INTRODUCTION

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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-erbA2) whose ligand(s), if any, are unknown (3-5).

The T₃Rs are encoded by two distinct but closely related genes ( and ) which, in humans (h), map to chromosomes 17 and 3, respectively (6). Each gene expresses several alternatively spliced isoforms. The T₃R gene in the rat (r) and man expresses the T₃-binding isoform T₃R1 along with c-erbA2, which does not bind T₃ because of alternative splicing at the COOH terminus (3, 7, 8). The closely related chicken (c)  gene expresses only cT₃R, which is more than 90% similar at the amino acid level to rT₃R1 and hT₃R1 (6, 9, 10). The T₃R gene expresses T₃R1 and T₃R2 that differ only in their NH₂-terminal A/B regions, which are distinct from the A/B region of T₃R1 (3, 11). Except for the A/B domains, the T₃R and T₃R 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 -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, T₃R binds more efficiently as monomers to these elements, and T₃R isoforms bind more efficiently as homodimers (33).

We reported previously that a 10-amino acid sequence within the A/B domain of cT₃R or rT₃R1 was essential for ligand-dependent activation of native HREs and for interaction of T₃R with TFIIB (34). Interestingly, deletion of the 50-amino acid A/B domain of cT₃R markedly reduced monomer binding and increased homodimer binding of the receptor, suggesting that the A/B domain of T₃R 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 cT₃R. 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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Plasmids-- MTV-TREp-CAT (14, 25), MTV-TRE-GH-CAT (25), and 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 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 TRE_1/2 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).

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-- cT₃R, cT₃R(21-408), cT₃R(21-408, 7/8), cT₃R(51-408), and the various cT₃R 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 cT₃R protein synthesized in the lysate was estimated by the relative amounts of the individual ³⁵S-proteins and quantitation of reticulocyte lysate-synthesized cT₃R by binding with L-[¹²⁵I]T₃ (24). Reticulocyte lysate-translated cT₃R proteins (about 10 fmol/µl of lysate) were incubated with 5 fmol (30,000 dpm) of the ³²P-labeled TREp or the TRE_1/2 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 cT₃R 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 cT₃R (14). Certain gel shift studies were quantitated using a PhosphorImager as indicated.

Influence of the NH₂ Terminus of cT₃R on the Formation of cT₃R·RXR Heterodimers in Solution in the Absence of DNA-- The binding of ³⁵S-cT₃R and mutants to GST and GST-RXR in vitro were carried out as described previously (34). L-[³⁵S]Cysteine-labeled wild-type cT₃R, cT₃R(21-408), and the cT₃R(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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Residues ²³KRKRK²⁷ of the NH₂-terminal A/B Domain of cT₃R 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 cT₃R indicated that amino acids 21-30 of cT₃R, which are also conserved in the NH₂-terminal A/B domains of rT₃R1 and hT₃R1, 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 cT₃R 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 cT₃R(21-408) and cT₃R(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 (MTV-TREp-CAT), cT₃R(21-408) and cT₃R (21-408, 7/8) showed similar activity (Fig. 1A). In contrast, with MTV-TRE-GH-CAT or MTV-TRE-Mal-CAT, which contain native TREs, cT₃R(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 cT₃R(21-408) (Fig. 1C). The ability of TFIIB to enhance the activity of cT₃R(21-408, 7/8) partially is not altogether unexpected and is similar to our previous results with cT₃R(51-408). This most likely stems from the low affinity of amino acids 119-154 of the cT₃R D region for TFIIB (34).

View larger version (22K): [in this window] [in a new window]   Fig. 1.   Maximal transcriptional activation of native TREs by cT3R 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 MTV-CAT (MTV-TREp-CAT) (14). Cells were cotransfected with 2 µg of either cT3R(21-408) or cT3R(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 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 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 cT3R(21-408) or cT3R(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 cT₃R 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 (cT₃R) 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).

View larger version (83K): [in this window] [in a new window]   Fig. 2.   Residues 23KRKRK27 affect DNA binding of cT3R. Wild-type cT3R, cT3R(21-408), cT3R(21-408, 7/8), and cT3R(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 cT3R (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 cT3R-expressing reticulocyte lysate (lanes 4-6, respectively). Monomer binding was detected using 1.5 µl of cT3R(21-408, 7/8)- and cT3R(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 cT3R(21-408) (lanes 1-3, respectively). Monomer binding was detected using 1.5 µl of lysate expressing cT3R(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 cT₃R 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).

View larger version (76K): [in this window] [in a new window]   Fig. 3.   T3 strongly inhibits DNA binding of cT3R(21-408, 7/8) and cT3R(51-408) to the TREp. Wild-type cT3R, cT3R(21-408), cT3R(21-408, 7/8), and cT3R(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 cT3R or cT3R(21-408) in the absence (lanes 1 and 3) or presence (lanes 2 and 4) of T3. cT3R(21-408, 7/8) and cT3R(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 cT3R(21-408, 7/8) or cT3R(51-408) as monomer and homodimer was inhibited strongly (lanes 6 and 8).

NH₂-terminal Mutants of cT₃R Bind Poorly to a TRE_1/2-- The preferential binding of cT₃R(21-408, 7/8) or cT₃R(51-408) to the TREp as a homodimer compared with the predominant monomeric binding of cT₃R(21-408) or wild-type cT₃R 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 cT₃R(21-408) and cT₃R(21-408, 7/8) with a TRE_1/2 in the absence and presence of T₃ (Fig. 4). The TRE_1/2 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 cT₃R(21-408) binds to the TRE_1/2 in the absence or presence of T₃ (lanes 1 and 2), cT₃R(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 cT₃R.

View larger version (34K): [in this window] [in a new window]   Fig. 4.   Binding of cT3R(21-408) and cT3R(21-408, 7/8) to the TRE1/2. cT3R(21-408) and cT3R(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. cT3R(21-408) bound TRE1/2 in both the absence (lane 1) and presence (lane 2) of 1 µM T3. cT3R(21-408, 7/8) did not bind TRE1/2 irrespective of T3 incubation (lanes 3 and 4).

The DNA Binding Properties of cT₃R 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 cT₃R, 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 cT₃R and cT₃R(21-408) bind the TREp predominantly as monomers (lanes 1 and 3), whereas cT₃R(51-408) and cT₃R(21-408, 7/8) bind predominantly as homodimers (lanes 2 and 4). Interestingly, mutant cT₃R(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 cT₃R(21-408, 11/12, lane 6), which has the last 3 basic amino acid residues changed (²³KRTIT²⁷), binds the TREp almost as efficiently as cT₃R(21-408, 7/8) or cT₃R(51-408) with slightly more monomer than homodimer. Finally, mutant cT₃R(21-408, 13/14) (lane 7), which has only the middle basic amino acid preserved (²³TIKIT²⁷), binds in a way similar that of cT₃R(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.

View larger version (55K): [in this window] [in a new window]   View larger version (100K): [in this window] [in a new window]   Fig. 5.   Binding of cT3R variants to the TREp and TRE1/2. Panel A, wild-type cT3R, cT3R(51-408), cT3R(21-408), cT3R(21-408, 7/8), cT3R(21-408, 9/10), cT3R(21-408, 11/12), and cT3R(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.

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 cT₃R(21-408) or cT₃R(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, cT₃R(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, cT₃R(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 cT₃R(21-408) forms heterodimeric complexes with RXR less efficiently than cT₃R(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.

View larger version (108K): [in this window] [in a new window]   Fig. 6.   Heterodimer formation between RXR and cT3R(21-408) or cT3R(21-408, 7/8). cT3R(21-408) and cT3R(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 cT3R(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 mRXR (5 ng), cT3R(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 cT3R(21-408, 7/8) in the absence of T3 (lane 7) was abolished by T3 (lane 8). In the presence of mRXR, cT3R(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).

View larger version (31K): [in this window] [in a new window]   Fig. 7.   Relative stability of various DNA-bound cT3R(21-408) and cT3R(21-408, 7/8) complexes. Panel A, cT3R(21-408) and cT3R(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 mRXR (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.

View larger version (80K): [in this window] [in a new window]   Fig. 8.   The NH2-terminal 23KRKRK27 motif influences the formation of cT3R/RXR heterodimers in solution. 25,000 dpm of 35S-labeled T3R(21-408) and cT3R(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

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Amino Acids ²³KRKRK²⁷ of the A/B Domain Are Required for Maximal Transcriptional Activation of Native TREs by cT₃R-- A conserved sequence of 10 amino acids from NH₂-terminal A/B domain of cT₃R, rT₃R1, and hT₃R1 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 T₃R is not found in the A/B domains of T₃R1 or T₃R2. Indeed, we have found that removal of the A/B domain of rat T₃R1 does not alter its ability to interact with TFIIB or to activate the rat TRE-GH or TRE-Mal in 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).

Amino Acids ²³KRKRK²⁷ Affect DNA Binding of cT₃R as Monomers and Homodimers-- Previous studies from our laboratory indicated that the NH₂-terminal region of cT₃R 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 hT₃R1 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 cT₃R in addition to its role in transcriptional activation and TFIIB binding. First, both cT₃R(21-408, 7/8), in which amino acids ²³KRKRK²⁷ were changed to ²³TITIT²⁷, and cT₃R(51-408) bind to the TREp more efficiently as homodimers than cT₃R (Fig. 2A). More direct evidence for the involvement of these residues in the DNA binding of receptor is provided by the finding that cT₃R(21-408) binds predominantly as a monomer even at high concentrations, whereas cT₃R(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 cT₃R and cT₃R(21-408) to the TREp is not affected significantly by T₃, the binding of cT₃R(21-408, 7/8) and cT₃R(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).

DNA Binding of cT₃R 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 -receptor. For example, cT₃R(21-408, 9/10), which has amino acids ²³TITRK²⁷, binds to the TREp very poorly and only as a monomer (Fig. 5). In contrast, cT₃R(21-408, 11/12), which has amino acids ²³KRTIT²⁷, binds to the TREp in a way similar to that of cT₃R(51-408) with slightly more monomer than homodimer. Finally, cT₃R(21-408, 13/14), which has amino acids ²³TIKIT²⁷, binds to the TREp in a way similar to that of cT₃R(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 cT₃R 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 cT₃R·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 T₃R. Fig. 6 indicates that cT₃R(21-408, 7/8) binds as a heterodimer with RXR to the TREp element much more efficiently than cT₃R(21-408). Thus, ~90% of the total TREp-bound cT₃R(21-408, 7/8) is bound as a heterodimer with RXR, whereas only ~20% cT₃R(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 cT₃R mutant results from the more efficient formation and/or binding cT₃R(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 cT₃R(21-408, 7/8) on the TREp does not result in enhanced activation of the MTV-TREp-CAT reporter compared with cT₃R(1-408) or cT₃R(21-408) (Fig. 1A). This discrepancy may reflect the weaker interaction of cT₃R(21-408, 7/8) with TFIIB, which could result in a number of transcriptionally active cT₃R(21-408, 7/8)·RXR·TFIIB complexes on the optimized element similar to those with cT₃R(21-408) or wild-type cT₃R(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 cT₃R) 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). RXR and RXR, but not RXR, 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 RXR A/B domain with that of RXR 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 ROR1, ROR2, and ROR3 depend on their distinct NH₂ termini, which, when fused to heterologous nuclear hormone receptors (e.g. hT₃R1), may impose novel DNA binding specificities. Finally, T₃R and T₃R bind differently to the same DNA element (33). Whereas T₃R binds predominantly as a monomer to the TREp (14), T₃R1 binds predominantly as a homodimer, which is thought to result in part from its increased ability to dimerize (33, 51, 55). Importantly, T₃R1 and T₃R show no homology in their NH₂-terminal A/B domains. In particular, T₃R1 does not contain a sequence in its A/B domain similar to the T₃R-specific KRKRK, which may account for the finding that mutant cT₃R(21-408, 7/8) exhibits certain DNA binding properties more like T₃R1 than wild-type cT₃R.