding that mutant cT3Ralpha (21-408, 7/8) exhibits certain DNA binding properties more like T3Rbeta 1 than wild-type cT3Ralpha .

In conclusion, the influence of the NH2 terminus of T3Ralpha on transcriptional activation, dimerization, and DNA and TFIIB binding supports the idea that this domain may play a role in the selective regulation of specific genes and impart distinct context-dependent transactivation potential to the individual receptor isoforms. The three-dimensional structural studies of the T3Rs performed thus far in either the absence (56) or in the presence of DNA (31) have not included the NH2-terminal A/B domain. The marked effect of the KRKRK residues of T3Ralpha on both DNA binding and transactivation suggests important effects of this sequence on receptor structure. Although this interaction may involve the DBD, our finding that mutation of the KRKRK residues alters the effect of ligand on DNA binding also suggests an interaction between the NH2 terminus and the ligand binding domain (Figs. 3 and 5). Thus, structural studies that analyze homo- or heterodimerization or other DNA binding properties of the T3Rs need to include receptor moieties containing the NH2-terminal A/B domain. Future studies that focus on the similarities and differences in the function of the NH2-terminal regions of the T3Rs and the related retinoid receptor isoforms should provide important insights into the roles and mechanisms of action of these diverse receptor forms in gene regulation and development.

    FOOTNOTES

* This research was supported in part by National Institutes of Health Grant DK16636 (to H. H. S.). Oligonucleotide synthesis was provided by the New York University Medical Center (NYUMC) General Clinical Research Center (National Institutes of Health Grant M01RR00096). Sequence analysis and data base searches were through the NYUMC Research Computing Resource, which received support from National Science Foundation Grant DIR-8908095). This study is in partial fulfillment of a Ph.D. degree from the Sackler Institute for Graduate Biomedical Sciences (for E. H.) and for the Clinical and Molecular Endocrinology Training Program, New York University School of Medicine (for I. H.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ Present address: Laboratory of Molecular Cell Biology, Rockefeller University, 1230 York Ave., New York, NY 10021.

Member of the NYUMC Cancer Center (Grant CA16087). To whom correspondence should be addressed: Dept. of Medicine and Pharmacology, TH-454, New York University Medical Center, 550 First Ave., New York, NY 10016. Tel.: 212-263-6279; Fax: 212-263-7701; E-mail: samueh01{at}mcrcr.med.nyu.edu.

1 The abbreviations used are: HRE(s), hormone response element(s); T3, triiodothyronine; T3R, triiodothyronine receptor; RAR, retinoic acid receptor; RXR, retinoid X receptor; h, human; r, rat; c, chicken; DR, direct repeat; IR, inverted repeat; ER, everted repeat; DBD, DNA binding domain; TFIIB, transcription factor IIB; MTV, mammary tumor virus; TREp, palindromic thyroid hormone response element; CAT, chloramphenicol acetyltransferase; GH, growth hormone; LTR, long terminal repeat; Mal, malic enzyme; TRE1/2, TREp with single G to C change in one of the half-sites; pEX, pEXPRESS; RSV, Rous sarcoma virus; GST, glutathione S-transferase; 9-cis-RA, 9-cis retinoic acid; ROR, retinoid orphan receptor.

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Top
Abstract
Introduction
Procedures
Results
Discussion
References

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