A Human Early Response Gene Homologous to Murine nur77 and Rat NGFI-B, and Related to the Nuclear Receptor Superfamily
Akira Nakai, Sreedharan Kartha, Akihiro Sakurai, F. Gary Toback, and Leslie J. DeGroot Thyroid Study Unit (A.N., A.S., L.J.D.) Section of Nephrology (S.K., F.G.T.) Department of Medicine University of Chicago Chicago, Illinois 60637
When a human fetal muscle cDNA library was screened with the human thyroid hormone receptor a2 cDNA at low stringency, we found a weakly hybridizing cONA. The sequence of the insert was 2498 basepairs, with an open reading frame of 1794 basepairs encoding a protein of 598 amino acids and a predicted molecular mass of 64 kDa. The DNAbinding domain and the ligand-binding domain are similar to those of steroid and thyroid hormone receptors. Moreover, this cDNA is highly homologous to mouse nur77 and rat NGFI-B, which are early response genes induced by nerve growth factor and other serum growth factors. We designated this gene NAK1. The modulation of expression of NAK1 during stimulation of cell growth was studied. The mRNA of NAK1 was induced rapidly and transiently by growth-stimulating agents, such as adenosine diphosphate, in monkey kidney cells (BSC-1), by phytohemagglutinin in human lymphocytes, and by serum stimulation of arrested fibroblasts. It is expressed in human fetal muscle and adult liver, brain, and thyroid. NAK1 could be a nuclear receptor. It will be of great interest to determine the ligand for NAK1 and the genes that are regulated by it. (Molecular Endocrinology 4: 1438-1443, 1990)
hormone and regulating the expression of specific genes (8). These hormones and their receptors are known to be related to proliferation and differentiation of cells (9). When a human fetal muscle cDNA library was screened with the human thyroid hormone receptor «2 (hTR«2) cDNA at low stringency to find other forms of hTR, we found a weakly hybridizing cDNA. We have sequenced this cDNA and compared it to other DNAbinding proteins. The DNA-binding domain and the ligand-binding domain are similar to those of steroid and thyroid hormone receptors. However, this cDNA is most homologous to mouse nur77 and rat NGFI-B, which are early response genes induced by nerve growth factor (NGF) and other serum growth factors (10, 11). The protein encoded by the cDNA we have cloned appears to be the human homolog of NGFI-B and nur77. It appears to be an early response gene which is rapidly and transiently induced by several growth factors and expressed in several tissues.
RESULTS
Two clones in the human fetal skeletal muscle cDNA library strongly hybridized with hTR«2 cDNA (12). One was a partial clone of hTRa2, and another was found to be hTR«1 (13). Four clones hybridized very weakly. These clones were analyzed by restriction mapping, and we sequenced the longest of them. The other three clones were identical in areas sequenced, but were shorter cDNAs. The sequenced insert was 2498 basepairs (bp) long, with an open reading frame of 1794 bp encoding a protein of 598 amino acids and a predicted molecular mass of 64 kDa (Fig. 1). This clone is highly homologous to rat NGFI-B and mouse nur77. NGFI-B and nur77 were described as genes that are very rapidly induced (in the mouse and rat) by NGF and several other growth factors (10,11). We have also compared
INTRODUCTION
The proliferation and differentiation of eukaryotic cells are regulated by growth factors, which act via intracellular messengers to cause rapid induction of certain genes (1-7). Some early response gene products are sequence-specific DNA-binding proteins. Steroid and thyroid hormone receptors are members of another gene family, which function by binding their cognate 0888-8809/90/1438-1443$02.00/0 Molecular Endocrinology Copyright © 1990 by The Endocrine Society
1438
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 04 June 2015. at 18:28 For personal use only. No other uses without permission. . All rights reserved.
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I TRA I
Ligand
100 bp
B
Met Pro ATG CCC Thr Cys lie Gin Ala Gin Tyr Gly Thr Pro Ala Pro TGI ATC CAA CCC CAA TAT GGG ACA CCA CCA CCG Thr Thr Pro Thr Met Asp Leu Ala Ser Pro Glu Ala Ala CCC ACC ATC GAC CTG GCC AGC CCC GAG GCA GCC Thr Glu Phe Asp Thr Phe Leu Tyr Gin Leu Pro Gly GAG TTT GAC ACC TTC CTC TAC CAG CTG CCA GGA Ala Thr Ser Pro Ala Ser Ala Ser Phe Lys Phe 366 GCC ACC TCC CCT GCC TCT GCT TCC TTC AAG TTC
Thr 20 Ser Pro Gly Pro Arg Asp His Leu Ala AGT CCG GGA CCC CGT GAC CAC CTG GCA Thr Ala Thr Thr Ala Pro Ser Pro Ala Ala ACT GCC CCC AGC CCC GCT GCC Ala Thr Thr Cys Thr 80 Cys Ser Ser Ser Thr Val Gin ACA GTC CAG TGC TCC TCA TCC Ala 110 Gly Cys Tyr Glu Asp Phe Gin Val GGC TGC TAC GAG GAC TTC CAG GTG
140 Gly Ser Pro Cys Ser Ala Pro Ser Pro Ser GGC AGC CCC TGC TCG GCC CCG TCG CCC TCC Pro 170 Trp Pro Ser Gin Thr Tyr Glu Gly Leu Arg Ala CCC AGC CAG ACT TAC GAA GGC CTG CGG GCA Val 200 Thr 190 Ser Leu Pro Ala Phe Phe Ser Phe Ser Pro Pro Thr Gly Pro Ser GCC TTC TTT TCC TTC AGT CCT CCC ACC GGC CCC AGC CCC AGC CTG Thr Ala 230 220 Leu Gly Glu Gly Glu Ser Tyr Ser Net Pro Thr Ala Phe Pro Gly CTG GGG CAG GGA CAG AGC TAT TCC ATG CCT ACG GCC TTC CCA GGT Val Ala Ser Ser Ala 250 Lys Ala Arg Gly Ala Pro Gly Thr Pro Val Thr Ser Thr GGG GCC CCA GGT ACA CCC GTG ACC TCA ACC AAG GCC CGG Ser Ser Ala 290 260 Gly Phe Phe Lys His Tyr Gly Val Arg Thr Cys Glu Gly Cys CAT TAT GGT GTC CGC ACA TGT GAG GGC TGC GGC TTC TTC AAG
Gly Ala Leu 30 Gly Ser Asp Pro Leu Thr Pro Glu Phe lie AGC GAC CCC CTG ACC CCT CAC TTC ATC Gly Ala Leu Ser 60 Phe Ser Thr Phe Met Asp Gly Tyr Thr TTC AGC ACC TTC ATG GAC^JjGC TAC ACA
Pro CCC
•Ser Ser. 90 Ser Ala Ser Ser Thr Ser Ser TCG GCC TCC TCC ACA^TCC TCG 'Set Ser' Thr Leu Gly Pro Leu Ser Gly Pro Val GCC CCC CTG AGC GGC CCA CTG Thr. Leu Ser 150 Asn Pro Ser Phe Gin Pro Pro Gin CCC AGC TTC CAG CCG CCC CAG Ser Asn 180 Thr Glu Gin Leu Pro Lys Ala ACA GAG CAG CTG CCC AAA GCC
Thr Glu Ala Leu Ser Ser Ser Gly Ser Asp Tyr Tyr 476 GCC CTG TCC TCC AGT GGC TCT GAC TAC TAT Thr 160 Pro Trp Asp Gly Ser Phe Gly His Phe Ser CCC TGC GAT GGC TCC TTC GGC CAC TTC TCG
Trp TGG
Pro Pro Pro Gin 656 CCC CCA CAG Pro Pro Gin Ala Thr 746 CAG GCC ACC Pro
Ser Ala Gin Ser Pro Leu Lys Leu GCC CAG AGC CCC CTG AAG TTG Ser Asn Leu Ala Pro Thr Ser Pro His TTG GCA CCC ACT TCT CCA CAC Asn
Pro Pro CCT CCA His Gin CAC CAG
Ser Gly lie Leu Asp 836 TCG GGG ATA CTG GAT Asn Ala Ser Cys Gin 926 AAC CCT TCA TGC CAG
310 Cys Leu Ala Asn Lys Asp Cys Pro Val Asp Lys Arg Arg Arg Asn Arg 1016 TGC CTG GCT AAC AAG GAC TGC CCT GTG GAC AAG AGG CGG CGA AAC CGC
Lys AAG Asn Gly CGA
Ser Ser TCC TCA Asp Glu GAT GAG
210 Phe TTC
Arg Leu CTT Arg 270 Ser Glu Gly Arg Cys Ala Val Cys AGT GAA GGC CGC TGT GCT GTG TGT
Ser 300 Arg Thr Val Gin Lys Asn Ala Lys Tyr H e CGC ACA GTG CAG AAA AAC GCC AAG TAC ATC Ser 330 320 Gin Phe Cys Arg Phe Gin Lys Cys Leu Ala Val Cly Met CAG TTC TGC CGC TTC CAG AAG TGC CTG GCG GTG GGC ATG
350 360 Thr 340 Val Lys Glu Val Val Arg Thr Asp Ser Leu Lys Gly Arg Arg Gly Arg Leu Pro Ser Lys Pro Lys Gin Pro Pro Asp Ala Ser Pro Ala 1106 GTG AAG GAA GTT GTC CGA ACA GAC AGC CTG AAG GGG CGG CGG GGC CGG CTA CCT TCA AAA CCC AAG CAG CCC CCA GAT*GCC TCC CCT CCC Thr 380 390 lie 370 Asn Leu Leu Thr Ser Leu Val Arg Ala His Leu Asp Ser Gly Pro Ser Thr Ala Lys Leu Asp Tyr Ser Lys Phe Gin Glu Leu Val Leu 1196 AAT CTC CTC ACT TCC CTG GTC CGT GCA CAC CTG GAC TCA GGG CCC AGC ACT GCC AAA CTG GAC TAC TCC AAG TTC CAG GAG CTG GTG CTG He Asn 410 Asp 420 Arg 400 Pro His Phe Gly Lys Glu Asp Ala Gly Asp Val Gin Gin Phe Tyr Asp Leu Leu Ser Gly Ser Leu Glu Val H e Arg Lys Trp Ala Glu 1286 CCC CAC TTT GGG AAG GAA GAT GCT GGG GAT GTA CAG CAG TTC TAC GAC CTG CTC TCC GGT TCT CTG GAG GTC ATC CGC AAG TGG GCG GAG Asp Arg 440 450 He 430 Cys Gly Lys lie Pro Gly Phe Ala Glu Leu Ser Pro Ala Asp Gin Asp Leu Leu Leu Glu Ser Ala Phe Leu Glu Leu Phe lie Leu Arg Leu Ala 1376 AAG ATC CCT GGC TTT GCT GAG CTG TCA CCG GCT GAC CAG GAC CTG TTG CTG GAG TCG GCC TTC CTG GAG CTC TTC ATC CTC CGC CTG GCG
He
470 Gin 480 460 Tyr Arg Ser Lys Pro Gly Clu Gly Lys Leu H e Phe Cys Ser Gly Leu Val Leu His Arg Leu Gin Cys Ala Arg Gly Phe Gly Asp Trp 1466 TAC
Akira Nakai, Sreedharan Kartha, Akihiro Sakurai, F. Gary Toback, and Leslie J. DeGroot Thyroid Study Unit (A.N., A.S., L.J.D.) Section of Nephrology (S.K., F.G.T.) Department of Medicine University of Chicago Chicago, Illinois 60637
When a human fetal muscle cDNA library was screened with the human thyroid hormone receptor a2 cDNA at low stringency, we found a weakly hybridizing cONA. The sequence of the insert was 2498 basepairs, with an open reading frame of 1794 basepairs encoding a protein of 598 amino acids and a predicted molecular mass of 64 kDa. The DNAbinding domain and the ligand-binding domain are similar to those of steroid and thyroid hormone receptors. Moreover, this cDNA is highly homologous to mouse nur77 and rat NGFI-B, which are early response genes induced by nerve growth factor and other serum growth factors. We designated this gene NAK1. The modulation of expression of NAK1 during stimulation of cell growth was studied. The mRNA of NAK1 was induced rapidly and transiently by growth-stimulating agents, such as adenosine diphosphate, in monkey kidney cells (BSC-1), by phytohemagglutinin in human lymphocytes, and by serum stimulation of arrested fibroblasts. It is expressed in human fetal muscle and adult liver, brain, and thyroid. NAK1 could be a nuclear receptor. It will be of great interest to determine the ligand for NAK1 and the genes that are regulated by it. (Molecular Endocrinology 4: 1438-1443, 1990)
hormone and regulating the expression of specific genes (8). These hormones and their receptors are known to be related to proliferation and differentiation of cells (9). When a human fetal muscle cDNA library was screened with the human thyroid hormone receptor «2 (hTR«2) cDNA at low stringency to find other forms of hTR, we found a weakly hybridizing cDNA. We have sequenced this cDNA and compared it to other DNAbinding proteins. The DNA-binding domain and the ligand-binding domain are similar to those of steroid and thyroid hormone receptors. However, this cDNA is most homologous to mouse nur77 and rat NGFI-B, which are early response genes induced by nerve growth factor (NGF) and other serum growth factors (10, 11). The protein encoded by the cDNA we have cloned appears to be the human homolog of NGFI-B and nur77. It appears to be an early response gene which is rapidly and transiently induced by several growth factors and expressed in several tissues.
RESULTS
Two clones in the human fetal skeletal muscle cDNA library strongly hybridized with hTR«2 cDNA (12). One was a partial clone of hTRa2, and another was found to be hTR«1 (13). Four clones hybridized very weakly. These clones were analyzed by restriction mapping, and we sequenced the longest of them. The other three clones were identical in areas sequenced, but were shorter cDNAs. The sequenced insert was 2498 basepairs (bp) long, with an open reading frame of 1794 bp encoding a protein of 598 amino acids and a predicted molecular mass of 64 kDa (Fig. 1). This clone is highly homologous to rat NGFI-B and mouse nur77. NGFI-B and nur77 were described as genes that are very rapidly induced (in the mouse and rat) by NGF and several other growth factors (10,11). We have also compared
INTRODUCTION
The proliferation and differentiation of eukaryotic cells are regulated by growth factors, which act via intracellular messengers to cause rapid induction of certain genes (1-7). Some early response gene products are sequence-specific DNA-binding proteins. Steroid and thyroid hormone receptors are members of another gene family, which function by binding their cognate 0888-8809/90/1438-1443$02.00/0 Molecular Endocrinology Copyright © 1990 by The Endocrine Society
1438
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 04 June 2015. at 18:28 For personal use only. No other uses without permission. . All rights reserved.
a>
o
2 £
5'ATGL
|
|
in a.
0.0.
II
II
o. co
a.
I TRA I
Ligand
100 bp
B
Met Pro ATG CCC Thr Cys lie Gin Ala Gin Tyr Gly Thr Pro Ala Pro TGI ATC CAA CCC CAA TAT GGG ACA CCA CCA CCG Thr Thr Pro Thr Met Asp Leu Ala Ser Pro Glu Ala Ala CCC ACC ATC GAC CTG GCC AGC CCC GAG GCA GCC Thr Glu Phe Asp Thr Phe Leu Tyr Gin Leu Pro Gly GAG TTT GAC ACC TTC CTC TAC CAG CTG CCA GGA Ala Thr Ser Pro Ala Ser Ala Ser Phe Lys Phe 366 GCC ACC TCC CCT GCC TCT GCT TCC TTC AAG TTC
Thr 20 Ser Pro Gly Pro Arg Asp His Leu Ala AGT CCG GGA CCC CGT GAC CAC CTG GCA Thr Ala Thr Thr Ala Pro Ser Pro Ala Ala ACT GCC CCC AGC CCC GCT GCC Ala Thr Thr Cys Thr 80 Cys Ser Ser Ser Thr Val Gin ACA GTC CAG TGC TCC TCA TCC Ala 110 Gly Cys Tyr Glu Asp Phe Gin Val GGC TGC TAC GAG GAC TTC CAG GTG
140 Gly Ser Pro Cys Ser Ala Pro Ser Pro Ser GGC AGC CCC TGC TCG GCC CCG TCG CCC TCC Pro 170 Trp Pro Ser Gin Thr Tyr Glu Gly Leu Arg Ala CCC AGC CAG ACT TAC GAA GGC CTG CGG GCA Val 200 Thr 190 Ser Leu Pro Ala Phe Phe Ser Phe Ser Pro Pro Thr Gly Pro Ser GCC TTC TTT TCC TTC AGT CCT CCC ACC GGC CCC AGC CCC AGC CTG Thr Ala 230 220 Leu Gly Glu Gly Glu Ser Tyr Ser Net Pro Thr Ala Phe Pro Gly CTG GGG CAG GGA CAG AGC TAT TCC ATG CCT ACG GCC TTC CCA GGT Val Ala Ser Ser Ala 250 Lys Ala Arg Gly Ala Pro Gly Thr Pro Val Thr Ser Thr GGG GCC CCA GGT ACA CCC GTG ACC TCA ACC AAG GCC CGG Ser Ser Ala 290 260 Gly Phe Phe Lys His Tyr Gly Val Arg Thr Cys Glu Gly Cys CAT TAT GGT GTC CGC ACA TGT GAG GGC TGC GGC TTC TTC AAG
Gly Ala Leu 30 Gly Ser Asp Pro Leu Thr Pro Glu Phe lie AGC GAC CCC CTG ACC CCT CAC TTC ATC Gly Ala Leu Ser 60 Phe Ser Thr Phe Met Asp Gly Tyr Thr TTC AGC ACC TTC ATG GAC^JjGC TAC ACA
Pro CCC
•Ser Ser. 90 Ser Ala Ser Ser Thr Ser Ser TCG GCC TCC TCC ACA^TCC TCG 'Set Ser' Thr Leu Gly Pro Leu Ser Gly Pro Val GCC CCC CTG AGC GGC CCA CTG Thr. Leu Ser 150 Asn Pro Ser Phe Gin Pro Pro Gin CCC AGC TTC CAG CCG CCC CAG Ser Asn 180 Thr Glu Gin Leu Pro Lys Ala ACA GAG CAG CTG CCC AAA GCC
Thr Glu Ala Leu Ser Ser Ser Gly Ser Asp Tyr Tyr 476 GCC CTG TCC TCC AGT GGC TCT GAC TAC TAT Thr 160 Pro Trp Asp Gly Ser Phe Gly His Phe Ser CCC TGC GAT GGC TCC TTC GGC CAC TTC TCG
Trp TGG
Pro Pro Pro Gin 656 CCC CCA CAG Pro Pro Gin Ala Thr 746 CAG GCC ACC Pro
Ser Ala Gin Ser Pro Leu Lys Leu GCC CAG AGC CCC CTG AAG TTG Ser Asn Leu Ala Pro Thr Ser Pro His TTG GCA CCC ACT TCT CCA CAC Asn
Pro Pro CCT CCA His Gin CAC CAG
Ser Gly lie Leu Asp 836 TCG GGG ATA CTG GAT Asn Ala Ser Cys Gin 926 AAC CCT TCA TGC CAG
310 Cys Leu Ala Asn Lys Asp Cys Pro Val Asp Lys Arg Arg Arg Asn Arg 1016 TGC CTG GCT AAC AAG GAC TGC CCT GTG GAC AAG AGG CGG CGA AAC CGC
Lys AAG Asn Gly CGA
Ser Ser TCC TCA Asp Glu GAT GAG
210 Phe TTC
Arg Leu CTT Arg 270 Ser Glu Gly Arg Cys Ala Val Cys AGT GAA GGC CGC TGT GCT GTG TGT
Ser 300 Arg Thr Val Gin Lys Asn Ala Lys Tyr H e CGC ACA GTG CAG AAA AAC GCC AAG TAC ATC Ser 330 320 Gin Phe Cys Arg Phe Gin Lys Cys Leu Ala Val Cly Met CAG TTC TGC CGC TTC CAG AAG TGC CTG GCG GTG GGC ATG
350 360 Thr 340 Val Lys Glu Val Val Arg Thr Asp Ser Leu Lys Gly Arg Arg Gly Arg Leu Pro Ser Lys Pro Lys Gin Pro Pro Asp Ala Ser Pro Ala 1106 GTG AAG GAA GTT GTC CGA ACA GAC AGC CTG AAG GGG CGG CGG GGC CGG CTA CCT TCA AAA CCC AAG CAG CCC CCA GAT*GCC TCC CCT CCC Thr 380 390 lie 370 Asn Leu Leu Thr Ser Leu Val Arg Ala His Leu Asp Ser Gly Pro Ser Thr Ala Lys Leu Asp Tyr Ser Lys Phe Gin Glu Leu Val Leu 1196 AAT CTC CTC ACT TCC CTG GTC CGT GCA CAC CTG GAC TCA GGG CCC AGC ACT GCC AAA CTG GAC TAC TCC AAG TTC CAG GAG CTG GTG CTG He Asn 410 Asp 420 Arg 400 Pro His Phe Gly Lys Glu Asp Ala Gly Asp Val Gin Gin Phe Tyr Asp Leu Leu Ser Gly Ser Leu Glu Val H e Arg Lys Trp Ala Glu 1286 CCC CAC TTT GGG AAG GAA GAT GCT GGG GAT GTA CAG CAG TTC TAC GAC CTG CTC TCC GGT TCT CTG GAG GTC ATC CGC AAG TGG GCG GAG Asp Arg 440 450 He 430 Cys Gly Lys lie Pro Gly Phe Ala Glu Leu Ser Pro Ala Asp Gin Asp Leu Leu Leu Glu Ser Ala Phe Leu Glu Leu Phe lie Leu Arg Leu Ala 1376 AAG ATC CCT GGC TTT GCT GAG CTG TCA CCG GCT GAC CAG GAC CTG TTG CTG GAG TCG GCC TTC CTG GAG CTC TTC ATC CTC CGC CTG GCG
He
470 Gin 480 460 Tyr Arg Ser Lys Pro Gly Clu Gly Lys Leu H e Phe Cys Ser Gly Leu Val Leu His Arg Leu Gin Cys Ala Arg Gly Phe Gly Asp Trp 1466 TAC
