Characterization of a Thyroid Hormone Receptor Expressed in Human Kidney and Other Tissues AKIRA NAKAI, SUSUMU SEINO, AKIHIRO SAKURAI, ILLYA SZILAK, GRAEME I

Home , Thyroid hormone receptor

Proc. Nail. Acad. Sci. USA Vol. 85, pp. 2781-2785, April 1988 Medical Sciences Characterization of a thyroid hormone receptor expressed in human kidney and other tissues AKIRA NAKAI, SUSUMU SEINO, AKIHIRO SAKURAI, ILLYA SZILAK, GRAEME I. BELL, AND LESLIE J. DEGROOT* Thyroid Study Unit, Department of Medicine, Howard Hughes Medical Institute, and Department of Biochemistry and Molecular Biology, The University of Chicago, Chicago, IL 60637 Communicated by Donald F. Steiner, December 18, 1987

ABSTRACT A cDNA encoding a specific form of thyroid brary, which, although clearly related to the thyroid hormone hormone receptor expressed in human liver, kidney, placenta, receptor type a that has been described by others, is a distinct and brain was isolated from a human kidney library. Identical molecule.t In addition, we have demonstrated that the protein clones were found in human placenta and HepG2 cDNA encoded by this cDNA corresponds to a high-affinity triiodo- libraries. The cDNA encodes a 490-amino acid protein (Mr, thyronine (T3) receptor and that its mRNA is expressed in 54,824). When expressed and translated in vitro, the protein several human tissues. product binds triiodothyronine with Ka of 2.3 X 109 M-1. This protein, designated human thyroid hormone receptor type a2 (hTRa2), has the same domain structure as other MATERIALS AND METHODS members of the v-erbA-related superfamily of receptor genes. It is similar to thyroid hormone receptor type a described in cDNA Cloning and Sequencing. Human adult kidney and chicken and rat and less similar to human thyroid hormone hepatoblastoma (HepG2) cDNA libraries in AgtlO were receptor type (3 (formerly referred to as c-erbA.8) from prepared as described (14). A human placenta cDNA library placenta. However, it is distinguished from these receptors by in Agtll was kindly provided by Mark R. Hughes (Baylor an extension of the C-terminal hormone binding domain College of Medicine). Phage encoding thyroid hormone making it 80 amino acids longer than rat thyroid hormone receptor-like sequences were identified by cross-hybridiza- receptor type al [Thompson, C. C., Weinberger, C., Lebo, tion with a 32P-labeled human c-erbA cDNA clone isolated R. & Evans, R. M. (1987) Science 237, 1610-1614]. Different his col- sizes of mRNA found in liver (2.5 kilobases) and kidney (2 from a K562 cDNA library by Carlo Croce and kilobases) suggest there may be tissue-specific processing of leagues (Wistar Institute, Philadelphia, PA) (13). Hybridiz- the primary transcript of this gene. Identification of human ing clones were plaque-purified, and their EcoRI inserts thyroid hormone receptor type a2 indicates that two or more were sequenced by using the Sanger dideoxy chain-termina- forms of thyroid hormone receptor exist in human tissues and tion procedure (15). Synthetic oligonucleotide primers were may explain the normal variation in thyroid hormone respon- used for sequencing some portions of the cDNA. siveness of various organs and the selective tissue abnormali- RNA Isolation and Blotting. RNA was isolated from human ties found in the thyroid hormone resistance syndromes. tissues obtained at surgery, with permission of the Commit- tee on Human Investigation, by using the guanidinium Thyroid hormone is believed to regulate gene expression thiocyanate procedure (16). Poly(A) + RNA was prepared by through binding of a hormone-receptor complex to the oligo(dT) chromatography. Poly(A)+ RNA was denatured promoter region of target genes (1-4). The receptor, which is with glyoxal and, after electrophoresis through a 1.0% located in the nucleus of responsive cells, is of similar size in agarose gel, transferred to a nylon membrane (GeneScreen- all the tissues that have been examined and may exist in two Plus, DuPont). The membranes were hybridized with a forms of -47 and 56 kDa (5-7). However, the low abun- nick-translated insert from Akel2 (see below) in a solution of dance of the thyroid hormone receptor has hampered its 50% (vol/vol) formamide, 5 x SSC, 1 x Denhardt's solution, isolation and characterization from various tissues by con- sonicated and denatured salmon testes DNA at 100 ,ug/ml, ventional biochemical procedures (7), and it is unknown if a 0.1% NaDodSO4, and 10% (wt/vol) dextran sulfate at 42°C single protein mediates the actions of thyroid hormone or if for 14-24 hr. (1 x SSC = 0.15 M NaCl/0.015 M sodium there are a family of receptors. Clinical studies of thyroid- citrate, pH 7.0, and 1 x Denhardt's solution = 0.02% hormone-resistant subjects have indicated apparent differ- polyvinylpyrrolidone/0.02% Ficoll/0.02% bovine serum ences in hormone responsiveness in various tissues, suggest- albumin.) Membranes were washed in 0.1 x SSC/0.1% ing that there is a family of proteins that can function as NaDodSO4 at 60°C before autoradiography. thyroid hormone receptors (8). The demonstrations that the In Vito Expression of the Cloned Thyroid Hormone Recep- thyroid hormone receptor was the protooncogene of v-erbA tor. The insert in the kidney cDNA clone Ake7 was isolated (9-12) and that there indeed appears to be a family ofthyroid by partial EcoRI digestion and cloned into the EcoRI site of hormone receptors prompted us to isolate and characterize pGEM4Z (Promega Biotec, Madison, WI). This plasmid, cDNA clones that cross-hybridized with a c-erbA probe pke711, was linearized with HindIII and used as a template isolated from a human leukemia cell line (13) and was for SP6 polymerase-catalyzed synthesis of RNA (17). mapped to human chromosome 17, for sequences that might represent forms of the thyroid hormone receptor. In this Abbreviation: T3, triiodothyronine. report, we describe the isolation and sequence of a distinct *To whom reprint requests should be addressed at: The University thyroid hormone receptor from a human kidney cDNA li- of Chicago, Thyroid Study Unit-Box 138, 5841 South Maryland Avenue, Chicago, IL 60637. tThe sequence reported in this paper is being deposited in the The publication costs of this article were defrayed in part by page charge EMBL/GenBank data base (Bolt, Beranek, and Newman Labora- payment. This article must therefore be hereby marked "advertisement" tories, Cambridge, MA, and Eur. Mol. Biol. Lab., Heidelberg) in accordance with 18 U.S.C. §1734 solely to indicate this fact. (accession no. J03239). 2781 Downloaded by guest on September 30, 2021 2782 Medical Sciences: Nakai et al. Proc. Natl. Acad. Sci. USA 85 (1988)

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FIG. 1. Restriction map and schematic representation of human thyroid hormone receptor a2 cDNA clones. Common restriction endonuclease cleavage sites are indicated above the linear map. The Akel2, Ake7, and Ake6 clones were isolated from a human kidney library; the Ape43 clone was from a human placenta library; and the Ahe2 was from a HepG2 library. Capped RNA was translated in a micrococcal nuclease- cDNA libraries (Fig. 1). The sequences of the inserts from treated rabbit reticulocyte lysate (Promega Biotec). The each of these indicated that they were identical in overlap- translated proteins were denatured and reduced, and their ping areas and encoded various portions ofthe same mRNA. sizes were determined by electrophoresis in a 10% NaDod- Two clones, Ake7 and -12, contained a long opening reading S04/polyacrylamide gel. The thyroid hormone binding ac- frame of 1470 base pairs (bp) preceded by a short 5'-un- tivity of the translated proteins was assayed at 0°C as translated region and an inframe stop codon just before the described (7, 18). Briefly, 7 ul of lysate (from a total of 210 beginning ofthe long open reading frame. The insert in Akel2 ,ul) was incubated in 0.3 ml of KMPD (0.3 M KCI/1 mM is 2015 bp exclusive of the poly(A) tract (Fig. 2). The larger MgCl2/10 mM potassium phosphate, pH 7.85/1 mM dithio- 5' EcoRI fragment, from the insert in Akel2 spanning the threitol) and 6 pg of [1251]T3 (DuPont) for 2 hr. Bound and region from the 5' EcoRI linker to the internal EcoRI site, free T3 were separated with Dowex resin, and 30 ,l ofboiled hybridizes to a prominent adult kidney transcript of =z2 rat liver nuclear extract was added to reduce nonspecific kilobases (kb), and thus it is likely that this cDNA sequence binding of thyroid hormone receptor to resin (7). represents most of the mRNA (Fig. 3). The open reading frame encodes a protein of 490 amino acids and a molecular weight of 54,824 (Fig. 2). Comparison of this sequence, with RESULTS those of other thyroid hormone receptors, indicates 89% Isolation and Characterization of cDNA Clones Encoding DNA similarity and 99o amino acid similarity in the region the Thyroid Hormone Receptor. Phage hybridizing to the coding residues 1-370 of the receptor derived from rat brain human c-erbA clone isolated by Dayton and coworkers (13) designated rat thyroid hormone receptor type al (Figs. 4 and were isolated from human kidney, placenta, and HepG2 5); however, interestingly, the C-terminal regions of the two

1 GTGGCCCACCCCAGTCTCTTGGCGTGCTGGAGGGCATCCTGGhMGAATTGAAGTGA 838 CTGCGGGCGGCTGTCCGCTACGACCCTGAGAGCGACACCCTGACGCTGAGTGGGGAGATG 1 281 MetGl uGlnLysProSerLysValGl uCysGlySerAspProGluGluAsnSerAlaArg AlaValLysArgGluGlnLeuLysAsnGlyGlyLeuGlyValValSerAspAlaIlePhe 58 ATGGAACAGAAGCCAAGCAAGGTGGAGTGTGGGTCAGACCCAGAGGAGAACAGTGCCAGG 898 GCTGTCAAGCGGGAGCAGCTCAAGAATGGCGGCCTGGGCGTAGTCTCCGACGCCATCTTT 21 301 Se rProAspGlyLysArgLysArgLysAsnGlyGlnCysSerLeuLysTh rSerMetSer GluLeuGlyLysSerLeuSerAlaPheAsnLeuAspAspThrGluValAlaLeuLeuGln 118 TCACCAGATGGAAAGCGAAAAAGAAAGAACGGCCAATGTTCCCTGAAAACCAGCATGTCA 958 GAACTGGGCAAGTCACTCTCTGCCTTTAACCTGGATGACACGGAAGTGGCTCTGCTGCAG 41 321 GlyTyrIleProSerTy rLeuAspLysAspGluGlnCysValValCysGlyAspLysAla AlaValLeuLeuMetSerThrAspArgSerGlyLeuLeuCysValAspLysIleGluLys 178 GGGTATATCCCTAGTTACCTGGACAAAGACGAGCAGTGTGTCGTGTGTGGGGACAAGGCA 1018 GCTGTGCTGCTAATGTCAACAGACCGCTCGGGCCTGCTGTGTGTGGACAAGATCGAGAAG 61 341 ThrGlyTyrHisTyrArgCysIleThrCysGl uGlyCysLysGlyPhePheArgArgTh r SerGlnGluAlaTyrLeuLeuAlaPheGl uHisTyrValAsnHisArgLysHisAsnIle 238 ACTGGTTATCACTACCGCTGTATCACTTGTGAGGGCTGCAAGGGCTTCTTTCGCCGCACA 1078 AGTCAGGAGGCGTACCTGCTGGCGTTCGAGCACTACGTCAACCACCGCAAACACAACATT 81 361 IleGlnLysAsnLeuHisProThrTyrSerCysLysTyrAspSerCysCysValIleAsp ProHisPheTrpProLysLeuLeuMetLysGl uArgGluValGlnSe rSerIleLeuTy r 298 ATCCAGAAGAACCTCCATCCCACCTATTCCTGCAAATATGACAGCTGCTGTGTCATTGAC 1138 CCGCACTTCTGGCCCAAGCTGCTGATGAAGGAGAGAGAAGTGCAGAGTTCGATTCTGTAC 101 381 LysI leTh rArgAsnGl nCysGlnLeuCysArgPheLysLysCysIleAlaValGlyMet LysGlyAlaAlaAlaGluGlyArgProGlyGlySerLeuGlyValHisProGluGlyGln 358 AAGATCACCCGCAATCAGTGCCAGCTGTGCCGCTTCAAGAAGTGCATCGCCGTGGGCATG 1198 AAGGGGGCAGCGGCAGAAGGCCGGCCGGGCGGGTCACTGGGCGTCCACCCGGAAGGACAG 121 401 AlaMetAspLeuValLeuAspAspSerLysArgValAlaLysArgLysLeuIleGluGln GlnLeuLeuGlyMetHisValValGlnGlyProGlnValArgGlnLeuGluGlnGlnLeu 418 GCCATGGACTTGGTTCTAGATGACTCGAAGCGGGTGGCCAAGCGTAAGCTGATTGAGCAG 1258 CAGCTTCTCGGAATGCATGTTGTTCAGGGTCCGCAGGTCCGGCAGCTTGAGCAGCAGCTT 141 421 AsnArgGluArgArgArgLysGluGluMetIleArgSerLeuGlnGlnArgProGluPro GlyGluAlaGlySerLeuGlnGlyProValLeuGlnHisGlnSerProLysSerProGln 478 AACCGGGAGCGGCGGCGGAAGGAGGAGATGATCCGATCACTGCAGCAGCGACCAGAGCCC 1318 GGTGAAGCGGGAAGTCTCCAAGGGCCGGTTCTTCAGCACCAGAGCCCGAAGAGCCCGCAG 161 441 ThrProGluGluTrpAspLeuIleHisIleAlaThrGluAlaHisArgSerThrAsnAla GlnArgLeuLeuGluLeuLeuHi sArgSerGlyIleLeuHi sAlaArgAlaValCysGly 538 ACTCCTGAAGAGTGGGATCTGATCCACATTGCCACAGAGGCCCATCGCAGCACCAATGCC 1378 CAGCGTCTCCTGGAGCTGCTCCACCGAAGCGGAATTCTCCATGCCCGAGCGGTCTGTGGG 181 461 GlnGlySerHisTrpLysGlnArgArgLysPheLeuProAspAspIleGlyGlnSerPro GluAspAspSerSerGl uAlaAspSerProSe rSerSe rGl uGluGluProGluValCys 598 CAGGGCAGCCATTGGAAACAGAGGCGGAAATTCCTGCCCGATGACATTGGCCAGTCACCC 1438 GAAGACGACAGCAGTGAGGCGGACTCCCCGAGCTCCTCTGAGGAGGAACCGGAGGTCTGC 201 481 490 IleValSerMetProAspGlyAspLysValAspLeuGluAlaPheSerGluPheThrLys G1 uAspLeuAlaGlyAsnAlaAlaSerProEnd 658 ATTGTCTCCATGCCGGACGGAGACAAGGTGGACCTGGAAGCCTTCAGCGAGTTTACCAAG 1498 GAGGACCTGGCAGGCAATGCAGCCTCTCCCTGAAGCCCCCCAGAAGGCCGATGGGGAAGG 221 1558 AGAAGGAGTGCCATACCTTCTCCCAGGCCTCTGCCCCAAGAGCAGGAGGTGCCTGAAAGC IleIleThrProAlaIleThrArgValValAspPheAlaLysLysLeuProMetPheSer 16 18 TGGGAGCGTGGGCTCAGCAGGGCTGGTCACCTCCCATCCCGTAAGACCTCCTTCCCTTCC 718 ATCATCACCCCGGCCATCACCCGTGTGGTGGACTTTGCCAAAAAACTGCCCATGTTCTCC 1678 TCAGCAGGCCAAACATGGCCAGACTCCTTGCTTTTTGCTGTGTAGTTCCCTCTGCCTGGG 241 1738 ATGCCCTTCCCCCTTTCTCTGCCTGGCAACATCTTACTTGTCCTTTGAGGCCCCAACTCA GluLeuProCysGl uAspGlnIl eIleLeuLeuLysGlyCysCysMetGl uIleMetSe r 1998 AGTGTGCACCTCCTTCCCCAGCTCCCCCAGGCAGAAAATAGTTGTCTGTGCTTCCTTGGT 778 GAGCTGCCTTGCGAAGACCAGATCATCCTCCTGAAGGGGTGCTGCATGGAGATCATGTCC 1858 TCATGCTTCTACTGTGACACTTATCTCAGCTGTTTTATAATTAGTCGGGCATGAGTCTGT 261 1918 TTCCCAAGCTAGACTGTGTCTGAATCATGTCTGTATCCCCAGTGCCCGGTGCAGGGCCTG LeuArgAlaAlaValArgTyrAspProGluSerAspThrLeuThrLeuSerGlyGluMet 1978 GCATAGAGTAGGTACTCCATAAAAGGTGTGTTGAATTGAAAAAAAAAAAAAAAAAAAAAA FIG. 2. Nucleotide and deduced amino acid sequence of human thyroid hormone receptor type a2 cDNA. The initiation and termination codons of a short open reading frame in the 5'-untranslated region are underlined. The variant polyadenylylation signal (CATAAA) is also underlined. The nucleotide sequence of the coding region was determined on both strands. Downloaded by guest on September 30, 2021 Medical Sciences: Nakai et al. Proc. Natl. Acad. Sci. USA 85 (1988) 2783

1 2 3 4 thyroid hormone receptor as human thyroid hormone receptor type a2 to indicate that its sequence is not completely homologous to that reported (12) for a rat a-type receptor. In Vitro Synthesis of Thyroid Hormone Receptor Activity. Translation of SP6 polymerase generated transcripts of the FIG. 3. RNA gel blot analysis kidney thyroid hormone receptor resulted in the synthesis of 28S of human poly(A)+ RNA sam- a major protein of -58 kDa that is almost identical to the size ples probed with a 32P-labeled (55 kDa) predicted from the sequence of the protein; in 1417-bp EcoRI fragment of the addition, three other minor proteins of 54, 46, and 42.5 kDa human thyroid hormone receptor type a2. Lanes: 1, 1 ,ug of adult were observed (Fig. 6). The translated proteins were tested 18S- brain mRNA; 2, 4 ,ug of term for T3 binding. They bound T3 with an association constant placenta mRNA; 3, 4 pZg of adult of 2.3 x 109 M-1 (Fig. 7 Left), which is identical to the Ka kidney mRNA; 4, 16 ,ug of adult of thyroid hormone receptor extracted from a number of liver mRNA. Note that the abun- different human and rat tissues (5, 7). The Ka for tetraio- dance of the 2-kb transcript in brain and placenta is presumably dothyronine (T4) was -0.5 x 108 M-1 (Fig. 7 Right). Triac closer to that present in liver, (triiodothyroacetic acid) was less effective than unlabeled T3 since less mRNA was used from in displacing [125I]T3 from receptor (data not shown). brain and placenta. Expression of the Human Thyroid Hormone Receptor Type a2 Gene. On RNA gel blots of RNA prepared from several proteins are of different size and sequence. By contrast, the human tissues, the human thyroid hormone receptor type a2 human kidney receptor sequence has only 58% DNA simi- probe hybridized to a 2-kb transcript in kidney, placenta, larity with that of a human placenta receptor, designated and brain, and to a 2.5-kb transcript in liver (Fig. 3). In human thyroid hormone receptor type j31. There is 78% addition, this probe also hybridized to a transcript in these DNA similarity between the human kidney and chicken tissues of -5 kb. As we observe increased relative hybrid- embryo receptor type al. Thus it appears that the cDNAs ization to the larger transcript, when total rather than that we have isolated encode a protein different from the poly(A)+ is used, we believe that some of the signal may be described thyroid hormone receptors. The greater identity of due to cross-hybridization of the probe with 28S rRNA. this protein with rat thyroid hormone receptor type al than Also, cross-hybridization to unknown but related human human thyroid hormone receptor type 81 suggests that the thyroid hormone receptor type a forms cannot be excluded, kidney receptor is of the a type. We have adopted the but since we recovered identical clones from placenta and nomenclature suggested by Thompson et al. (12), designat- liver, it is certain that human thyroid hormone receptor type ing the receptors by species and by relatedness to forms a2 is expressed in these tissues. Cross-hybridization with present on chromosome 17 (type a) or on chromosome 3 human thyroid hormone receptor type j3 is unlikely under the (type S3). Thus we have designated this type of human stringency of washing we used.

hTRa2 1 [MEQKPSKVECGSDPEE] n [SARSPDGKRKRKNGQC] s [LK] t [SMSGYIPSYLDKDEQCVVC rTRal 1 [MEQKPSKVECGSDPEE] d [SARSPDGKRKRKNGQC] p [LK] s [SMSGYIPSYLDKDEQCVVC hTR a2 57 GDKATGYHYRCITCFGCKGFFRRTIQKNLHPTYSCKYDSCCVIDKITRNQCQLCRFKKCIAVGMAMDLVL rTR cil 57 GDKATGYHYRCITCEGCKGFFRRTIQKNLHPTYSCKYDSCCVIDKITRHIQCQLCRFKKCIAVGMAMDLVL hTR c2 127 DDSKPVAKRKL.IEQNRERRRKEEMIRSLQQRPEPTPEEWJDLIH] i [ATEAHRSTNAQGSHWKQRRKFL rTR al 127 DDSKRVAKRKLIEQNRERRRKEEMIRSLQQRPEPTPEEWDLIH] v [ATEAHRSTNAQGSHWKQRRKFL hTR c2 193 PDDIGQSPIVSMPDGDKVDLEAFSEFTKIITPAITRVVDFAKKLPMFSELPCEDQIILLKGCCMEIMSLR rTRal 193 PDDIGQSPIVSMPDGDKVDLEAFSEFTKIITPAITRVVDFAKKLPMFSELPCEDQIILLKGCCMEIMSLR hTRci2 263 AAVRYDPESDTI,TLSGEM] a [VKR] e [QLKNGGLGVVSDAIFELGKSLSAFNLDDTEVALLQAVLL rTR al 263 AAVRYDPESDTLTLSGEMI t IVKR] k [QLKNGGLGVVSDAIFELGKSLSAFNLDDTEVALLQAVLL hTRa2 325 MSTDRSGLLCVDKIEKSQEAYLLAFEHYVNHRKHNIPHFWPKLLMK] erevqssilykgaaaegrpggs rTRal 325 MSTDRSGLLCVDKIEKSQEAYLLAFEHYVNHRKHNIPHFWPKLLMK] vtdlrmigachasrflhmkvec hTR 2 393 lgvhpegqqllgmhvvqgpqvrqleqqlgeagslqgpvlqhqspkspqqrllellhrsgilharavcged rTRoil 393 ptelfpplflevfedqev hTR c2 463 dsseadspssseeepevcedlagnaasp

FIG. 4. Amino acid sequence comparison between the human thyroid hormone receptor type a2 (hTRa2) and rat thyroid hormone receptor al (rTRal). One-letter abbreviations for amino acids are used. Identical residues are indicated by upper case letters.

NH21 102 169 243 419 456 COOH hTRi3 12% | 90% | 73% 85% 1 53 120 194 370 490 hTRa2 | DNA T3/T4 53 120 194 370 410 rTRaI 94%01 100%] 99% 94 %

FIG. 5. Schematic comparison of the human thyroid hormone receptor type a2 protein (hTRa2) with the human thyroid hormone receptor type f31 (hTRP1) and the rat thyroid hormone receptor type al (rTRal). Numbers above the boxes indicate amino acid residues. Numbers inside the boxes indicate the percent amino acid identity within the enclosed region with human thyroid hormone receptor type a2. Downloaded by guest on September 30, 2021 2784 Medical Sciences: Nakai et al. Proc. Natl. Acad. Sci. USA 85 (1988)

2 by Thompson et al. (12) and chicken thyroid hormone receptor type al cDNA described by Sap et al. (10), human k FL thyroid hormone receptor type a2 codes for a protein with an 80-amino acid C-terminal extension. Because of the differ- ence in size, we have identified our clone as thyroid hor- ---92.5 mone receptor type a2, to distinguish it from the rat and chicken clones. It is of interest that the cognate mRNAs - 66.2 detected by the human thyroid hormone receptor type a2 probe in human kidney, placenta, and brain are -2 kb, whereas in liver, the major form is 2.5 kb. This may suggest alternative processing of the mRNA, which could produce 4 5 FIG. 6. Synthesis ofthe human thyroid thyroid hormone receptor forms with various affinities for hormone receptor type a2 protein. T3 or its analogs in various tissues. Rat thyroid hormone Capped mRNA synthesized in vitro with receptor type al hybridizes with sequences present on human SP6 RNA polymerase was translated in a chromosome 17 (12). The probe used to isolate our human rabbit reticulocyte lysate. The [355]me- thionine-labeled products were sepa- thyroid hormone receptor type a2 cDNA clones has also been 31 rated on a 1o NaDodSO4/polyacryl- localized to human chromosome 17 by Dayton and coworkers amide gel and visualized by fluorogra- (13). We have partially sequenced the clone isolated by phy. Lanes: 1, translation with RNA Dayton and coworkers (13) and found it to be identical (data from pke711 that contains the entire cod- not shown) to human thyroid hormone receptor type a2 in ing region; 2, no added RNA. Size of sequenced areas and to be a nearly full-length clone, possibly protein markers: phosphorylase b, 92.5 lacking some of the 5' end. Thus the thyroid hormone 21.5 kDa; bovine serum albumin, 66.2 kDa; receptor a2 gene is on chromosome 17. It is, however, ovalbumin, 45 kDa; carbonic anhydrase, uncertain whether more than one gene exists in the human 31 kDa; soybean trypsin inhibitor, 21.5 thyroid hormone receptor type a series. Since the human kDa. thyroid hormone receptor type 81 gene (11) is on chromosome 3, our data demonstrate that at least two, and possibly DISCUSSION three, forms of thyroid hormone receptor are present in humans. Whereas the human thyroid hormone receptor type We have cloned from human kidney a unique cDNA (human ,81 mRNA has only been reported in placenta and several cell thyroid hormone receptor type a2) which, when expressed lines, human thyroid hormone receptor type a2 mRNA ap- and translated in vitro, codes for a thyroid hormone receptor pears more widely distributed, and in fact may be present at with Ka for T3 of 2.3 x 109 M -1, typical of human nuclear low levels in most human tissues. The distribution of human thyroid hormone receptors (5, 7), and nearly identical in size thyroid hormone receptor type a2 mRNA also contrasts with (56 kDa) to one of the two reported forms of thyroid rat thyroid hormone receptor type al mRNA that is present in hormone receptor in mammalian tissues (6). While highly high levels in the brain and at much lower levels in other similar to the rat thyroid hormone receptor type a described tissues (12).

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10 20 30 40 50 Bound (pM) lodothyronine Concentration (M) FIG. 7. Thyroid hormone binding to human thyroid hormone receptor type a2 synthesized in vitro. (Left) Scatchard analysis of T3 binding. Samples (7 ,ul) from the in vitro translation mixture (210 Al, total volume) were assayed for specific T3 binding activity. (Right) Competition of thyroid hormone analogs for ['251]T3 binding to the human thyroid hormone receptor type a2. Samples from programmed lysates (7 ,l) were incubated, with [1251]T3 and with various concentrations of unlabeled T3 or tetraiodothyronine. (o) L-T3. (0) L-Tetraiodothyronine. Bound and free [1251]T3 were separated by using the resin method. Downloaded by guest on September 30, 2021 Medical Sciences: Nakai et al. Proc. Natl. Acad. Sci. USA 85 (1988) 2785

The human thyroid hormone receptor type a2 gene is 3. Ye, Z.-S. & Samuels, H. H. (1987) J. Biol. Chem. 262, 6313-6317. clearly another product of the v-erbA superfamily of nuclear 4. Gustafson, T. A., Markham, B. E., Bahl, J. J. & Morkin, E. hormone receptor genes. Like the other thyroid hormone (1987) Proc. Natl. Acad. Sci. USA 84, 3122-3126. 5. Oppenheimer, J. H. (1985) Ann. Int. Med. 102, 374-384. and steroid hormone receptors (9, 19), the human thyroid 6. Pascual, A., Casanova, J. & Samuels, H. H. (1982) J. Biol. hormone receptor type a2 has an N-terminal domain of Chem. 257, 9640-9647. unassigned function, a central cysteine-rich DNA-binding 7. Ichikawa, K. & DeGroot, L. J. (1987) Proc. Natl. Acad. Sci. region, and a C-terminal domain that presumably binds USA 84, 3420-3424. thyroid hormone. 8. Refetoff, S., DeGroot, L. J., Benard, B. & DeWind, L. T. Our laboratory has reported a "sibship" with several (1972) Metabolism 21, 723-756. 9. who were to Green, S. & Chambon, P. (1986) Nature (London) 324, 615-617. members resistant thyroid hormone action, had 10. Sap, J., Munoz, A., Damm, K., Goldberg, Y., Ghysdael, J., retarded growth, deaf mutism, and abnormal bones, but had Leutz, A., Beug, H. & Vennstrom, B. (1986) Nature (London) normal intellect and sexual maturation as well as augmented 324, 635-640. cardiovascular activity (8). This variegated clinical picture 11. Weinberger, C., Thompson, C. C., Ong, E. S., Lebo, R., suggested differential hormone responsiveness in various Gruol, D. J. & Evans, R. M. (1986) Nature (London) 324, tissues. Subsequent studies revealed that indicated abnormal 641-646. T3 nuclear receptors were present in blood cells (20) and 12. Thompson, C. C., Weinberger, C., Lebo, R. & Evans, R. M. fibroblasts (21) from these children. The availability of (1987) Science 237, 1610-1614. cDNAs encoding the various thyroid hormone receptors will 13. Dayton, A. I., Selden, J. R., Laws, G., Dorney, D. J., Finan, be useful in J., Tripputi, P.,. Emanuel, B. S., Rovera, G., Nowell, P. C. & determining the underlying genetic defect in this Croce, C. M. (1984) Proc. Natl. Acad. Sci. USA 81,4495-4499. family as well as studying the long recognized differential 14. Bell, G. I., Fong, N. M., Stempien, M. M., Wormsted, M. A., sensitivity of various tissues to thyroid hormone action. Caput, D., Ku, L., Urdea, M. S., Rall, L. B. & Sanchez- Pescador, R. (1986) Nucleic Acids Res. 14, 8427-8446. Note. Since this work was completed, Benbrook and Pfahl (22) 15. Sanger, F., Coulson, A. R., Barrell, B. G., Smith, A. J. H. & reported the isolation of a distinct human thyroid hormone receptor Roe, B. A. (1980) J. Mol. Biol. 143, 161-178. type a sequence, from a testis library. Although they did not present 16. Ullrich, A., Shine, J., Chirgwin, J., Pictect, R., Tischer, E., the cDNA sequence, complete protein sequence, or tissue distribu- Rutter, W. J. & Goodman, M. (1977) Science 196, 1313-1319. tion of the mRNA, the predicted size of the receptor protein and 17. Krieg, P. A. & Melton, D. A. (1984) Nucleic Acids Res. 12, partial protein sequence suggest that their cDNA may be identical to 7057-7070. the one we have described. 18. Torresani, J. & DeGroot, L. J. (1975) Endocrinology 96, 1201-1209. This research was supported in part by Public Health Service 19. Arriza, J. L., Weinberger, C., Cerelli, G., Glaser, T. M., Hand- Grants DK13377 and DK27384, the David Wiener Research Fund, elin, B. L., Housman, D. E. & Evans, R. M. (1987) Science and the Howard Hughes Medical Institute. 237, 268-274. 20. Bernal, J., Refetoff, S. & DeGroot, L. J. (1978) J. Clin. Endo- 1. Larsen, P. R., Harney, J. W. & Moore, D. D. (1986) J. Biol. trinol. Metab. 47, 1266-1272. Chem. 261, 14373-14376. 21. Ichikawa, K., Hughes, I. A., Horwitz, A. L. & DeGroot, L. J. 2. Catanzaro, D. F., West, B. L., Baxter, J. D. & Reudelhuber, (1987) Metabolism 36, 392-399. T. L. (1987) Mol. Endocrinol. 1, 90-96. 22. Benbrook, D. & Pfahl, M. (1987) Science 238, 788-791. Downloaded by guest on September 30, 2021