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• W2031036179 abstract "The thyroid hormone receptors (TR) bind to cis-acting DNA elements as heterodimers with the retinoid X receptors (RXR). These heterodimers display distinct specificities in mediating the hormonal response to target gene transcription. We characterized the interaction between TRα1 and RXRα via their ligand binding domains (LBDs) and the effect of ligands on the interaction using a yeast two-hybrid system. The DNA binding domain (BD) of yeast Gal4 fusion to the LBD of TRα1 had no transcriptional activity on its own, but when it was coexpressed with the activation domain (AD) of yeast Gal4 fusion to LBD of RXRα conferred activation to a reporter gene harboring a Gal4 binding site, indicating that LBDs of TRα1 and RXRα interact with each other in solution. Furthermore, T3 and 9-cis-RA increased the reporter activity, and an additive effect was observed when both ligands were added, indicating that the TRα1·RXRα heterodimerization is augmented by their respective ligands in vivo. Using anin vitro pull-down experiment, we confirmed the ligand-dependent interaction observed in the yeast system. Matrix-bound glutathione S-transferase-RXRα specifically coprecipitated the 35S-labeled TRα1 above the control, and associated 35S-labeled TRα1 was increased by the addition of T3 and 9-cis-RA. These results imply a complex, sensitive cross-talk in vivo among nuclear receptors and their respective ligands through distinct hormonal signaling pathways. The thyroid hormone receptors (TR) bind to cis-acting DNA elements as heterodimers with the retinoid X receptors (RXR). These heterodimers display distinct specificities in mediating the hormonal response to target gene transcription. We characterized the interaction between TRα1 and RXRα via their ligand binding domains (LBDs) and the effect of ligands on the interaction using a yeast two-hybrid system. The DNA binding domain (BD) of yeast Gal4 fusion to the LBD of TRα1 had no transcriptional activity on its own, but when it was coexpressed with the activation domain (AD) of yeast Gal4 fusion to LBD of RXRα conferred activation to a reporter gene harboring a Gal4 binding site, indicating that LBDs of TRα1 and RXRα interact with each other in solution. Furthermore, T3 and 9-cis-RA increased the reporter activity, and an additive effect was observed when both ligands were added, indicating that the TRα1·RXRα heterodimerization is augmented by their respective ligands in vivo. Using anin vitro pull-down experiment, we confirmed the ligand-dependent interaction observed in the yeast system. Matrix-bound glutathione S-transferase-RXRα specifically coprecipitated the 35S-labeled TRα1 above the control, and associated 35S-labeled TRα1 was increased by the addition of T3 and 9-cis-RA. These results imply a complex, sensitive cross-talk in vivo among nuclear receptors and their respective ligands through distinct hormonal signaling pathways. Effects of thyroid hormone on a wide variety of tissues are mediated via specific nuclear thyroid hormone receptors (TRs) 1The abbreviations used are: TR(s), thyroid hormone receptor(s); RAR(s), retinoic acid receptor(s); PPAR(s), peroxisome proliferator-activated receptor(s); RXR(s), retinoid X receptor(s); DBD, DNA binding domain; LBD, ligand binding domain; T3, 3,3′,5-triiodo-l-thyronine; 9-cis-RA, 9-cis-retinoic acid; PCR, polymerase chain reaction; AD, activation domain; CPRG, chlorophenol red-β-d-galactopyranoside; GST, glutathioneS-transferase; Triac, 3,3′,5-triiodothyroacetic acid; T4, l-thyroxine. (1Oppenheimer J.H. Ann. Intern. Med. 1985; 102: 374-384Crossref PubMed Scopus (72) Google Scholar) which are the cellular homologs of v-erbA (2Weinberger C. Thompson C.C. Ong E.S. Lebo R. Gruol D.J. Evans R.M. Nature. 1986; 324: 641-646Crossref PubMed Scopus (1154) Google Scholar). Several isoforms of TRs and TR variants have been isolated (2Weinberger C. Thompson C.C. Ong E.S. Lebo R. Gruol D.J. Evans R.M. Nature. 1986; 324: 641-646Crossref PubMed Scopus (1154) Google Scholar, 3Thompson C.C. Weinberger C. Lebo R. Evans R.M. Science. 1987; 237: 1610-1614Crossref PubMed Scopus (337) Google Scholar, 4Nakai A. Sakurai A. Bell G.I. DeGroot L.J. Mol. Endocrinol. 1988; 2: 1087-1092Crossref PubMed Scopus (92) Google Scholar, 5Benbrook D. Pfahl M. Science. 1987; 238: 788-791Crossref PubMed Scopus (166) Google Scholar, 6Nakai A. Seino S. Sakurai A. Szilak I. Bell G.I. DeGroot L.J. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 2781-2785Crossref PubMed Scopus (105) Google Scholar) and shown to be the members of a gene superfamily that includes steroid receptors, retinoic acid receptors (RARs), vitamin D receptors, peroxisome proliferator-activated receptors (PPARs), and numbers of proteins with high homology but as yet unidentified ligands. Based on sequence homology and functional analysis, the nuclear receptors exhibit a modular structure with functionally separable domains. Members of the superfamily are characterized by a highly conserved cysteine-rich DNA binding domain containing two zinc finger structures necessary for sequence-specific DNA interaction (7Evans R.M. Hollenberg S.M. Cell. 1988; 52: 1-3Abstract Full Text PDF PubMed Scopus (487) Google Scholar). The complex carboxyl-terminal region of the receptors contains ligand binding, receptor dimerization, and putative transcriptional activation functions (8Carson J.M. Schrader W.T. O'Malley B.W. Endocr. Rev. 1990; 11: 201-220Crossref PubMed Scopus (740) Google Scholar). The TRs, as well as other members of the superfamily, regulate transcription by binding to response elements containing two or more copies (often degenerate) of the consensus motif AGGTCA (9Brent G.A. Harney J.W. Chen Y. Warne R.L. Moore D.D. Larsen P.R. Mol. Endocrinol. 1989; 3: 1996-2004Crossref PubMed Scopus (182) Google Scholar, 10Glass C.K. Holloway J.M. Devary O.V. Rosenfeld M.G. Cell. 1988; 54: 313-323Abstract Full Text PDF PubMed Scopus (463) Google Scholar). Recently it was shown that RARs, TRs, vitamin D receptors, and PPARs form heterodimers with the RXRs on bipartite hormone response elements composed of nonsymmetrical head-to-tail tandem AGGTCA half-sites (11Yu V.C. Delsert C. Andersen B. Holloway J.M. Devary O.V. Näär A.M. Kim S.Y. Boutin J.-M. Glass C.K. Rosenfeld M.G. Cell. 1991; 67: 1251-1266Abstract Full Text PDF PubMed Scopus (1060) Google Scholar, 12Kliewer S.A. Umesono K. Mangelsdorf D.J. Evans R.M. Nature. 1992; 355: 446-449Crossref PubMed Scopus (1239) Google Scholar, 13Zhang X.K. Hoffmann B. Tran P.B. Graupner G. Pfahl M. Nature. 1992; 355: 441-446Crossref PubMed Scopus (793) Google Scholar). To date two distinct dimerization surfaces were proposed in the DNA binding domain (DBD) and ligand binding domain (LBD) of TR. The surface in the DNA binding domain conferred selective power in DNA-dependent dimer formation (14Rastinejad F. Perlmann T. Evans R.M. Sigler P.B. Nature. 1995; 375: 203-211Crossref PubMed Google Scholar). In contrast to the interface within the DBDs, dimerization motifs in the LBDs permit the heterodimeric complex subsequently to interact with response elements. The carboxyl-terminal LBD is responsible for DNA-independent dimerization that in vitro allows performation of certain dimers in solution before DNA targeting. This dimerization function is believed to stabilize the complex and promote the recognition of DNA. Several heptad repeats in LBD are well conserved among the members of the erbA-related nuclear receptor family and have been proposed to form a hydrophobic surface that might act as a receptor dimerization interface (15Forman B.M. Yang C.R. Au M. Casanova J. Ghysdael J. Samuels H.H. Mol. Endocrinol. 1989; 3: 1610-1626Crossref PubMed Scopus (230) Google Scholar, 16Evans R.M. Science. 1988; 240: 889-895Crossref PubMed Scopus (6326) Google Scholar), which is structurally similar to the leucine zipper dimerization domain found in Jun-Fos (17Landschulz W.H. Johnson P.F. McKnight S.L. Science. 1988; 240: 1759-1764Crossref PubMed Scopus (2538) Google Scholar). The heterodimerization influences the recognition of DNA targets and confers specificity for a defined spacing between two directly repeating hexameric sequences. Deletion of heptad repeats abolished the trans-activation function of the receptor (18Glass C.K. Lipkin S.M. Devary O.V. Rosenfeld M.G. Cell. 1989; 59: 697-708Abstract Full Text PDF PubMed Scopus (353) Google Scholar, 19Holloway J.M. Glass C.K. Adler S. Nelson C.A. Rosenfeld M.G. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 8160-8164Crossref PubMed Scopus (44) Google Scholar). Furthermore, the formation of heterodimers results in cross-talk among different ligands potentially to affect a range of physiological processes. The effect of ligand on the formation of TR·RXR heterodimers in solution has not been demonstrated clearly. To investigate the role of ligand in the process of receptor dimerization before binding to the target DNA, we have used the yeast two-hybrid system that allows the measurement of specific protein-protein interaction in solution. In the present study, we demonstrated that TR/RXR interaction occurred in solution before DNA targeting and was augmented by T3 and 9-cis-RA additively both in vivo and in vitro. The genotype of theSaccharomyces cerevisiae reporter strain SFY526 is MATa, ura3–52, his3–200, ade2–101, lys2–801, trp1–901, leu2–3, 112, canr, gal4–542, gal80–538, URA3::GAL1-lacZ (obtained fromCLONTECH). The genotype of HF7c strain is MATa, ura3–52, his3–200, lys2–801, ade2–101, trp1–901, leu2–3, 112, gal4–542, gal80–538, LYS2::GAL1-HIS3::URA3::(GAL4 17-mers)3-CYC1-lacZ (obtained from CLONTECH). Both yeast host strains carry a lacZ reporter gene under the control of GAL4 binding site. HF7C contains a second reporter gene (HIS3), also under the control of GAL4 response elements. Yeast strains were grown at 30 °C in YPD medium (1% yeast extract, 2% Bacto-Peptone, 2% dextrose) or in synthetic selection medium with appropriate supplements. Gal4BD fusion proteins were expressed by transfecting pGBT9 (CLONTECH) vector harboring the cDNA encompassed TRα1 LBD (amino acids 120–410), or RXRα LBD (amino acids 200–461) (Fig.1). The pGBT9-TRα1(LBD) (amino acids 120–410) and pGBT9-RXRα(LBD) (amino acids 200–461) plasmids were constructed by inserting the cDNA sequence encompassing the hinge and the LBD of human TRα1 or RXRα (20Mangelsdorf D.J. Ong E.S. Dyck J.A. Evans R.M. Nature. 1990; 345: 224-229Crossref PubMed Scopus (1257) Google Scholar) intoBamHI-SalI sites orEcoRI-BglII sites of pGBT9, respectively. To introduce the EcoRI and BamHI sites into 5′- and 3′-ends of the LBD of TRα1, pMe21 (4Nakai A. Sakurai A. Bell G.I. DeGroot L.J. Mol. Endocrinol. 1988; 2: 1087-1092Crossref PubMed Scopus (92) Google Scholar), which contains the entire coding region of hTRα1 cDNA, was amplified by polymerase chain reaction (PCR) using following synthetic oligonucleotide primers: 5′-ggatccgtATGGCCATGGACTTGGTTCT-3′ (forward primer) and 5′-gtcgacTTAGACTTCCTGATCCTCAA-3′ (reverse primer). The forward primer contains the BamHI site (ggatcc) and an additional 2 base pairs (gt) to adjust the reading frame followed by the coding strand sequence just after DBD of hTRα1. The reverse primer includes theSalI site (gtcgac) and the coding sequence at the carboxyl terminus of hTRα1. The hinge and LBD of hRXRα were amplified by PCR using following forward and reverse oligonucleotide primers: 5′-gaattcATGAAGCGGGAAGCCGTGCA-3′ and 5′-agatctCTAAGTCATTTGGTGCGGC-3′, containing EcoRI (gaattc) and BglII (agatct) sites, respectively. Standard PCR conditions were used. The sequences of the PCR products were verified by sequencing. The resulting plasmids express the hinge and LBD of hTRα1(120–410) or hRXRα(200–461) as a fusion protein to the DBD of Gal4 transcription factor (BD-TRα1(LBD) and BD-RXRα(LBD)). For construction of expression plasmids for BD-TRβ1(LBD) (amino acids 174–461) and their mutant receptors, EcoRI-BamHI sites of the pGBT9 were used as a cloning site. The hinge and LBD (amino acids 174–461) of human TRβ1 cDNA were amplified by PCR using following forward and reverse oligonucleotide primers: 5′-gaattcATGAAGCGGGAAGCCGTGCA-3′ and 5′-agatctCTAAGTCATTTGGTGCGGC-3′, containing EcoRI (gaattc) and BglII (agatct) sites, respectively. pGAD424 (CLONTECH) was used for the expression vector for the Gal4 activation domain (AD)-TRα1 LBD or RXRα LBD fusion protein, pGAD424-TRα1(LBD) and pGAD424-RXRα(LBD). The BamHI and SalI insert from pGBT9-TRα1(LBD) or EcoRI and BglII fragment from pGBT9-RXRα(LBD) were inserted intoBamHI-SalI sites andEcoRI-BglII sites of pGAD424 in-frame, respectively. Resulting plasmids express the hinge and LBD of hTRα1(120–410) or hRXRα(200–461) as a fusion protein to the AD of Gal4 transcription factor (AD-TRα1(LBD) and AD-RXRα(LBD)). Yeast expression vector, which lacks the Gal4AD, pRXRα(LBD), was generated by replacing the KpnI-EcoRI fragment, which contains the Gal4AD sequence of the wild type pGAD424-RXRα(LBD) with synthetic oligonucleotides in-frame. Oligonucleotides used were 5′-CGCCGCCTCTAGAGAGATCG-3′ (sense) and 5′-AATT CGATCTCTCTAGAGGCGGCGGTAC-3′ (antisense). The nuclear localization signal was not removed in the above procedures. β-Galactosidase activity, which was the product of the lacZ reporter gene, was used to indicate the interaction between the two hybrid proteins in vivo, which reconstitutes Gal4 function. Recombinant strains were grown overnight at 30 °C in 5 ml of synthetic medium lacking leucine and tryptophan to select for the maintenance of plasmids. The yeast cultures were initially diluted to an A 600 nmof 0.05 and incubated in the presence of various concentrations of T3 and/or 9-cis-RA at 30 °C overnight in separate tubes. The A 600 nm was determined. 700 μl of Z buffer (60 mm Na2HPO4, 40 mm NaH2PO4, 10 mm KCl, 1 mm MgSO4, 50 mmβ-mercaptoethanol, pH 7.0) was added to 0.1 ml of culture. The cells in the reaction mixtures were permeabilized by adding 50 μl of 0.1% SDS and 50 μl of chloroform and vortex vigorously for 30 s. The reaction were started with the addition of 0.16 ml of chloroform red-β-galactopyranoside (CPRG) (4 mg/ml) at 30 °C and stopped by adding 0.5 ml of 3 mm ZnCl2, and theA 570 nm was read. β-Galactosidase activity was determined with the values at A 570 nm andA 600 nm using the equation U = (1,000 × A 570 nm)/(v ×t × A 600 nm), wheret is the time of reaction (min), and v is the volume of yeast culture used in the reaction mixture (ml) (21Miller J.H. Experiments in Molecular Genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1972Google Scholar). To express the fusion protein with glutathione S-transferase (GST), PCR-amplified full-length RXRα cDNA was inserted in-frame into BamHI and EcoRI cloning sites of the pGEX-2T vector (Pharmacia Biotech Inc.). The following oligonucleotides were used to amplify the full-length hRXRα: 5′-agatctcatATGGACACCAAACATTTCCTG-3′ (forward primer) and 5′-gaattcTAAGTCATTTGGTGCGGC-3′ (reverse primer). Overnight cultures of Escherichia coli JM109 carrying the recombinant GST-RXRα and GST control plasmid were diluted 100-fold, cultured for 5–6 h, and then induced with 0.1 mm isopropyl β-d-thiogalactopyranoside. After another 3 h, bacteria were collected and washed with phosphate-buffered saline. Pellets were suspended in phosphate-buffered saline containing 1% (v/v) Triton X-100 and sonicated. Debris was removed by centrifugation. The fusion protein or the GST control protein was bound to glutathione-Sepharose (Pharmacia) and washed extensively with phosphate-buffered saline containing 1% (v/v) Triton X-100. Matrix-bound proteins were used for interaction experiments. 35S-Labeled TRα1 protein was produced by in vitro translation (Promega TNT, Madison, WI) using a T7 expression vector containing full-length TRα1 cDNA (pET TRα1) (22Miyamoto T. Sakurai A. DeGroot L.J. Endocrinology. 1991; 129: 3027-3033Crossref PubMed Scopus (40) Google Scholar). In vitro translated35S-labeled TRα1 (1–2 μl) was incubated for 20 min at room temperature with glutathione-Sepharose (10 μl) preloaded with GST-RXRα fusion or GST control protein in 250 μl of binding buffer (20 mm Tris-Cl, pH 7.8, 100 mm NaCl, 10% glycerol, 1 mm dithiothreitol, 1 mm EDTA, 1 mm phenylmethanesulfonyl fluoride, 1 mmleupeptin, 1 mm pepstatin) in the presence or absence of 10−6m T3 and/or 9-cis-RA. After extensive washing with binding buffer, bound proteins were eluted in 25 μl of Laemmli sample buffer, boiled for 10 min, and resolved by SDS-polyacrylamide gel electrophoresis (10%) followed by autoradiography. The results of the in vitroreactions and the amount of 35S-labeled protein bound by GST or GST-RXRα were visualized and quantified using a PhosphoImager (Fuji BAS 1500). To investigate the ability of TRα1 and RXRα to heterodimerize through their LBD, the interaction of Gal4BD-TRα1(LBD) and Gal4AD-RXRα(LBD) fusion proteins in yeasts was examined using the yeast two-hybrid system. The yeast reporter strain HF7c was cotransformed with pGBT9-TRα1(LBD) or pGAD424-RXRα(LBD) or both plasmids and selected with tryptophan and leucine dropout medium. Transformed colonies were streaked onto tryptophan and leucine dropout medium with or without histidine. In this assay, the formation of a complex between TRα1(LBD) fused to Gal4BD and RXRα(LBD) fused to Gal4AD confers histidine auxotrophy and β-galactosidase activity. As shown in Fig. 2, the yeasts expressing Gal4BD-TRα1(LBD) and Gal4AD-RXRα(LBD) are allowed to grow in the absence of histidine. The yeast cotransformed with pairs of Gal4BD-TRα1(LBD) and Gal4AD, or Gal4BD and Gal4AD-RXRα(LBD), did not permit growth in the absence of histidine, indicating that histidine auxotrophy is the result of the interaction between TRα1(LBD) and RXRα(LBD). Similar results were obtained in the reverse experiment using cotransformants expressing Gal4BD-RXRα(LBD) and Gal4AD-TRα1(LBD) (data not shown). First, we examine the ligand-dependent transcriptional activity of Gal4BD-TRα1(LBD) or Gal4BD-RXRα(LBD) alone (Fig.3). pGBT9-TRα1(LBD) or pGBT9-RXRα(LBD) was introduced into SFY526 yeast strain to express Gal4BD-TRα1(LBD) or Gal4BD-RXRα(LBD) protein. Yeast colonies were selected with tryptophan dropout medium. Five independent yeast colonies were cultured overnight in the absence or presence of T3 (10−6m) and/or 9-cis-RA (10−6m), and β-galactosidase activities were determined. No increase of β-galactosidase activity was observed in the yeasts transformed with Gal4BD-TRα1(LBD) or Gal4BD-RXRα(LBD) expressing vector. T3 or 9-cis-RA did not show a significant increase of β-galactosidase activity (Fig. 3). The LBD of TRα1 or RXRα cannot function as a transcriptional activator even in the presence of ligands in the yeasts. Neither Gal4BD-TRα1(LBD) nor Gal4BD-RXRα(LBD) alone activates the transcription of GAL1promoter. Then we tested the ability of the heterodimers between LBDs of TR and RXR as a transcriptional activator for Gal4 DBD. pRXRα(LBD), just an expression vector for RXRα(LBD), was cointroduced into SFY526 with pGBT9-TRα1(LBD). No increase of β-galactosidase activity was observed in the yeasts transformed with Gal4BD-TRα1(LBD) and RXRα(LBD) expressing vector, and T3 or 9-cis-RA did not show a significant increase of β-galactosidase activity (Fig. 3). It is concluded, therefore, that the heterodimers of LBDs of TR and RXR cannot function as a transcriptional activator in the yeasts. When a combination of Gal4BD-TRα1(LBD) and Gal4AD-RXRα(LBD), or Gal4BD-RXRα(LBD) and Gal4AD-TRα1(LBD), were introduced into yeast strain SFY526, a significant increase in β-galactosidase activities was observed. Both T3 and 9-cis-RA increased the β-galactosidase activity, and an additive effect was observed when both ligands were present. Dose dependence of ligands for activation of TR/RXR interaction was assessed. Yeast colonies were cultured overnight in the presence of various concentration of T3 or 9-cis-RA. As shown in Fig. 4, T3 and 9-cis-RA increased the β-galactosidase activity in a dose-dependent manner. A half-maximal increase was observed at 10−7m for T3 and 10−6m for 9-cis-RA. We examined further the effect of 9-cis-RA (10−6m) on the T3-dependent TR/RXR interaction. As shown in Fig. 5 A, the presence of T3 further activated the β-galactosidase activity without changing the sensitivity to 9-cis-RA. In Fig. 5 B, the reverse experiments were performed to show the T3dose-response curve in the presence or absence of 10−6m T3. Similarly, the presence of 9-cis-RA further activated the β-galactosidase activity without changing the sensitivity to T3. All fusion proteins were expressed at approximately the same level in the transformed yeasts, as determined by ligand binding assay (data not shown). The above results were also verified by cotransforming the yeast host strain HF7c with the two hybrid vectors. In HF7c, the lacZreporter gene is under the control of a promoter different from that used to control the lacZ gene in SFY526. These two promoter share only GAL4 response elements in common; the rest of the promoter sequences differ significantly.Figure 5Additive effects of T3 and 9-cis-RA on the TRα1(LBD)/RXRα(LBD) interaction. A combination of pGBT9-TRα1(LBD) (BD-TRα) and pGAD424-RXRα(LBD) (AD-RXRα) was introduced into yeast SFY526 and selected with tryptophan and leucine dropout medium. Yeast colonies were cultured overnight in the presence of various concentrations of T3 with or without 9-cis-RA (10−6m) (panel A) or in the presence of various concentrations of 9-cis-RA with or without T3 (10−6m) (panel B). β-Galactosidase activities were determined using CPRG (“Materials and Methods”). Values are the mean ± S.D. for at least three independent experiments performed in duplicate.View Large Image Figure ViewerDownload Hi-res image Download (PPT) We next tested the effect of T3 analogs on activating the BD-TRα1(LBD)·AD-RXRα(LBD) complex. As shown in Fig.6, the order of potency of each analog to induce β-galactosidase activity is consistent with previously determined affinity constant of binding of the ligands to the TR (23Schueler P.A. Schwartz H.L. Strait K.A. Mariash C.N. Oppenheimer J.H. Mol. Endocrinol. 1990; 4: 227-234Crossref PubMed Scopus (123) Google Scholar). Half-maximal activation was obtained at 10−8mfor Triac, 10−7m for L-T3, D-T3, and 10−6m for L-T4 (Triac < L-T3 < D-T3 < L-T4). These compounds did not affect the growth property of the yeasts. The GST-RXRα fusion protein was used to investigate the effects of ligands on the interaction between the LBD of RXR and TR in vitro. Complexes with GST-RXRα were retained on glutathione-Sepharose, the beads were washed and pulled down by centrifugation, and associated proteins were analyzed by SDS-polyacrylamide gel electrophoresis and autoradiography. TRα1 was labeled with [35S]methionine by in vitrotranslation and incubated with GST (Fig.7, upper panel, second lane) or a GST-RXR fusion protein (third throughsixth lanes) bound to glutathione-Sepharose beads, as indicated. 35S-Labeled TRα1 was specifically retained in the presence of GST-RXRα but did not bind to the GST control protein (second lane). Approximately 7.5% of the total input of TRα1 was specifically bound to the GST-RXR fusion protein. Addition of T3 or 9-cis-RA increased the amount of35S-labeled TRα1 that associated with GST-RXRα, and further increase was observed when both ligands were present. We next investigated the ability of TRβ1(LBD) to interact with RXRα(LBD). Similar to the results with TRα1 and RXRα, both ligand-independent (constitutive) and ligand-induced interactions were observed with TRβ1 and RXRα (Fig.8). In addition, we also examined the heterodimerizing properties of specific mutant receptors, TRβ1(G345R) and TRβ1∧422, to compare ligand-dependent transcriptional activity with their heterodimerizing activity. TRβ1(G345R), which was isolated from a patient with resistance to thyroid hormone (24Sakurai A. Miyamoto T. Refetoff S. DeGroot L.J. Mol. Endocrinol. 1994; 4: 1988-1994Crossref Scopus (111) Google Scholar), has no detectable T3 binding activity because of a glycine to arginine substitution at amino acid 345 in the hormone binding domain. TRβ1∧422 is an artificial mutant receptor with 4 amino acids insertion in the 9th heptad region, which is known to be essential for heterodimerization with RXR (18Glass C.K. Lipkin S.M. Devary O.V. Rosenfeld M.G. Cell. 1989; 59: 697-708Abstract Full Text PDF PubMed Scopus (353) Google Scholar). The ligand binding-defective mutant TRβ1(G345R) revealed the constitutive and 9-cis-RA-induced interaction but did not show the T3-induced interaction. Heterodimerization-defective mutant TRβ1∧422 failed to support both constitutive- and ligand-induced interaction in the yeasts (Fig. 8). The TRs are believed to function as heterodimers with RXR and exert their physiological activities through binding to the thyroid hormone response elements in DNA (16Evans R.M. Science. 1988; 240: 889-895Crossref PubMed Scopus (6326) Google Scholar). Dimerization of receptors through the specific interfaces that exist in the LBD has been demonstrated for the glucocorticoid receptor, estrogen receptor, and progesterone receptor (26Kumar V. Chambon P. Cell. 1988; 55: 145-156Abstract Full Text PDF PubMed Scopus (958) Google Scholar, 27Tsai S.Y. Carlstedt D.J. Weigel N.L. Dahlman K. Gustafsson J.A. Tsai M.J. O'Malley B.W. Cell. 1988; 55: 361-369Abstract Full Text PDF PubMed Scopus (414) Google Scholar, 28Fawell S.E. Lees J.A. White R. Parker M.G. Cell. 1990; 60: 953-962Abstract Full Text PDF PubMed Scopus (485) Google Scholar). Evidence for heterodimer formation between TR and RXR (20Mangelsdorf D.J. Ong E.S. Dyck J.A. Evans R.M. Nature. 1990; 345: 224-229Crossref PubMed Scopus (1257) Google Scholar) and TR homodimer formation (19Holloway J.M. Glass C.K. Adler S. Nelson C.A. Rosenfeld M.G. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 8160-8164Crossref PubMed Scopus (44) Google Scholar) has been suggested based on TR/RXR interaction in cotransfection studies and gel mobility shift assay or cross-linking studies (11Yu V.C. Delsert C. Andersen B. Holloway J.M. Devary O.V. Näär A.M. Kim S.Y. Boutin J.-M. Glass C.K. Rosenfeld M.G. Cell. 1991; 67: 1251-1266Abstract Full Text PDF PubMed Scopus (1060) Google Scholar, 12Kliewer S.A. Umesono K. Mangelsdorf D.J. Evans R.M. Nature. 1992; 355: 446-449Crossref PubMed Scopus (1239) Google Scholar, 13Zhang X.K. Hoffmann B. Tran P.B. Graupner G. Pfahl M. Nature. 1992; 355: 441-446Crossref PubMed Scopus (793) Google Scholar). To examine in detail the interaction between TR and RXR in solution and the effect of their ligands in vivo, we have established the yeast two-hybrid system as an in vivo approach to analyze the heterodimerization between the LBD of TR and RXR. In this report, we provide evidence that the TRs are able to heterodimerize with RXR in the absence of DNA, and T3 and 9-cis-RA augment this interaction in vivo, supporting the physiological importance of the heterodimer formation in solution. LBDs of TRα1 and RXRα act as transcriptional regulators in mammalian cells when fused to the heterologous DBD of the transcription factors (29Forman B.M. Umesono K. Chen J. Evans R.M. Cell. 1996; 81: 541-550Abstract Full Text PDF Scopus (567) Google Scholar). Therefore, it is important to know whether Gal4BD-TRα1(LBD) or Gal4BD-RXRα(LBD) alone can activate the reporter gene in a ligand-dependent manner in the yeast system. If the ligand-induced activity of the reporter gene could be caused by activation of Gal4BD-TRα1(LBD) or Gal4BD-RXRα(LBD) by the ligands, the activities of the reporter gene do not exactly reflect the interaction of the two proteins. In Fig. 3, we showed that neither of Gal4BD-TRα1(LBD) nor Gal4BD-RXRα(LBD) alone activates the reporter gene, irrespective of the presence of ligands. Moreover, using pRXRα(LBD), which lacks Gal4AD, we further analyzed the capability of the TRα(LBD)·RXRα(LBD) heterodimer to function as a ligand-dependent transcriptional activator in the yeasts. As shown in Fig. 3, a combination of Gal4BD-TRα1 and RXRα(LBD) did not activate the reporter gene even in the presence of ligands. These results indicate that neither TRα1(LBD) alone nor a combination of TRα1(LBD) and RXRα(LBD) confers ligand-dependent activation to the reporter gene in the yeast system. Therefore, it is concluded that the activity of the reporter gene indicates the reconstitution of the Gal4 molecule, allowing the measurement of the strength of interaction between TRα1(LBD) and RXRα(LBD). Because the yeast two-hybrid system is extremely sensitive and can detect very transient interactions, we utilized the in vitropull-down experiment as a second more stringent assay to confirm the ligand-induced heterodimerization seen in the yeast system. As shown in Fig. 7, it is demonstrated that the dimerization can occur in solution and that it is augmented by ligands in a pull-down experiment using the bacterially expressed RXRα fused to GST. It has been shown in mammalian systems that cotransfection of a Gal4-RXR expression vector and an expression vector for the LBD of the TR can confer T3 responsiveness to a promoter containing a Gal4 binding site (29Forman B.M. Umesono K. Chen J. Evans R.M. Cell. 1996; 81: 541-550Abstract Full Text PDF Scopus (567) Google Scholar). This experiment indicates that TR/RXR can form functional heterodimers in vivo in the absence of DBDs. Our results in the yeast system are consistent with the reported data in mammalian systems. In addition, augmentation of the TR/RXR interaction by T3 analogs correlated with the biological potency and ligand affinity of thyroid hormone analogs (Triac > T3 > T4), suggesting a physiological importance of ligand-induced heterodimerization in solution. In mammalian cells, the enhancement of T3-dependent reporter gene activation by TR·RXR heterodimers is well established, and the binding of TR to thyroid hormone response elements is much more efficient in the presence of RXR. Herein, we provide more direct evidence that TR and RXR form heterodimers before DNA targeting in vivo, and ligand binding clearly enhances receptor associations with each other. Previous studies have shown that ligand binding induced conformational change in TR in solution (30Ichikawa K. Hashizume K. Miyamoto T. Nishii Y. Yamauchi K. Ohtsuka H. Yamada T. J. Endocrinol. 1988; 119: 431-437Crossref PubMed Scopus (11) Google Scholar, 31Ichikawa K. Hashizume K. Furuta S. Osumi T. Miyamoto T. Yamauchi K. Takeda T. Yamada T. Mol. Cell. Endocrinol. 1990; 70: 175-184Crossref PubMed Scopus (9) Google Scholar, 32Miyamoto T. Suzuki S. DeGroot L.J. Mol. Endocrinol. 1993; 7: 224-231PubMed Google Scholar, 33Wagner R.L. Apriletti J.W. McGrath M.E. West B.L. Baxter J.D. Fletterick R.J. Nature. 1995; 378: 690-697Crossref PubMed Scopus (811) Google Scholar). The structures of the LBD of RARγ (34Renaud J.P. Rochel N. Ruff M. Vivat V. Chambon P. Gronemeyer H. Moras D. Nature. 1995; 378: 681-689Crossref PubMed Scopus (1030) Google Scholar) and TRα (33Wagner R.L. Apriletti J.W. McGrath M.E. West B.L. Baxter J.D. Fletterick R.J. Nature. 1995; 378: 690-697Crossref PubMed Scopus (811) Google Scholar) crystallized in the presence of their respective ligands revealed that helix 12 was aligned over the ligand binding pocket, in contrast to its position in unliganded receptors, in which it protrudes away from the LBD. In liganded receptors, hydrophobic residues within helix 12 face toward the pocket, perhaps contacting the ligand, whereas the negatively charged residues are exposed on the protein surface. Thus, as suggested by Renaud et al. (34Renaud J.P. Rochel N. Ruff M. Vivat V. Chambon P. Gronemeyer H. Moras D. Nature. 1995; 378: 681-689Crossref PubMed Scopus (1030) Google Scholar), realignment of helix 12 over the ligand binding pocket when the receptor binds ligand may generate a novel surface for interaction with the partner proteins or bridging proteins. Ligand binding seems to stabilize protein-protein interactions that lead to high affinity DNA binding and trans-activation. Although it is clear that ligand induces conformational change in the DNA-bound receptors, the precise molecular mechanism by which the TRs regulate transcription in response to the binding of ligands remain enigmatic. Ligand-dependent heterodimerization of TR and RXR is involved, at least in part, in the ligand-induced gene activation. It has been suggested recently that the DBD of TR and RXR can bind to a thyroid hormone response element sequence as a heterodimer in vitro, and a dimer interface found in the DBD has selective power for recognition of specific DNA interaction (36Rastinejad F. Perlmann T. Evans R.M. Sigler P.B. Nature. 1995; 375: 203-211Crossref PubMed Scopus (473) Google Scholar). On the other hand, we demonstrated that DNA binding is not necessary for heterodimer formation via the surface of the LBD, suggesting that several domains cooperate for forming heterodimers on target DNA in vivo. These results are consistent with the two-step hypothesis for binding of heterodimers to DNA (37Mangelsdorf D.J. Evans R.M. Cell. 1995; 83: 841-850Abstract Full Text PDF PubMed Scopus (2837) Google Scholar). In the first step, TR would form solution heterodimers with RXR through their LBDs. In the second step, the DBDs, by virtue of their proximity, would be able to bind a high affinity site in DNA. Once bound to DNA, the receptors are capable of modulating transcription. Since RXR plays a central role in mediating many hormonal signals, including retinoids, thyroid hormone, vitamin D3, and peroxisome proliferator activators, ligand (T3)-induced heterodimerization leads to an influence on other nuclear receptor signaling by squelching out the common partner, RXR. Recently, Chuet al. (25Chu R. Madison L.D. Lin Y. Kopp P. Rao M.S. Jameson J.L. Reddy J.K. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 11593-11597Crossref PubMed Scopus (89) Google Scholar) reported that TR inhibits PPAR signaling by squelching out RXR. They observed that T3 enhances the inhibition of PPAR activity by TR. Our results explain this phenomenon well, because T3 increases the number of TR·RXR heterodimers, resulting in a decrease in the number of active PPAR·RXR heterodimers. Recently the apparent hormone dependence was observed in an estrogen receptor dimerization experiment (26Kumar V. Chambon P. Cell. 1988; 55: 145-156Abstract Full Text PDF PubMed Scopus (958) Google Scholar). Wang et al. (35Wang H. Peters G.A. Zeng X. Tang M. Ip W. Khan S.A. J. Biol. Chem. 1995; 270: 23322-23329Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar) studied extensively estrogen receptor homodimerization using a yeast two-hybrid system. A particularly useful feature of the yeast two-hybrid system is the ability to study the heterodimerization activity of the various receptor forms. It would be interesting to analyze solution interaction among nuclear receptors including orphan receptors. This approach may lead to an understanding the role of ligand in transcriptional activation by nuclear receptors. We thank Dr. R. M. Evans for providing RXRα cDNA and Dr. Klaus for providing 9-cis-RA." @default.
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• W2031036179 title "Ligand-dependent Heterodimerization of Thyroid Hormone Receptor and Retinoid X Receptor" @default.
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