Proc. Natl. Acad. Sci. USA Vol. 85, pp. 8444-8448, November 1988 Biochemistry A gene inducible by serum growth factors encodes a member of the steroid and thyroid hormone receptor superfamily (transcription factors /DNA-binding proteins /zinc fingers /growth-related genes) THomas G. HazeL_*, DANIEL NATHANSt, AND LESTER F. Lau*# *Department of Molecular Biology, Northwestern University Medical School, Chicago, IL 60611; and tHoward Hughes Medical Institute Laboratory and the Department of Molecular Biology and Genetics, Johns Hopkins University School of Medicine, Baltimore, MD 21205 Contributed by Daniel Nathans, August 12, 1988 ABSTRACT We previously identified, by cDNA cloning, a set of genes that are expressed during the G,/G, transition (cell cycle reentry) in mouse fibroblasts. These immediate early genes are transcriptionally activated within minutes of addition of serum or purified growth factors, and their mRNAs are superinduced in the presence of protein-synthesis inhibitors. We report here that one of these genes, represented by nur/77 cDNA (originally called 3CH77), encodes a member of the superfamily of ligand-binding transcription factors that in- cludes the steroid and thyroid hormone receptors. The nur/77 cDNA sequence encodes a protein of 601 amino acids containing two regions of sequence similarity to members of this nuclear receptor superfamily, corresponding to their DNA-binding and ligand-binding domains. These results suggest that the growth factor-inducible immediate early gene nur/77 encodes a ligand- binding protein that regulates the genomic response to growth factors. The proliferation of animal cells is initiated and regulated by polypeptide growth factors. The interaction of growth factors with their specific receptors generates a cascade of intracel- lular biochemical events (1), leading to the sequential expres- sion of specific genes. By analogy to the developmental program of viruses, some of the genes activated early by the actions of growth factors are likely to regulate the expression of other genes necessary for the onset of DNA replication (2). We previously identified, by cDNA cloning, a set of genes that are transiently activated within minutes after quiescent BALB/c mouse 3T3 cells are stimulated with serum, platelet- derived growth factor, or fibroblast growth factor (3, 4). These ‘immediate early’’ genes are regulated at the tran- scriptional and posttranscriptional levels, and their mRNAs are superinduced in the presence of protein-synthesis inhib- itors (4). In this communication we report the nucleotide sequence of one of these cDNA clones, 3CH77 (hereafter referred to as nur/77), and the amino acid sequence it encodes.§ Sequence comparison shows that the nur/77- encoded protein is related to the nuclear receptor superfamily that includes the receptors for such compounds as giucocor- ticoids (5), mineralocorticoids (6), estrogen (7), testosterone (8, 9), progesterone (10), thyroid hormone (11, 12), retinoic acid (13-15), and vitamin D (16). These receptors function as modulators of gene expression; upon binding to their respec- tive ligands, they exhibit increased affinity for enhancer-like DNA sequence elements associated with target genes (17). Interaction of the receptor-ligand complex with the DNA sequence elements results in altered expression of these genes (17). The nur/77 protein and the known nuclear receptors share two regions of sequence similarity. For several receptors the first region has been shown to be the DNA-binding domain The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked ‘‘advertisement”’ in accordance with 18 U.S.C. §1734 solely to indicate this fact. 8444 (13, 14, 18-20), and the second region the ligand-binding domain (18, 19, 21, 22). These results suggest that nur/77 cDNA encodes a ligand-dependent nuclear receptor; in the presence of its ligand, the nur/77 protein may act as a specific DNA-binding protein that regulates the genomic response to growth factors. MATERIALS AND METHODS Cell Culture. BALB/c 3T3 cell clone A31 was cultured as described (4). cDNA Screening and Sequence Analysis. A cDNA library containing near-full-length copies of immediate early mRNAs (4) was screened with a nur/77 cDNA probe as described (3). Near-fuli-length nur/77 cDNAs were cloned into pGEM plasmids (Promega Biotec, Madison, WI). Nested 5’ and 3’ deletions were created by using BAL-31 nuclease (Bethesda Research Laboratories) and were sequenced by the dideoxy- nucleotide chain-termination procedure (23) with [a- (?°S)thio]dATP. , Primer Extension. Synthetic *?P-labeled oligonucleotides complementary to nucleotides 1-25 or 24-43 of nur/77 were hybridized to 30 yg of total cellular RNA from either quiescent cells or cells stimulated with serum for 3 hr in the presence of cycloheximide (10 »g/ml). Hybridization and subsequent reverse transcription were carried out as de- scribed (24), and reaction products were resolved by elec- trophoresis in 8% polyacrylamide gels containing 8 M urea. RESULTS Nucleotide Sequence of nur/77 cDNA and its Encoded Protein. A cDNA clone representing the 3’ protein of nur/77 (clone 3CH77 in ref. 3) was used to select a nearly full-length cDNA clone from a library prepared by using poly(A)* mRNA isolated from BALB/c 3T3 cells stimulated with serum for 3 hr in the presence of cycloheximide (4). Several cDNA clones containing inserts of about 2.7 kilobases were independently isolated (approximately full-length as esti- mated by electrophoresis of nur/77 mRNA) and found to have identical restriction maps. Various restriction fragments of the cDNA clones hybridized to the same RNA species as detected by RNA blot analysis. One of these clones was chosen for sequence analysis. The cDNA analyzed is 2456 nucleotides in length, exclud- ing the poly(A) tail (Fig. 1). It contains a major open reading frame initiating at position 112 that is 1803 nucleotides in length and encodes a protein of molecular weight 64,738 with a calculated isoelectric point of 6.72. This reading frame is Abbreviation: NGF, nerve growth factor. To whom reprint requests should be addressed. §The sequence reported in this paper is being deposited in the EMBL/GenBank data base (IntelliGenetics, Mountain View, CA, and Eur. Mol. Biol. Lab., Heidelberg) (accession no. J04113). 112 202 292 382 472 562 652 742 832 922 1012 1102 1192 1282 1372 1462 1552 1642 1732 1822 1912 2029 2148 2267 2386 Biochemistry: Hazel et al. ATG Met GGC Gly ACC Thr tcc Ser CCA Pro Tec Ser AAG Lys AAA Lys ccG Pro GCA Ala AGC Ser CTG Leu GAT Asp CAG Gln CGA Arg ATC Ile GGC Gly GCT Ala CAC His CAA Glin TTC Phe ccc Pro AAG Lys GGA Gly TCA Ser TTA Leu CAG Gln ccT Ala TTG Leu AAC Asn oTc val ccc Ala GcT Ala 6cc Ala GAA Glu AAG Lys cT¢c Leu TTT Phe cTG Leu ATG Met GGc Gly TGA TER On -Eco Ri Neo 1 (212) Sma i (455) Sca | (508) L-Xmni (745) Hae tl (1016) Pst l (1541) Pst | (1703) Pst! (1832) F Kpnl (1867) Proc. Natl. Acad. Sci. USA 85 (1988) rBst Ell (2105) rXhol (2296) -Eco Ri t{ | 3' GGCTGCGAAAGTTGGGGGAGTGTGCTAGAAGGACTGCGGAGCGGAGCGCACGCGGGACCAGGCTCGACTGGGTCGCTGGTCCCGGCCACAGGGAGTGGGAGCCGGCTGGAG TGT cys cect Pro GAG Glu TCC Ser GAT Aap CTT Leu TCT Ser TTC Phe CoT Arg TCT Cys AAG Lys GTG val TCC Ser cTG Leu TGG Trp ccc arg GGT Gly oTc val GcT Ala cTG Leu ATT Tle acc Thr TTT Phe TCG Ser GAG Glu TcT Ser TCA Ser CCA Pro Gac Asp GGT Gly TAC Tyr GGC Gly ccT Pro GTG Val GCA Ala cTG Leu GAT Asp etc Leu acc Thr CAG Gln CAA Gln ATG Met GAC Asp 6cc Ala ace Thr ccc Pro GGG Gly CCA Pro ACT Thr cac Asp ATC Tle ATG Met acc Thr CTG Leu GAA Glu GCA Ala TGG Trp ATC Tle GTG Val cece arg GcT Ala GAC Asp acc Thr acc Thr CTA Leu TGG Trp ccT Pro CCA Pro Tce Ser AAT Asn TGC cys GTG Val AAT Asn ccc Pro AAA Lys TAC Tyr ATT Tle acT Thr GCA Ala ATC Tle CAA Gin cTG Leu TTC Phe Tec Ser Tec Ser GAC Asp cece Pro Gcc Ala GGC Gly oct Ala cTG Leu AAG Lya CTT Leu cGc Arg ATC Tle CGA Arg GAC ASD GAT Asp GGA Gly TTT Phe TAT Tyr 6cc Ala ere Leu ¢ecc Pro tTc¢e Ser GGCc Gly CCA Pro ACC Thr ATT Ile TCG Ser GCA Ala GAA Glu etc Leu TTC Phe ccT Pro TCT Ser AAC Asn CGA arg GAC Asp TGC cys GGA Gly AGC Ser TAC Tyr ocT Ala AGC Ser TCA Ser ccT Pro CAC His cTG Leu TGOT Cys AAC Asn GTT val ACT Thr GGG Gly GGC Gly AAA Lys atc Tle cac His cCA Pro ete Leu ACA Thr ccc Pro CAG Gin TCG Ser GGc Gly TTT Phe CCA Pro CAG Gln Gac Asp CAG Glin AAG Lys GTA val TCC Ser AAG Lys TTC Phe cece Pro CTG Leu GGG Gly CAG Gln AAG Lys CCA Pro GAG Glu cTG Leu GCG Ala TCT Ser GGC Gly acc Thr cTT Leu GCA Ala cac His GAT Asp CGG Arg cTc Leu GAA Glu ATT Tle GGT Gly occ Ala ete Leu ccG Pro TTG Leu GCA Ala ACA Thr ccG Pro Tcc Ser GAG Glu Cac His TT¢c Phe GGG Gly ccc Pro TAT Tyr TGC cys ACA Thr aTc Ile GAT Asp GAG Glu GAG Glu Tre Phe CAG Gln 6cc Ala GAG Glu ACG Thr GCA Ala GGG Gly TTC Phe TAC Tyr TTC Phe TTC Phe GAG Glu GTG val GGG Gly ccT Pro GAC Asp CGG arg Gcc Ala cTT Leu GGG Gly TCA Ser GAC Aap AGC Ser GAC Aap AGC Ser cctT Pro ACG Thr AAG Lys TAT Tyr Tce Ser Tcc Ser GGG Gly acc Thr GT¢c Val GTG val AGC Ser GCA Ala GGT Gly TGC cys AAG Lys cGG Arg ccT Pro TGC Cys TTG Leu CCA Pro 6cc Ala acc Thr TTT Phe 6Gc Gly ccG Pro TTC Phe GAG Glu TCC Ser ccc Arg GAC Asp CTA Leu CAC His GAC Asp CCA Pro eTc Leu tcc Ser ccT arg CTS Leu GTA Val GGA Gly GCA Ala CAG Gin GAG Glu AGT Ser AGC Ser AGT Ser AGC Ser acc Thr acc Thr AAG Lys AAA Lys TTG Leu GTG val GGA Gly ATC Ile CTs Leu CGG arg TCA Ser ccc Pro ccG Pro ccr Pro ccG Pro GAC Asp ccc Pro CAG Gin ccT Pro TAT tyr AAG Lys TCT Cys aGG Arg GGG Gly GAC Aap CAA Gla GAC Asp TTc Phe CAC His GTG val CGT Arg ccT Pro CGT Arg 6cT Ala TGC Cys TTC Phe TGC cys ACT Thr ccc Pro TCC Ser tcc Ser GAG Glu cGG Arg CGG Arg Tec Ser CAA Gln CAA Gin TGC cys AGC Ser GAA Glu cTs Leu CCA Pro GAC Asp ACA Thr tec Ser CAG Gln TCA Ser TAT Tyr acT Thr ATG Met coc arg GGC Gly CGG arg CGG arg GGG Gly TTT Phe GAC Asp TCA Ser TTG Leu GAG Glu ctGc Leu ccT Pro Cac His CTG Leu TCA Ser GTG val 6ce Ala GRA Glu GGCc Gly CCA Pro AGC Ser TGC cys AAC Aan ccc Gly ccT Pro TAT Tyr cTG Leu GGC Gly GGT Gly cTG Leu occ Gly ATT Tle cTG Leu ccc Pro GcT Ala TAC Tyr ccc Pro GGCc Gly ccc Pro GCA Ala GGG Gly AAG Lys ccc Arg CcGG Arg AGC Ser GAC Asp TTG Leu cTG Leu GTT Val CAG Gln AAA Lys GTG Val acc Thr AGC Ser TGT cys GGC Gly TCG Ser ctc Leu AGC Ser GcT Ala GcT Ala ooc Gly TGC cys CTA Leu acT Thr TTG Leu CTA Leu GTA Val GAT Asp AAT Asn cTG Leu GAC Asp GGT Gly TTC Phe Tec Ser TGC Cys CCA Pro TGG Trp cece Pro TTC Phe TCA Ser TTC Phe CAG Gln cect Pro ccc Ala cTc Leu GAG Glu CTA Leu GTT Val ccc arg ect Pro AAG Lys GAT Aap AGC Ser TCT Ser TAC Tyr TCT Ser GCA Ala AGC Ser ccc Pro GGT Gly TTC Phe TTC Phe TCA Ser AAA Lys TCT Ser TCT Ser CAC His ece Pro ATT Tle GAG Glu ATC Tle cece Pro acc Thr 6cc Ala ccG Pro ACA Thr TGG Trp cTG Leu GGc Gly occ Gly AAG Lys TGC cys ALA Lys TTG Leu GGT Gly occ Ala CAG Glin @ce Ala ccT Ala CTT Leu TTT Phe cTG Leu TTC Phe Tec Ser ccc Gly ecc Pro ACA Thr ece Ala TITG Leu AGC Ser ccc arg cece arg ccc Pro GAC Asp TCC Ser TTC Phe CTG Leu TTT Phe AGC Ser cGG Arg ATG Met Gcc Ala ATG Met Tce Ser acc Thr AAC Asn GAG Glu CAG Gln GCA Ale GAG Glu ACA Thr TT¢c Phe AAG Lys TAT Tyr cTG Leu CTG Leu CAG Gln 6ce Ala TGT cys acc Thr GAC Asp CTT Leu Gac Asp acG Thr CTG Leu TTC Phe CAG Gin AGT Ser ccc Pro GG6C Gly GTA val CAG Gln CAG Gln Tcc Ser GAC Aap GAA Glu TGT cys TGC cys cts Leu cts Leu ACA Thr GAG Glu GGG Gly TCT Ser AGC Ser CAG Gin TTG Leu TCT Ser acc Thr coc Arg CAG Gin AAG Lys ccT Pro AAG Lys GTT Val eT¢c Leu occ Ala cTG Leu AAG Lys TGC Cys TTG Leu TTC Phe TAC Tyr TCT Ser GGC Gly ccG Pro ccT Pro CTs Leu TCT Ser TGT cys AAA Lys TGC cys CCA Pro TTc Phe aTc Tle TTC Phe cGT arg Tec Ser GAG Glu ACT Thr TCT Ser CCCCTGCCCTGAACATGTGTGCGCACACGTGGTGCTCTTCTGTCACCCATGTGCCTTTAAGCCTATAGCCCACGGACCCCCAGACCACCCTACCCCCAGCCTGGTTTTGAG CTAAGACTGACGTACCTCCTCACTCCAGAAGATGGACAGAGAACTCAAGACCTGGGGGAGGGTGTGTATTCACGGGGGTGACCCCACTATTTGTCTTATCCCTCCAGCTCAGTCCTGGC CTTCGTGTGTTTTTGTAAGATAAACCATTTTTAACACATACCACTCTGTTGTAAATAAGCTGACGCTACTGTAAATACAGAAAGGAAGAGGTTGAGATGGGGGTTGGGAGGAAGGGGTG GGGCTCCCACCAGCTGGGCGAGCCTCCAACTCGAGATCTCTTCCGCTCTCCTTCCATGTGTACATAACTGTCACTCAAGAAGGTGATTGACAGATICTGATTTATATTTGTGTATTTTC CTGGATITATAGGATGTGACTTTTCTGATTAATATATTITAATATATTGaat aaaAAATAGACATGTAGTTIG (A) s 8445 30 60 90 120 150 180 210 240 270 300 330 360 390 420 450 ago §10 540 §70 600 Fic. 1. Restriction map and nucleotide and deduced amino acid sequences of nur/77. (Upper) Schematic representation of the nur/77 cDNA showing cleavage sites of some common restriction enzymes. The open bar indicates the predicted coding region. (Lower) The complete nucleotide sequence and the predicted amino acid sequence of the nur/77 cDNA clone. Numbers at left refer to the first nucleotides on the lines, and numbers at right refer to the last amino acids on the lines. The ATTTA sequence motifs are underlined, and the polyadenylylation signal appears in lowercase letters. The 3’-terminal (A), denotes the poly(A) tail. followed by a 3' untranslated region of 542 nucleotides. A consensus polyadenylylation signal is located 18-23 bases upstream of the poly(A) tail. Like many other immediate early mRNAs, nur/77 mRNA has a short half-life (~20 min; ref. 4). Consistent with this observation, there exist within a 60-base-pair segment of the 3’ noncoding region three repeats of the ATTTA sequence motif thought to contribute to the instability of some mRNAs (25). In the deduced protein sequence, a potential N-linked glycosylation site is found at asparagine-276. Between the lysine residues at positions 32 and 104 there is a region rich in proline, glutamic acid, serine, and threonine. This type of ‘‘PEST’’ sequence is thought to be associated with proteins of short half-lives such as the fos and myc gene products and ornithine decarboxylase and is also found in members of the nuclear receptor superfamily (26, 27). That the cDNA is nearly full-length was demonstrated by primer extension analysis. Reverse transcription of BALB/c 3T3 cell RNA from an oligonucleotide primer complementary to nucleotides 1-25 of the cDNA sequence indicated that the 8446 Biochemistry: Hazel et al. 5.Oo 2 4 Fic. 2. Primer extension analysis. Reactions were performed with 30 ug of total RNA from either quiescent BALB/c 3T3 cells (lanes 1 and 3) or cells stimulated with serum for 3 hr in the presence of cycloheximide (lanes 2 and 4). Two **P-labeled oligonucleotides, one complementary to nucleotides 1-25 of the cDNA sequence (lanes 1 and 2) and the other complementary to nucleotides 24-43 (lanes 3 and 4), were used. Left and right four lanes show **S-labeled sequencing-reaction products as size markers. The positions of oligonucleotides used as primers are indicated by arrows. 5’ end of the nur/77 mRNA occurs heterogeneously 9-17 nucleotides upstream of the primer sequence (Fig. 2, lanes 1 and 2). The predominant mRNA start site occurs 16 nucle- otides upstream of the 5‘ end of the sequence shown in Fig. 1. These results were confirmed by primer extension using an oligonucleotide complementary to nucleotides 24-43 of the cDNA; this reaction yielded products 32-40 nucleotides long (Fig. 2, lanes 3 and 4). There are three in-frame ATG codons at the 5’ end of the nur/77 cDNA sequence occurring at positions 112, 214, and 280. In addition, an out-of-frame ATG occurs at position 134. The first ATG (position 112) has a poor flanking sequence for translation initiation according to the consensus sequence derived by Kozak (28), whereas the ATG at position 214 has a more favorable flanking sequence. However, if the ATG at position 112 is not used, the next ATG (position 134), which does have features of the consensus sequence for initiation, would be used (29), leading to translation of 13 codons of a different reading frame. Although nur/77 mRNA purified by hybrid selection is inefficiently translated in the rabbit retic- ulocyte lysate system (3), it does yield two detectable protein products with molecular weights of 64,000 and 58,000, consistent with initiation at positions 112 and 280 (data not Proc. Natl. Acad. Sci. USA 85 (1988) shown). Further analysis will be required to determine the correct initiation site in the cell. Relationship of nur/77 to the Nuclear Receptor Superfamily. The deduced amino acid sequence of nur/77 was compared to known protein sequences in the National Biomedical Research Foundation data base (release 14.0) and was found to share significant amino acid sequence similarity with the superfamily of nuclear ligand-binding transcription factors that includes the steroid and thyroid hormone receptors (S- 16). This similarity occurs in two regions. One is a highly conserved 66-amino acid region demonstrated for some of these receptors to be the DNA-binding domain (Fig. 3; refs. 13, 14, 18-20). Most notable in this region is the presence of eight strictly conserved cysteine residues thought to form two DNA-binding ‘‘fingers,’’ each coordinated by a zinc ion (30). The extensive conservation of these cysteines and other residues in this domain is characteristic of the members of the nuclear receptor superfamily (31). The nur/77 sequence contains 50-58% sequence identity in this domain when compared to the human nuclear receptors (Fig. 3). A second region of sequence similarity occurs carboxyl to the DNA- binding domain (Fig. 4). This region has been shown for several of the nuclear receptors to function as the ligand- binding domain (18, 19, 21, 22). Although they bind struc- turally distinct ligands, all members of the nuclear receptor superfamily analyzed to date share moderate sequence sim- ilarity in this region. The sequence similarity shared between nur/77 and known nuclear receptors is comparable to that shared among the known receptors. Thus the homology between nur/77 and known nuclear receptors indicates that nur/77 is a member of this superfamily. DISCUSSION The interaction of serum growth factors with their membrane receptors results in the sequential activation of specific genes. Some of these genes are transcriptionally activated within minutes of growth factor stimulation, even in the absence of protein synthesis (3, 4, 32-34). Among these immediate early genes are several that encode known or probable transcription factors: the protooncogenes c-fos (33) and c-jun (2, 35), jun-B (36), Krox-20 (37), zif/268 (NGFI-A, Egr-1, Krox-24) (38-41), and fra-/ (42). In this communica- tion we report that the immediate early gene represented by nur/77 cDNA is another member of this group, encoding a protein that is related to steroid receptors and other ligand- dependent transcription factors. Such hormones as glucocorticoids, mineralocorticoids, estrogen, progesterone, testosterone, the morphogen reti- 270 [claiy.c G DIN C QIH YG VJR Clu I]¢ G DIE cin y Gfa Lb 185 IciA chyply yJH YG VW 567 |cjL I]c G DIE clH YG v[L 421 |c|L/V c[S]pjE clH YG v|L 603 {c|L|v c & DJE clH ¥ G viv 102 |c|viv cc piKla[T]ciy|H y[RC I 88 iclF c[g}e|xs ylw y GV¥js cjolv c @ piR{a] FlH M 302 A Kly|jx[c|L AN c|P K|R QRiy|[L[cja sR c|t K|F 217 NDiy|[Mic|PAT cjr KIN 599 HN|YILICIAGR c}1 K|z 453 HNIYILICIAGR c]z K|I 635 HNIY|LICIAGR clr RII 136 P S/y|S|C|K ¥ BE civ K\v 120 Mm vily|Ti[cjH Rk D c|1 K\v AMP T|cCj]P FN c|K K|D aagaaaaagaan Rr R R R R R R R R a AAAANAAAA aoagqccar <4 me) HH] my og ) NARA AAARRR ADMD DWDDAA nny ee ny 9 KRHH CEH eS ACO aA BO aaagaagagaaaaa N K K K K K N N R nur/77 hAR hER hPR hoR bMR aTR hRAR cVDR RPAMAAAGAQAA PrBoeprvge Pree rague AZROOOTAA aga nur/77 hAR hER hPR hGR nhMR hTR hRAR cVDR RIC nic sjc NIC]P Nic NIC Qic RI]c HIC aaanaaanan DW DW Www mw aagaannaanaa mH erage om» Sore eH CHS AD ADD AW RO DARA AAR AAA Fic. 3. Homology between the predicted nur/77 sequence and the DNA-binding domain of hormone receptors. Numbers on the left refer to the first amino acids (one-letter code) of the lines. Amino acids that are identical in five or more sequences are boxed. hAR, human androgen receptor (8, 9); hER, human estrogen receptor (7); hPR, human progesterone receptor (10); hGR, human glucocorticoid receptor (5); hMR, human mineralocorticoid receptor (6); hTR, human thyroid hormone receptor (11, 12); ARAR, human retinoic acid receptor (13-15); cVDR, chicken vitamin D receptor (16). Biochemistry: Hazel et al. Proc. Natl. Acad. Sci. USA 85 (1988) 8447 412 Llu so siujp VIR E I CPG D L[L] nur/77 349 [LJ/A DR ELLJV HM IN K Vv DILIT LH Vv H/L| hER 566 |LIGGRQVIAAV K R N/L|H LD M T/L| hGR 772 |uU|AGKRQMIOQVY K KNILIP LE I TL] MR 721 |uLIG ER Q[]tsvv SK R NIL{H I D I TIL| PR 231 [LIS TRC IIKTVEFIAIK ToL[T Ia I T/L| nRAR 270 TTP AITRV YD FAIR c PCE I I}u| mtr 442 sae F yfr s|x p c[E] - 1 -{F]c s nur/77 379 clajw TL wIR S|MEH P- Vv L -|F|A P hER 596 OY Sw F{tM wIR SJY RQSS AN|LJL C/FIA P hoR 802 oy sw cts wiR s|¥ KH TNS QFLY|FIA P hMR 751 1QYS WM S(L|M wR S|Y K HV S[GJ]QMLY/FIA P hPR 261 Lx afa}c[L |p Tb tIR[- y T P[E]QD TMT -|F/S D hRAR 300 |L|K GC c H[E]I w VjR|- Y DP SE T(L)T - NG htTR 470 -H Q R o[p]- - - wafo|w rfp ajr[s|R nurs77 407 R NIQ(G Kic{[V E[G/M v E - - - 1 F[DjM LIL Al T|s|/S_ ER 626 E-[Q|RMTLPCMY[PJQCKH--MLYV S{s|E hoR 832 E-EKMHQSAMYELCQG--MHQISLQ HMR 781 E-fOIRMRES S[F]Y¥SLCL-~-TMWQIPQE hPR 289 RTIOIMHN[A]JG FGPLTDLVFAFANQLLP hRAR 328 RG KNGGLGVVSDATIF[D]UGMH Ss L{s]s hTR 495 SL [vJp v Pp alE]A s a{i]v[uJ1 - -------- nur/77 434 RF LOG EEIF|V KSTIULIDNSGVYTFLS hER 653 LH VJs YEEYuciM KT[LILILIL SS VPKDGULK HGR 859 FV LT FEEYTEMREKVILILILJIL STIPKDGULK AMR gos FV [VJs QE E[FJ]L[C]M K V[LILILILNTIPLEGLR hPR 319 LEM---7>-- DD-~--AETGL|LISAICLICGDR hRAR 358 FNL------ DD---TEVALILIQAVLLMS SDR hTR 516 th}e a cfelope rev eleluone sc EB H]M A)? V_ nur/77 466 STLEKS|LIEEKDHIHRVLD RIIff DT|L[t oH L[M AJK A hER 682 SQELFDEIRMTYIKELGK-~----- IVEKRE hGR 888 SQAAFEEMRTNYIKRELRK------ MVTKC P HMR 837 SQTQOFEEMRSSYIRELIK----- = AIGULRQ hPR 340 OfJL EQPoRVOML ofl Pub efaju- -[vy- VRE hRAR 379 POLACVERIEKYQODSFLLUAFE--AHYINYRK NTR 546 a - [elole op as clils 8 6 pele alrect osu nur/77 494 G- LTL QIOIQH OQ R{LIA OC L SH I|[R|H MSN K|G[M hER 706 GNSS --|QOINWQRFYQ-|LIT LDSHHE VV ENT) nGR 912 ww s(@]- -Igjs¥QREYO- T LDSMHDLVsS DLL} hMR 828 KGVVSSS--QRFYQ-|LIT LDNLHDLVKQ- hPR 366 RRP S R[PJHM- -- FP K M/LIM role ele ie a cba hRAR 407 HHVTHF- - - - - WPK M tp p|L R|MIGACHA hTR 574 olelrfe ce elelelo uly eye ele)- rv ox x(elufolty se nur/77 $23 EHLY S MIKJIC KNViVP|LYD-LLLEML........ 595 hER 733 LN-Y{C)FOTFLD-KTMSIEFPEML........ 177 hGR 939 LE - FYTFRESHALKVEFPAML........ 984 hMR 854 LHL Y NTFIQSRALSVEFPEMMHM........ 933 hPR 393 E[R]V I MfEJI PGSM -[P]PLIQEML........462 hRAR 432 sin(F ou v CPTELLPPLFLeE VFEp 456 hTR Fic. 4. Sequence comparison by a computer-generated alignment of nur/77 with the ligand-binding domains of hormone receptors. Numbers on the left refer to the first amino acids of the lines. Those amino acids of nur/77 that are identical to at least one other receptor sequence are boxed. Numbers at the ends of sequences refer to the total numbers of amino acids in those sequences. Receptor designations are as given in the legend of Fig. 3. noic acid, thyroid hormone, and vitamin D are known to activate the transcription of specific genes through a recep- tor-mediated mechanism (17). The receptors for these com- pounds comprise a superfamily of intracellular proteins that, in contrast to cell-surface receptors, directly modulate tran- scription by interaction with their target genes. Binding to their respective ligands, which enter the cell by diffusion, increases affinity for specific enhancer-like DNA sequence elements associated with the target genes, resulting in acti- vation or repression of transcription (6, 14, 20, 43-45). Whereas the amino-terminal domain, which is thought to be responsible in part for modulating gene expression (18, 46, 47), exhibits little sequence similarity among the different receptors, the central DNA-binding domain is highly con- served (13, 14, 18-20), and the carboxyl-terminal ligand- binding domain is moderately conserved (18, 19, 21, 22). The nur/77 protein shares all of these common structural fea- tures. A ligand for the nur/77 protein has not been identified. Since both retinoic acid and thyroid hormone bind multiple tissue-specific receptors (12, 15, 48), it is possible that nur/77 is another receptor for one of the known ligands. An additional possibility is that nur/77 may bind an intracellular molecule generated by growth factor action, rather than a ligand that diffuses into the cell. It has also been suggested that thyroid hormone-related and cholesterol-derived com- pounds, some of which are known to alter gene expression at the transcriptional level (49), may act through binding to as yet unknown receptors (31). One such compound may be the ligand for nur/77. The appearance of nur/77 mRNA is not restricted to growth factor-stimulated fibroblasts. This mRNA also ap- pears in mouse liver within 1 hr after partial hepatectomy (2) and thus appears to play a role in the proliferative response in vivo. Arelated RNA appears in the rat pheochromocytoma cell line PC12 after exposure to nerve growth factor (NGF) 8448 Biochemistry: Hazel et al. (ref. 50; M. E. Greenberg and L.F.L., unpublished observa- tion), which induces neuronal differentiation of PC12 cells (51). Recently, a cDNA (NGFI-B) derived from mRNA of NGF-stimulated PC12 cells has been reported to encode a protein closely related to nur/77 (50). Since the nur/77 protein sequence contains only 22 amino acid substitutions when compared to the NGFI-B sequence, it is likely that nur/77 is the murine homologue of NGFI-B. It is striking that NGF and platelet-derived growth factor (or fibroblast growth factor) acting on different cell types to induce differentiation and growth, respectively, activate a subset of identical genes that encode probable transcription factors: the putative ligand-binding nuclear receptor described in this report, the protein encoded by the fos gene (33, 52), and another protein with zinc-finger sequences (38-41). The same regulatory proteins thus appear to mediate a variety of cellular re- sponses to external signals, suggesting that some of the target genes of immediate early transcription factors are cell-type- specific. We thank Richard Hall for excellent technical assistance. This work was supported by a grant from the National Institutes of Health to L.F.L. (R01 CA46565). T.G.H. is supported by a predoctoral training grant from the Public Health Service. L.F.L. is a recipient of the American Cancer Society Junior Faculty Research Award and is a Pew Scholar in the Biomedical Sciences. 1. Rozengurt, E. (1986) Science 234, 161-166. 2. Nathans, D., Lau, L. F., Christy, B., Hartzell, S., Nakabeppu, Y. & Ryder, K. (1988) Cold Spring Harbor Symp. Quant. Biol., in press. Lau, L. F. & Nathans, D. (1985) EMBO J. 4, 3145-3151. Lau, L. F. & Nathans, D. (1987) Proc. Natl. Acad. Sci. USA 84, 1182-1186. 5. Hollenberg, S. M., Weinberger, C., Ong, E. S., Cerelli, G., Oro, A., Lebo, R., Thompson, E. B., Rosenfeld, M. G. & Evans, R. M. (1985) Nature (London) 318, 635-641. 6. Artiza, J. L., Weinberger, C., Cerelli, G., Glaser, T. M., Handelin, B. L., Housman, D. E. & Evans, R. M. (1987) Science 237, 268-275. 7. Green, S., Walter, P., Kumar, V., Krust, A., Bornert, J., Argos, P. & Chambon, P. (1986) Nature (London) 320, 134-139. Chang, C., Kokontis, J. & Liao, S. (1988) Science 240, 324-326. Lubahn, D. B., Joseph, D. R., Sullivan, P. M., Willard, H. F., French, F. S. & Wilson, E. M. (1988) Science 240, 327-330. 10. Misrahi, M., Atger, M., d’Auriol, L., Loosfelt, H., Meriel, C., Fridlansky, F., Guiochon-Mantel, A., Galibert, F. & Milgrom, E. (1987) Biochem. Biophys. Res. Commun. 143, 740-748. 11. Weinberger, C., Thompson, C.C., Ong, E. S., Lebo, R., Gruol, D. J. & Evans, R. M. (1986) Nature (London) 324, 641- 646. 12. Thompson, C. C., Weinberger, C., Lebo, R. & Evans, R. M. (1987) Science 237, 1610-1614. 13, Petkovich, M., Brand, N. J., Krust, A. & Chambon, P. (1987) Nature (London) 330, 444-450. 14. Giguere, V., Ong, E. S., Segui, P. & Evans, R. M. (1987) Nature (London) 33, 624-629. 15. Brand, N., Petkovich, M., Krust, A., Chambon, P., de The, H., Marchio, A., Tiollais, P. & Dejean, A. (1988) Nature (London) 332, 850-853. 16. McDonnell, D. P., Mangelsdorf, D. J., Pike, J. W., Haussler, M. R. & O'Malley, B. W. (1987) Science 235, 1214-1217. 17. Yamamoto, K. R. (1985) Annu. Rev. Genet. 19, 209-252. aw we oe 18. 19, 20. 21. 22. 23. 24, 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38. 41. 42. 43. 45. 47. 49. 50. 51. 52. Proc. Natl. Acad. Sci. USA 85 (1988) Giguere, V., Hollenberg, S. M., Rosenfeld, M. G. & Evans, R. M. (1986) Cell 46, 645-652. Gronemeyer, H., Turcotte, B., Quirin-Stricker, C., Bocquel, M. T., Meyer, M. E., Krozowski, Z., Jeltsch, J. M., Lerouge, T., Garnier, J. M. & Chambon, P. (1987) EMBO J. 6, 3985- 3994. Green, S. & Chambon, P. (1987) Nature (London) 325, 75-78. Rusconi, S. & Yamamoto, K. R. (1987) EMBO J. 6, 1309-1315. Kumar, V., Green, S., Staub, A. & Chambon, P. (1986) EMBO J. 5, 2231-2236. Sanger, F., Nicklen, S. & Coulson, A. R. (1977) Proc. Natl. Acad. Sci. USA 74, 5463~5467. Linzer, D. H. & Mordacgq, J. C. (1987) EMBO J. 6, 2281-2288. Shaw, G. & Kamen, R. (1986) Cell 46, 659-667. Rogers, S., Wells, R. & Rechsteiner, M. (1986) Science 234, 364-368. Rechsteiner, M., Rogers, S. & Rote, K. (1987) Trends Biochem. Sci. 12, 390-394. Kozak, M. (1986) Cell 44, 283-292. Kozak, M. (1980) Cell:22, 7-8. Evans, R. M. & Hillenberg, S. M. (1988) Cell 52, 1-3. Evans, R. M. (1988) Science 240, 889-895. Cochran, B. H., Reffel, A. C. & Stiles, C. D. (1983) Cell 33, 939-947. Greenberg, M. E., Hermanowski, A. L. & Ziff, E. B. (1986) Mol. Cell Biol. 6, 1050-1057. Almendral, J. M., Sommer, D., MacDonald-Bravo, H., Burck- hardt, J., Perera, J. & Bravo, R. (1988) Mol. Ceil. Biol. 8, 2140- 2148. Lamph, W. W., Wamsley, P., Sassone-Corsi, P. & Verma, I. M. (1988) Nature (London) 334, 629-631. Ryder, K., Lau, L. F. & Nathans, D. (1988) Proc. Natl. Acad. Sci. USA 85, 1487-1491. Chavrier, P., Zerial, M., Lemaire, P., Almendral, J., Bravo, R. & Charnay, P. (1988) EMBO J. 7, 29-35. Christy, B. A., Lau, L. F. & Nathans, D. (1988) Proc. Natl. Acad. Sci. USA 85, 7857-7861. Milbrandt, J. (1987) Science 238, 797-799. Sukhatme, V. P., Cao, X., Chang, L. C., Tsai-Morris, C., Stamenkovich, D., Ferreira, P. C. P., Cohen, D. R., Edwards, S. A., Shows, T. B., Curran, T., Le Beau, M. M. & Adamson, E. D. (1988) Ceil 53, 37~43. Lemaire, P., Revelant, O., Bravo, R. & Chamay, P. (1988) Proc. Natl. Acad. Sci. USA 85, 4691~4695. Cohen, D. R. & Curran, T. (1988) Mol Cell. Biol. 8, 2063-2069. Klein-Hitpass, L., Schorpp, M., Wagner, U. & Ryffel, G. U. (1986) Cell 46, 1053-1061. Cato, A.C. B., Miksicek, R., Schiitz, G., Aremann, J. & Beato, M. (1986) EMBO J. 5, 2237-2240. Adler, S., Waterman, M. L., He, X. & Rosenfeld, M. G. (1988) Cell §2, 685-695. Kumar, V., Green, S., Stack, G., Berry, M., Jin, J. & Chambon, P. (1987) Cell 51, 941-951. Tora, L., Gronemeyer, H., Turcotte, B., Gaub, M. & Cham- bon, P. (1988) Nature (London) 333, 185-188. Nakai, A., Seino, S., Sakurai, A., Szilak, I., Bell, G. I. & De-Groot, L. J. (1988) Proc. Natl. Acad. Sci. USA 85, 2781- 2785. Sudhof, T. C., Russell, D. W., Brown, M. S. & Goldstein, J. L. (1987) Cell 48, 1061-1069. Milbrandt, J. (1988) Neuron 1, 183-188. Greene, L. A. & Tischler, A. S. (1976) Proc. Natl. Acad. Sci. USA 73, 2424-2428. Kruijer, W., Schubert, D. & Verma, I. M. (1985) Proc. Natl. Acad. Sci. USA 82, 7330-7334.