Identification of a novel human thyroid hormone receptor β isoform as a transcriptional modulator

Biochemical and Biophysical Research Communications 396 (2010) 983–988

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Identification of a novel human thyroid hormone receptor b isoform as a transcriptional modulator q Tetsuya Tagami a,*, Hiroyuki Yamamoto b, Kenji Moriyama b, Kuniko Sawai a, Takeshi Usui a, Akira Shimatsu a, Mitsuhide Naruse a a b

Division of Endocrinology, Metabolism and Hypertension, Clinical Research Institute, Kyoto Medical Center, National Hospital Organization, Kyoto 612-8555, Japan Department of Pharmacology, Graduate School of Pharmaceutical Sciences, Mukogawa Women’s University, Nishinomiya 663-8179, Japan

a r t i c l e

i n f o

Article history: Received 28 April 2010 Available online 12 May 2010 Keywords: Thyroid hormone receptor b Molecular cloning Tissue distribution Dominant negative effect

a b s t r a c t Thyroid hormone exerts a pleiotropic effect on development and homeostasis. A novel thyroid hormone receptor b isoform (hereafter referred to as TRb4) was cloned using PCR from a human pituitary cDNA library as a template. Analysis of the PCR products revealed a 137-bp insertion, which contains a stop codon in the middle, between the 5th and 6th exons that encode the ligand-binding domain of TRb. The corresponding sequence of this insertion exists within the 5th intron of the human TRb gene and consensus splice sequences were found at the junction sites. RACE analysis revealed that TRb4 is a carboxylterminal splicing variant of TRb1. RT-PCR and Northern blot analyses indicate that TR b4 mRNA is expressed in various human tissues, and especially abundant in testis and skeletal muscle. The TRb4 protein was unable to bind thyroid hormone (T3) and transient transfection assays demonstrate that TRb4 construct does not mediate T3-dependent gene regulation. TRb4 weakly but significantly inhibited transcription mediated by functional TR. Thus, this novel isoform may modulate hormone action as an endogenous antagonist in the tissue or cellular context. Ó 2010 Elsevier Inc. All rights reserved.

1. Introduction Thyroid hormone (T3) receptors (TRs), members of the large family of nuclear receptors, regulate transcription of specific target genes involved in development, differentiation and metabolism [1]. TRs exhibit functionally separable domains as also seen in other nuclear receptors. The DNA-binding domain (DBD) and the ligand-binding domain (LBD) of the TRs are highly conserved. The LBD is involved in homo- and heterodimerization [2] and in transcriptional activation or repression through interactions of the TR with coactivators (CoA) or corepressors (CoR) [3]. Two different genes, TRa (NR1A1) and TRb (NR1B1), encode the TRs, which are expressed as several isoforms [4]. The TRa locus generates the TRa1 [5] and several non-T3 binding proteins: TRa2 and TRDas, which result from internal promoter usage or Abbreviations: CoA, coactivator protein; CoR, corepressor protein; DBD, DNAbinding domain; FBS, fetal bovine serum; LBD, ligand-binding domain; Luc, luciferase; RTH, resistance to thyroid hormone; TK, thymidine kinase; TR, thyroid hormone receptor; TRE, thyroid hormone response element. q

All authors have nothing to declare. * Corresponding author. Address: Division of Endocrinology, Metabolism and Hypertension, Clinical Research Institute, National Hospital Organization Kyoto Medical Center, Mukaihata-cho 1-1, Fukakusa, Fushimi-ku, Kyoto 612-8555, Japan. Fax: +81 75 645 2781. E-mail address: [email protected] (T. Tagami). 0006-291X/$ - see front matter Ó 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2010.05.038

alternative splicing of the TRa primary transcript [6–11]. The TRa2 is identical to a functional receptor TRa1 for the first 370 amino acids, but the carboxyl (C)-terminal 40 amino acids are replaced by an entirely distinct sequence of 120 residues. Therefore, the C-terminal variant TRa2 has an inability to bind hormone and to function as a T3-dependent transcription factor and, in addition, the TRa2 can act as an antagonist to functional TRs such as TRa1 and TRbs, at least in transfected cells [12–15]. The TRb locus generates the TRb1 [16], TRb2 [17], rat TRb3 and TRDb3 [18] by using different promoters and alternative splicing. TRb1, TRb2 and TRb3 have an identical LBD and act as functional receptors. The expression of TRb2 is restricted to some specific organs, including the pituitary and hypothalamus, whereas the tissue distributions of TRa1, TRb1 and TRb3 are relatively ubiquitous [19–20] and the expression of these proteins begins early in development [21–25]. Truncated forms of TR isoforms, TRDa1, Da2 and Db3, lack the DBD [11,18]. Numbers of mouse models containing TR mutations in various combinations have been developed to understand the functions of each isoform [26]. TRa is crucial for post-natal development and cardiac function, whereas TRb mainly controls inner ear and retina development, liver metabolism and thyroid hormone levels. Besides mediating T3 action, the unliganded TR aporeceptor can exert a negative effect on transcription by recruiting CoR complexes with histone deacetylase activity [27]. The mutant TRs

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found in the human syndrome of resistance to thyroid hormone (RTH) behave as constitutive aporeceptor to exert dominant negative activity to wild type TRs, because most of the RTH mutants do not bind T3 [28]. The in vivo function of TRa2, TRDa1and Da2 is still unclear but may have some biological activity [26,29,30]. We report here that the identification of a novel human thyroid hormone receptor (designated TRb4), which does not bind T3, but may inhibit hormone action mediated by functional TRs. 2. Materials and methods 2.1. Primers and RACE-PCR Three oligonucleotides were designed on completely conserved regions of the DBD (primer 1) and LBD (primer 2 and primer 3) between human TRa and TRb. PCR was performed using the human pituitary gland 50 -STRETCH cDNA library (Clontech, CA) as a template. 50 -RACE was performed using the Marathon-Ready cDNA kit (Clontech) with gene-specific primers (primers 4 and 5) and adaptor primers, AP1 and AP2 (provided by the manufacturer). Similarly, 30 RACE was performed with gene-specific primers (primers 6 and 7) and adaptor primers, AP1 and AP2. The PCR was carried out at 94 °C for 5 s and 68 °C for 4 min for 30 cycles on GeneAmp System 9700 (Perkin-Elmer Applied Biosystems, Japan). Primer Primer Primer Primer Primer Primer Primer

1: 2: 3: 4: 5: 6: 7:

50 -TATCACTACCGCTGTATCAC-30 50 -AGGGACATGATCTCCATGCAGCA-30 50 -AGGAGGATGATCTGGTCTTC-30 50 -TGTCTTCTGGCTGTGTTCCTC-30 50 -TGCAGCCTTCAGATATCCCTGCTCTTCTTT-30 50 -GAAATTCCTGTTGATCTTCAT-30 50 -GACTTGTCAAGTAGACAAGGTCAG-30

Underlined sequences are TRb4-specific. 2.2. RT-PCR RT (reverse transcription)-PCR was performed using the AMV RNA PCR kit (Takara, Japan) with human multiple tissue poly A + mRNAs (Clontech, and OriGene, MD). The TRb4 and TRb1 mRNAs were co-amplified using the primers 1 and 8 and the products were verified by nucleotide sequencing. As controls, G3PDH and b-actin mRNA was amplified using the G3PDH primers provided by the kit (Clontech) and the b-actin primers (9 and 10), respectively. The PCR was carried out at 94 °C for 5 s and 68 °C for 4 min for 30 cycles on GeneAmp System 9700 (Perkin-Elmer Applied Biosystems). Primer 8: 50 -CTCTGGTAATTGCTGGTG-30 Primer 9: 50 -CACTCTTCCAGCCTTCCTTCC-30 Primer 10: 50 -CGGACTCGTCATACTCCTGCTT-30 2.3. Northern blot analysis Human multiple tissue Northern Blots (Clontech) were prehybridized for 1 h at 65 °C in ExpressHyb (Clontech) hybridization buffer. 32P-labeled hTRb4-specific probe, which was prepared as the PCR products using the primers 4 and 6, was added to the prehybridization solution and incubated for 1 h at 65 °C. Washing conditions were: 3  SSC at room temperature, followed by 3  SSC at 65 °C and then 1  SSC at 65 °C. 2.4. Plasmid constructions The numbering of the amino acid residues of TRb1 is based on a consensus nomenclature [31]. The expression plasmids of

hTRb4, pCMX-hTRb4, were prepared by exchanging the NsiIBstXI fragments of the PCR products with pCMX-hTRb1, driven by the cytomegalovirus (CMV)/T7 promoter [32]. The Gal4 constructs for hTRb1 contain the LBD of the hTRb1 (common among TRb1, 2 and 3) downstream of the Gal4-DBD in frame in pSG424, driven by the simian virus 40 early (sv40) promoter [33] and exchanged with the specific sequences of TRb4 to create Gal4TRb4. The reporter plasmid TREp-tk-Luc contains two copies of a palindromic TRE upstream of the thymidine kinase (tk) promoter in the pA3 luciferase vector [33] and DR4-sv40-Luc contains four copies of a direct repeat TRE upstream of the sv40 promoter in the pGL3 luciferase vector [15]. F2-tk-Luc, which contains the chick lysozyme TRE, (nucleotides 2358 to 2326; half-sites arranged as an inverted palindrome with a nucleotide gap of 6), was provided by P.M. Yen (National Institute of Health, Bethesda) [34]. The Gal4 reporter plasmid, UAS-tk-Luc, contains two copies of the Gal4 recognition sequence (UAS) upstream of tk109 in pA3Luc. 2.5. Western blotting NEC-8 cells, a clone of human testicular germ cell tumor, were purchased from The Japanese Collection of Research Bioresources and Health Science Research Resources Bank and were grown in RPMI1640 medium containing 10% FBS, penicillin (100 U/mL) and streptomycin (100 lg/mL). Nuclear extracts (10 lg) from transfected TSA-201 cells or NEC-8 cells prepared using Nuclear Extract kit (Active Motif, Carlsbad, CA) were analyzed by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) with 10% acrylamide gel. The proteins were electroblotted onto nitrocellulose membranes, followed by reaction with monoclonal antibodies (J52 or C4) as described previously [35]. The J52 was raised against the amino (N)-terminal domain of TRb1 and the C4 was against the C-terminal domain of TRb1 [36]. 2.6. Electrophoretic mobility shift assay TRb or RXRa was transcribed and translated using TNT-coupled reticulocyte lysate system labeled with Transcend non-radioactive translation detection system (Promega, WI). Reticulocyte lysates expressing TR (2 ll) with or without RXRa (2 ll) were preincubated at room temperature in a 20 ll reaction with a binding buffer consisting of 20 mM HEPES, pH 7.8, 50 mM KCl, 1 mM EDTA, 10% glycerol, 1 mM DTT, and 50 lg/mL poly(dI-dC) for 15 min. Unlabeled LAP-TREs (sense strand, agtcTGACCTgacgtcAGGTCActcga) were added, and the mixture was incubated for an additional 20 min. The protein-DNA complexes were analyzed by electrophoresis through a 5% polyacrylamide gel containing 2.5% glycerol in 0.5 TBE (45 mM Tris borate, 1 mM EDTA). The proteins were electroblotted onto nylon membranes and detected using the streptavidin alkaline phosphatase substrate. 2.7. Transient expression assays TSA-201 cells, a clone of human embryonic kidney 293 cells [37], were grown in Dulbecco’s modified essential medium (DMEM) containing 10% fetal bovine serum (FBS), penicillin (100 U/mL) and streptomycin (100 lg/mL) and transfected by the calcium phosphate method as described [33]. The transfection efficiencies were corrected with the internal control. Results are expressed as the mean ± SD from at least three transfections, each performed in triplicate. Data were analyzed by ANOVA with post hoc Dunnett’s tests to compare with the control.

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3. Results

3.2. RT-PCR and Northern blot analysis

3.1. Cloning of TRb4

To determine the tissue distribution of TRb4, RT-PCR was performed using human multiple tissue poly A + mRNAs. To see the relative expression ratio of TRb4 and TRb1, the primers were used that recognize TRb4 and TRb1 but not TRas. TRb1 mRNA was ubiquitously expressed as previously reported [38]. In contrast, TRb4 was less expressed in many tissues tested (left panel of Fig. 2A), but it was relatively abundant in skeletal muscle and brain. When RT-PCR was performed using a different set of RNA, it revealed that TRb4 mRNA was expressed higher than TRb1 in testis (right panel of Fig. 2A). Next, multiple tissue Northern blots were hybridized to the TRb4-specific probe (Fig. 2B). TRb4 was expressed predominantly as a 7.0-kb and was most abundant in the skeletal muscle, followed by the brain and heart.

The PCR primers were designed on completely conserved regions of the DBD and LBD between human TRa and TRb. Using a human pituitary gland cDNA library as a template, we cloned a PCR fragment, which contains novel sequences at the junction of the 5th and 6th exons that encode the LBD of TRb1. The fragment that contains the same insert was obtained using another set of primers (see Section 2). As shown in Fig. 1A, the length of insertion is 137 base pairs and the corresponding region is found on the reported genomic sequence of the human chromosome 3 clone (GenBank Accession No. AC093927). The consensus splice sequences were found at the junction site of both ends. The insertion encodes 13-amino acids before encountering a stop codon. To determine the whole sequences of the fragment, 50 - and 30 -RACEs were performed. The both of 50 and 30 ends coincided with those of TRb1. The predicted TRb4 protein contains 259-amino acids with a molecular mass of 29.5 kDa. Structural arrangement of the TRb gene is also shown (Fig. 1B). The specific exon for the TRb4 is within the 5th intron. The nuclear localization signal, common to TRb1, is preserved in the amino terminal of LBD of TRb4.

3.3. Western blot analysis The expression plasmids of TRb4 were created and were transcribed and translated using a reticulocyte lysate system. A 30kDa protein was detected by SDS–PAGE for TRb4 (Fig. 3A). Next, transient expression experiments were performed using TSA-201 cells, which are derivatives of 293 cells. To test the protein expression of the TRb4 in the nucleus, we performed Western blotting

---AAA TTC CTG Lys Phe Leu 244 246

Exon 5 (TRβ1 &4)

Intron 5 (TRβ4)

gtaaggcttc---cttttatcag TTG ATC TTC ATA AAA GAC TTG TCA AGT AGA CAA GGT CAG TGA Leu Ile Phe Ile Lys Asp Leu Ser Ser Arg Gln Gly Gln stop 247 259 GAT GAG CCT CAG TTT CAG GAA TGA GAA GAA ATT ATA AAG AAG AGC AGG GAT ATC TGA AGG CTG CAG GGA GCA GCA TT A CCA AAG GAG GAA CAC* AG gt acttttac---tcctccttag

Exon 6 (TRβ1)

CCA GAA GAC---

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Fig. 1. PCR cloning of TRb4 and structure of TRb4. (A) Nucleotide sequence and deduced amino acid sequence of the specific region of TRb4. The end of exon 5, intron 5 and the beginning of exon 6 of TRb1 are shown. Exon sequences are indicated in uppercase and intron sequences are in lowercase. Exons are based on the numbering of TRb1. The corresponding sequences of insertion are found within the intron 5 of TRb1 and the insertion is indicated as capital letters. Codon numbers are shown under the amino acid sequence. The consensus splice sequences were underlined at the junction site of both ends of the intron 5. A stop codon is indicated as bold letters. *’G’ in the reported genomic sequence (GenBank Accession No. AC093927) (B) TRb4 predicted proteins of 259 amino acids (29.5 kDa). The TRb4 is identical to a functional receptor TRb1 for the first 246 amino acids, but the C-terminal 215 amino acids are replaced by an entirely distinct sequence of 137 residues, that encodes 13-amino acids before encounter a stop codon. Structural arrangement of the TRb gene [23]) is shown at the bottom of the figure. Exons are shown as shaded boxes and introns are shown as lines. The specific exon for the TRb4 is shown as ‘x’ within the 5th intron and the estimated sizes of the adjacent introns are noted.

T. Tagami et al. / Biochemical and Biophysical Research Communications 396 (2010) 983–988

Co

n He trol ar Br t ai Pl n ac Lu enta n Li g ve Sk r ele Ki tal dn Mu e Pa y scle nc rea s

A

Sk ele t Sp al M lee us cle Te n sti s

986

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TRβ β-actin G3PDH

kb 9.5

3.4. DNA binding and RXR binding of TRb4

He a Br rt Pl ain a Lu cent n a Li g ve Sk r ele Ki tal M d Pa ney usc nc le rea s

B

The DNA binding and RXR binding were examined using EMSA. The TR protein expressed using a reticulocyte lysate system was incubated with LAP oligonucleotides. As shown in Fig. 3C, the homodimerization of TRb1 and its heterodimerization with RXR were clearly observed. In the case of TRb4, weak but significant homodimers but not heterodimers with RXR were observed.

7.5

4.4

3.5. Functional properties of TRb4

2.4 1.4

The functional characteristics of TRb4 were assessed based on their abilities to modulate expression of the T3 responsive reporter genes in TSA-201 cells. Using the TREp-tk-Luc, TRb1, TRb2 and TRa1 exhibited T3-dependent transcriptional stimulation, whereas TRb4 and TRa2 showed no apparent activation (Fig. 4A). The absence of transcriptional stimulation was not surprising because TRb4 lacks the majority of T3 binding domain and significant T3 binding was not seen in the T3 binding experiment (data not shown) [39]. The function of the LBD of TRb4 was also examined with a heterologous DBD (Fig. 4B). The LBD of TRb1, which is identical among TRb1-3, or that of TRb4 was fused to the DBD of the yeast transcription factor Gal4. The reporter gene, UAS-tk-Luc, which contains two Gal4 binding sites, was used to assess the transcriptional function of TRb4-LBD. Relative to the Gal4-TRb, the TRb4-LBD (Gal4-TRb4) conferred no T3-dependent stimulation.

Fig. 2. Tissue distribution of TRb4 mRNA. (A) RT-PCR was performed using human multiple tissue poly A + mRNAs (left: Clontech, right: OriGene) with common primers to TRb1 and TRb4. b-actin and/or G3PDH was amplified using the aliquots as a control. (B) Multiple tissue Northern blots (Clontech) were hybridized to the 32 P-labeled TRb4-specific probe, which was prepared as the PCR products using the TRb4-specific primers. RNA size markers are indicated on the left.

V

V

ec to TR r β1 TR β4 NE C8

B ec TR tor β TR 1 (rl β4 ) (rl )

A kDa

3.6. Dominant negative effect of TRb4

75

50

TRβ1

TRβ1

TRβ4

TRβ4

37

25 C4

ec to r TR β1 TR β β1 4 +R β4 X R +R α RX X R Rα α

J52

Because TRa2 is supposed to be an endogenous antagonist, we next examined the dominant negative activity of TRb4 and compared with those of TRa2. Because the protein expression of TRb4 construct was lower than that of TRb1 in the transfected cells (Fig. 3B) with unknown reason(s), a 1:5 ratio of functional TRs to TR isoforms was used to clearly illustrate the dominant negative properties of the receptors [39]. TRb4 as well as TRa2 inhibited TRb1- and TRa1-regulated expressions of TREp-tk-Luc (Fig. 4C) in the presence of 10 nM T3. Similar inhibitions were observed in the context of DR4-sv40-Luc (Fig. 4D) and F2-tk-Luc (Fig. 4E). 4. Discussion

V

C

using anti-TR antibody, which recognizes the N-terminal region of TRb1 and TRb4 (J52) [36]. The protein expression of each construct in the nuclear fraction of transfected cells was ascertained (left panel of Fig. 3B). Finally, the endogenous TRb4 protein was examined by Western blotting using a testicular tumor cell line because TRb4 mRNA was most abundant in testis. The corresponding size of protein with in vitro translated TRb4 was detected by J52 but not by C4 antibody (right panel of Fig. 3B). C4 recognizes the C-terminal of TRb1, which is truncated in TRb4. The significance of two bands corresponding to TRb4 seen in the NEC-8 is uncertain at this time.

β1/RXRα β1/β1 β4/β4 Fig. 3. Expression of TRb4 protein and interactions of TRb4 with DNA and RXR. (A) TRb1 or TRb4 was transcribed and translated using a reticulocyte lysate system labeled with a non-radioactive translation detection system, and analyzed by the SDS–PAGE with 10% acrylamide gel. (B) The nuclear extracts (10 lg) from transfected TSA-201 cells or nuclear extracts (10 lg) from NEC-8 cells were analyzed by SDS–PAGE and were electroblotted onto nitrocellulose membranes, followed by reaction with J52- or C4 mAbs. (C) Reticulocyte lysates expressing TR (2 ll) with or without RXRa (2 ll) were incubated with unlabeled LAP-TREs. The protein-DNA complexes were analyzed by EMSA. The positions of the TR/TR homodimer and TR heterodimer with RXR are indicated.

A novel human TR isoform, TRb4, has been identified. The fragment from the 5th intron of TRb1 gene was inserted between 5th and 6th exons, resulting in the presence of a C-terminal variant of TRb1. TRb4 mRNA encodes a 259-amino acid, 29.5-kDa protein that contains a unique 13-amino acid C terminus. It retains DNA binding but does not bind T3 and RXR, because it lacks a majority of LBD of TRb1 [40]. TRb4 mRNA was expressed at low levels in various tissues, but relatively abundant in testis, skeletal muscle, and, at the lower level, brain and heart among others. The protein, which is supposed to be an endogenous TRb4, was detected in the testicular tumor cell line. Although TRb4 did not mediate T3dependent transcription, it behaved as a weak antagonist of functional TRs in transfected cells. The homologues in mouse or other

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A TREp-tk-Luc

UAS-tk-Luc †

†

†

†

100

100

80

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40

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0 l ro nt o C

β1 TR

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4 1 BD TRβ Rβ -D -T 4 4 4 l l l Ga Ga Ga

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α1 TR

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DR4-sv40-Luc

F2-tk-Luc

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40

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40

40

−T3 +T3

100

TRβ1

* †

TRβ4

100

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100

Control

Luciferase Activity

C

TRα2

Luciferase Activity

−T3 +T3

B

TRβ1

Fig. 4. Function of TRb4 or TR isoforms and dominant negative activity of TRb4 and TRa2. (A) TR expression plasmids (10 ng) for the indicated isoforms were transfected into TSA-201 cells together with 100 ng of the positively regulated reporter gene, TREp-tk-Luc.  P < 0.01 vs. control. (B) Gal4 expression plasmids (10 ng) for the indicated isoforms were transfected into TSA-201 cells together with 100 ng of the Gal4 responsive reporter gene, UAS-tk-Luc.  P < 0.01 vs. control. (C-E) TRb4 or TRa2 expression plasmids (50 ng) were cotransfected with 10 ng of TRb1 or TRa1 into TSA-201 cells together with 100 ng of TREp-tk-Luc (C), DR4-sv40-Luc (D), or F2-tk-Luc (E). *P < 0.05 and  P < 0.01 compared with the activity in the absence of T3. Cells were incubated in the absence or presence of 10 nM T3. Results are the mean ± SD from at least three transfections performed in triplicate.

species were not detected using EST and genomic sequence database. TRb4 is the first identified C-terminal variant of TRb isoforms and it is comparable to TRa2, which is a C-terminal variant of TRa isoforms. The in vivo function of TRa2 is still unclear even in the context of several knockout mice [26]. The TRa2 is highly expressed in several tissues, including brain, kidney and testis [21]. Because the metabolic effects of T3 are relatively modest in these tissues, it has been proposed that TRa2 could play a role as an endogenous antagonist. The inhibitory activity of TRa2 is relatively weak, at least when compared to the dominant negative activity of mutant TRs that occur in patients with RTH in transient expression assays [14] because it weakly interacts with RXR or CoRs [15]. The dominant negative activity of TRb4 was similar to TRa2. Although the physiological role of TRb4 remains to be elucidated, it may also be an endogenous antagonist in some tissues such as testis and skeletal muscle. Since the TRa2: TRa1 ratio may be high enough to suggest a possible inhibitory effect in a number of tissues such as testis and brain, TRb4 may also modulate TRb1 action in tissues such as testis and skeletal muscle. Thus, the expression of func-

tional TRs (TRb1 and TRa1) may be switched to non-functional TRs (TRb4 and TRa2, respectively) by the alternative splicing as a buffer. In conclusion, this novel isoform, TRb4, may modulate T3 action as an endogenous antagonist in the tissue or cellular context, like TRa2. However, ultimately, gene knockouts or overexpression in transgenic animals may be required to understand physiological significance of TRb4. Acknowledgments The authors are grateful to R.M. Evans for kindly providing plasmids and to Drs. S.-Y. Cheng and F. Furuya for the mAbs of TRb. We thank Dr. D. Nagata, Dr. N. Kanamoto, Ms. S. Sakaguchi, Ms. K. Kushii, Ms. K. Matsuda and Mr. Y. Tsukuda for their technical assistance. We are deeply grateful to Dr. J. Larry Jameson for critical reading of the manuscript. This study was supported by the grants from the Ministry of Education and Science (No. 17590973 and 20591106), the Yamaguchi Endocrine Research Foundation and the Novo Nordisk Growth and Development Research Foundation.

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