Identification of a New Member of the Steroid Hormone Receptor Superfamily That Is Activated by a Peroxisome Proliferator and Fatty Acids
Azriel Doron
Schmidt, Naoto Endo, Su Jane Shinar, and Gideon A. Rodan
Rutledge,
Robert
Vogel,
Department of Bone Biology and Osteoporosis Research Merck Research Laboratories West
Point,
Pennsylvania
19486
We have identified a novel member of the steroid hormone receptor superfamily by cDNA cloning from a human osteosarcoma SAOS-S/B10 cell library. Sequence analysis predicts a protein of 441 amino acids, which includes the conserved amino acid residues characteristic of the DNA- and ligand-binding domains of nuclear receptors. Amino acid sequence alignment and transcriptional activation experiments revealed that the new protein is closely related to the mouse peroxisome proliferator activated receptor. The overall homology is 62%, and the highest similarity is seen in the DNAand ligand-binding domains, 86% and 71%, respectively. Northern blot analysis showed that in mature rats, the receptor is highly expressed in heart, kidney, and lung as a transcript of approximately 3500 nucleotides. In human cells, the size of the mRNA is approximately 4000 nucleotides. Transcription assays using hybrid receptors consisting of the ligandbinding domain of the new protein and the DNAbinding domain of the glucocorticoid receptor showed weak stimulation by the peroxisome proliferator activator WY14643, suggesting a relationship to that receptor. Similar stimulation was observed with arachidonic and oleic acid (loo-250 PM). (Molecular Endocrinology 6: 1634-1641, 1992)
vealed two important regions of these receptors that exhibit a high degree of amino acid residue conservation. The highest level of similarity among the receptors is found in a region that contains nine cysteine residues that bind zinc atoms to form two “zinc fingers,” which interact with the cognate steroid response elements of DNA (4,5). The second region, which is less conserved, is the ligand-binding domain responsible for the interaction with the hormone (6, 7). Recent studies have attributed additional functions to other domains in these receptors, such as protein-protein interactions of domains that participate in transcriptional regulation (8IO). The amino acid conservation in the DNA-binding domain has led to the identification of new members of the steroid receptor superfamily. For example, hER1 and hER2 have been cloned by low stringency hybridization of cDNA libraries with a DNA probe coding for the DNA-binding domain of the estrogen receptor (11). Similar approaches have led to the discovery of multiple retinoic acid receptors and the mouse peroxisome proliferator activator receptor (mPPAR) (12, 13). We report here the use of polymerase chain reaction (PCR) to identify a new, as yet unreported member of this receptor family.
RESULTS INTRODUCTION Cloning Retinoids, steroid and thyroid hormones, and possibly other molecules produce their biological effects by binding to proteins of the steroid receptor superfamily. These receptors interact with specific DNA sequences and modulate gene expression (for reviews, see Refs. l-3). Sequence analysis and functional studies re0888-8809/92/1634-1641$0300/O Molecular Endocmology CopyrIght 1992 by The Endocrine
of NUCI cDNA
The PCR method was used to identify new members of the nuclear hormone receptor superfamily from bonederived cells. Degenerate oligonucleotides were synthesized according to the amino acid sequence of two conserved segments shared by members of the nuclear receptor superfamily (2). The 5’-end primers, ESI’I and ES12, were designed according to a segment of the DNA-binding domain (Fig. 1A). The primer at the 3’end, ES15 was prepared according to a conserved
Scmty
1634 The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 03 February 2015. at 04:55 For personal use only. No other uses without permission. . All rights reserved.
New
Member
of Steroid
Hormone
Receptor
Superfamily
1635
A SENSE ES11
CGAATTC
C E G C K TGT GAG GGC TGC AAD
A/G
5’
SENSE ES12
CGAATTC
CEGCK TGT GAG GGC TGC AAG
A/G
5’
ANTISENSE ES15
5’
GGMTTC
GCC A G
GCC A G
PGPXXKAF AAA ACC AGG IA1 G C C T T G G
III
1
P
F
TTC
TTC
2
3
MW
4 1078
CTT TGC lvuL T A G G C
-
603
-
310
4 234 - 194 4 118
B 917
950 TCACCGGCAAA II,, AACGTCGGGAGTGT~TTAAGC
5 ’ AAAAAGAAGGCCCGCAGCATCC 3’ IIIIIIIII III
CTTCTTCCGG
II
3 ’ 5’
ES12
Fig. 1. Amplification
of NUCI DNA by PCR The DNA sequences of the primers used in the PCR reaction are shown in A. The corresponding amino acid residues are indicated (upper line). On the right, the products of the second round of PCR amplification, separated on 5% polyacrylamide gel and stained by ethidium bromide, are shown. The alignment of primer ES12 to the actual cDNA sequence of NUCI is shown in B.
amino acid sequence in the ligand-binding domain of the retinoid receptor subfamily and the vitamin D receptor. Since thisconserved region contains two nonconserved amino acid residues, inosine nucleotides were used as part of this primer (Fig. 1A). Amplification of cDNA prepared from RNA of human osteosarcoma cells SAOS-2/BlO, amplified with the primers ES1 1 and ES15 yielded multiple DNA fragments with various sizes after the first round of amplification (data not shown). A portion of the reaction was subjected to a second round of amplification using the nested primer ES12 and the same 3’-end primer ES15. This amplification produced two DNA fragments of 270 and 320 basepairs (bp), respectively (Fig. 1 A), which were cloned into pUC 18 and sequenced (data not shown). The amino acid residues predicted by the DNA sequences indicated that each of these DNA fragments coded for a segment of a genuine and novel member of the steroid hormone receptor superfamily. The focus of this work is the cloning and characterization of one of these novel receptors, NUCI. To obtain the complete cDNA sequence we screened a human SAOS-2/BlO cell cDNA library with the amplified 320-bp fragment of NUCI. All the strongly hybridizing clones were identical and matched the sequence of the amplified 320-bp DNA fragment. Analysis of the deduced amino acid sequence revealed a long open reading frame, which starts at the putative initiation methionine codon at nucleotide 338 and codes for a protein of 441 amino acid residues (Fig. 2). The putative protein contains a cysteine-rich region at the amino-terminus, which probably represents the conserved DNA-binding domain of this receptor. The carboxyl-terminus of the protein contains conserved amino acid residues, which may be part of a ligandbinding domain (l-3). The DNA-binding region contains
11 cysteine residues, compared to 9 residues in other steroid receptors (Fig. 2) suggesting the potential formation of an alternative Zn’-dependent finger structure(s). As in the peroxisome proliferator-activated receptor, the conserved loop structure of the second Zn+ finger (Cys”’ to Cys”‘) contains only three amino acid residues, compared to five in other members of the receptor family (12). Comparison of the amino acid sequence of NUCI with those of other members of the nuclear receptor family revealed best overall similarity (62%) to the mouse peroxisome proliferator-activated receptor (Fig. 3) (12). The highest similarity is in the putative DNA-binding domain, where 86% of the 66 amino acid residues are identical (Fig. 4). The ligand-binding domain of NUCI also exhibited close similarity with the corresponding region of the mPPAR. These domains share 71% of the amino acid residues (Fig. 4). Outside these two regions, the similarity between NUCI and the mPPAR is lower. While 57% of the amino acid residues in the region between the DNA- and ligand-binding domains are identical, in the N-terminal portion only 27% are the same. Sequence comparisons between NUCI and the three X,enopus receptors, xPPARcu, xPPARP, and xPPARy, exhibited the same approximate similarities (14). The DNA- and ligand-binding domains of NUCI receptor show 81-86% and 71-73% similarity to the parralel domains of the Xenopus receptors, respectively (Fig. 4). None of the less conserved domains of these receptors showed closer sequence relationship to NUCI receptor. These observations support the idea that NUCI is a new member of this receptor family and not the human homolog of any of these receptors. Although the similarity between NUCI and PPAR is substantial, it is much lower than the similarity among members of the retinoic acid receptor family (2). NUCI shows the
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 03 February 2015. at 04:55 For personal use only. No other uses without permission. . All rights reserved.
MOL 1636
ENDO.
1992
Vol6
1 61 121 181 241 301 361 421 481 541 601 661 721 781 841 901 961 1021 1081 1141 1201 1261 1321 1381 1441 1501 1561 1621 1681 1741 1801 1861 1921 1981 2041 2101 2161 2221 2281 2341 2401 2461 2521 2581 2641 2701 2761 2821 2881 2941 3001 3061 3121 3241 3301
GAATTCTGCGGAGCCTGCGGGACGGCGGCGGGTTGGCCCGTAGGCAGCCGGGACAGTGTT GTACAGTGTTTTGGGCATGCACGTGATACTCACACAGTGGCTTCTGCTCACC~CAGATG AAGACAGATGCACCAACGAGGGTCTGGAATGGTCTGGAGTGGTCTGGAAAGCAGGGTCAG ATACCCCTGGAAAACTGAAGCCCGTGGAGCAATGATCTCTACAGGACTGCTTCAAGGCTG ATGGGAACCACCCTGTAGAGGTCCATCTGCGTTCAGACCCAGACGATGCCAGAGCTATGA CTGGGCCTGCAGGTGTGGCGCCGAGGGGAGATCAGCCATGGAGCAGCCACAGGAGG~GC MEQPQEEA CCCTGAGGTCCGGGAAGAGGAGGAGAAAGAGGAGGCAGGAGCCCCAGA PEVREEEEKEEVAEAE GAPE GCTCAATGGGGGACCACAGCATGCACTTCCTTCCAGCAGCTACACAGACCTCTCCCGGAG LNGGPQHALPSSSYT DLSRS CTCCTCGCCACCCTCACTGCTGGACCAACTGCAGATGCAGATGGGCTGTGACGGGGCCTCATGCGG s s P P SLLDQLQMGCDGASCG GTGCCGGGTGTGCGGGGACAAGGCATCGGGCTTCCACTACGGTGT S L N M @RV@G DKASGFHYGV TCATGCATGTGAGGGGTGCAGGGCTTCTTCCGTCGTACGATCCGCATG~GCTGGAGTA HA@EG@KGFFRRT I RMK L E Y CGAGAAGTGTGAGCGCAGCTGCAAGATTCAGAAGAAGAACCGCAACAGTGCCAGTACTG EK@ERS@K IQKKNRNK@QY@l28 CCGCTTCCAGAAGTGCCTGGACTGGGCATGTCACACAACT RF Q K CLALGMSHNAIRF G R M GCCGGAGGCTGAGAAGAGGAGCTGGTGGCAGGGCTGACTGC~CGAGGGGAGCCAGTA P EAEKRKLVAGLTANEGSQY CAACCCACAGGTGGCCGACCTGAAGGCCTTCTCCAAGCACATCTACAATGCCTACCTGAA S K H I YNAYLK N P Q VADLKAF AAACTTCAACATGACCAG~GGCCCGCAGCATCCTCACCGGC~GCCAGCCACAC NFNMTKKKARSI LTGKASHT GGCGCCCTTTGTGATCCACGACATCGAGACATTGTGGCAGGCAGAGIlAGGGGCTGGTGTG AP FV I H D I ET LWQAEK G LVW GAAGCAGTTGGTG~TGGCCTGCCTCCCTACAGGAGATCAGCGTGCACGTCTTCTACCG KQLVNGLPPYKEI SV HV F Y R CTGCCAGTGCACCAC GTGGAGACCGTGCGGGAGCTCACTGAGTTCGCCAAGAGCATCCC C QC T TVE TVRE LT EF AK S I P CAGCTTCAGCAGCCTCTTCCTCAACGACCAGGTTACCCTTCTCILAGTATGGCGTGCACGA S F S SLFLNDQVTLLK Y G V H E GGCCATCTTCCCGATGCTGGCCTCTATCGTCAACAAGGACGGGCTGCTGGTAGCCAACGG A IFPMLASIVNKDGLLVANG CAGTGGCTTTGTCACCCGTGAGTTCCTGCGCAGCCTCCGCAAACCCTTCAGTGATATCAT SGFVTREFLRS I L R K P F S D I TGAGCCTAAGTTTGAATTTGCTGTCAAGTTCAACGCCCTGGAACTTGATGACAGTGACCT EPKFEFAVKFNALELDD S D L GGCCCTATTCATTGCGGCCATCATTCTGTGTGG~~~~~~~~~~~~~~~~~~~~~~~~~C A L F I A A I ILCGDRPGLMNVP ACGGGTGGAGGCTATCCAGGACACCATCCTGCGTGCCCTCGAATTCCACCTGCAGGCCAA R V E A I Q D T I L RA L E F H L Q A N CCACCCTGATGCCCAGTACCTCTTCCCCAAGCTGCTGCAGGATGGCTGACCTGCGGCA HPDAQYLFPKL L Q K MA D L R Q ACTGGTCACCGAGCACGCCCAGATGATGCAGCGGATCAAGAAGACCGAAACCGAGACCTC LVTEHAQMMQRI KKTETETS GCTGCACCCTCTGCTCCAGGAGATCTACAAGGACATGTACTAACGGCGGCACCCAGGCCT ILHPLLQE I Y K D M Y/END CCCTGCAGACTCCAATGGGGCCAGCACTGGAGGGGCCCACCCACATGACTTTTCCATTGA CCAGCTCTCTTCCTGTCTTTGTTGTCTCCCTCTTTCTCAGTTCCTCTTTCTTTTCTAATT CCTGTTGCTCTGTTTCTTCCTTTCTGTAGGTTTCTCTCTTCCCTTCTCCCTTCTCCCTTG CCCTCCCTTTCTCTCTCCTATCCCCACGTCTGTCCTCCTTTCTTATTCTGTGAGATGTTT TGTATTATTTCACCAGCAGCATAGAACAGGACCTCTCTGCTTTTGCACACCTTTTCCCCAGG AGCAGAAGAGAGTGGGCCTGCCCTCTGCCCCATCATTGCACCTGCAGGCTTAGGTCCTCA CTTCTGTCTCCTGTCTTCAGAGCAAAAGACTTGAGCCATCCAAAGAAACACTAAGCTCTCTC TGGGCCTGGGTTCCAGGGAAGGCTAAGCATGGCCTGGACTGACTGCAGCCCCCTATAGTC ATGGGGTCCCTGCTGCAAAGGACAGTGGCAGACCCCGGCAGTAGAGCCGAGATGCCTCCC CAAGACTGTCATTGCCCCTCCGATCGTGAGGCCACCCACTGACCCAATGATCCTCTCCAG CAGCACACCTCAGCCCCACTGACACCCAGTGTCCTTCCATCTTCACACTGGTTTGCCAGG CCAATGTTGCTGATGGCCCCTCCAGCACACACACATAAGCTCACTTTACCTGC AGGCACCATGCACCTCCCTTCCCTCCCTGAGGCAGGTGAGAACCCAGAGAGAGGGGCCTG CAGGTGAGCAGGCAGGGCTGGGCCAGGTCTCCGGGGAGGCAGGGGTCCTGCAGGTCCTGG TGGGTCAGCCCAGCACCTCGCCCAGTGGGAGCTTCCCGGGAT~CTGAGCCTGTTCATT CTGATGTCCATTTGTCCCAATAGCTCTACTGCCCTCCCCTTCCCCTTTACTCAGCCCAGC TGGCCACCTAGAAGTCTCCCTGCACAGCCTCTAGTGTCCGGGGACCTTGTGGGACCAGTC CCACACCGCTGGTCCCTGCCCTCCCCTGCTCCCAGGTTGAGGTGCGCTCACCTCAGAGCA GGGCCAIlAGCACAGCTGGGCATGCCATGTCTGAGCGGCGCAGAGCCCTCCAGGCCTGCAG GGGCAAGGGGCTGGCTGGAGTCTCAGAGCACAGAGGTAGGAGAACTGGGGTTGCCCA GGCTTCCTGGGTCCTGCCTGGTCCTCCCTCCCAAGGAGCCATTCTATGTGACTCTGGGTG GAAGTGCCCAGCCCCTGCCTGACGGXXXXXXXGATCACTCTCTGCTGGCAGGATTCTTCC CGCTCCCCACCTACCCAGCTGATGGGGGTTGGGGTGCTTCTTTCAGCC~GGCTATG~G GGACAGCTGCTGGGACCCACCTCCCCCCTTCCCCGGCCACATGCCGCGTCCCTGCCCCCA CCCGGGTCTGGTGCTGAGGATACAGCTCTTCTCAGTGTCTG~C~TCTCC~TTG~ ACTGACGAAACTTTAAATAAATGGGAATTAAATATTAAATATTT WGCGGCCGCGAATT c 3301
8 28 48 68 88 108
148 168 188 208 228 248 268 288 308 328 348 368 388 408 428 441
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 03 February 2015. at 04:55 For personal use only. No other uses without permission. . All rights reserved.
No. 4
New Member
of Steroid
Hormone
Receptor
Superfamily
1637
base transcript in human SAOS-2/BlO cells (Fig. 5A). In mice and rats the transcript for NUCI is about 500 nucleotides shorter than that observed in human cell lines and baboon tissues. In mature rats we found high levels of NUCI expression in heart, lung, and kidney (Fig. 5B). About 5-fold lower levels were found in skin and bone RNA prepared from tibia. High expression levels were also observed in spleen and ovaries (data not shown). This result suggests that NUCI is expressed in many tissues and organs, and is most abundant in heart, kidneys, and lungs. Unlike mPPAR, which is highly expressed in liver, the concentration of NUCI RNA is lowest in adult liver.
273
LFLNDa”TLLKYG”.HEAIFPMLASIVNKDGLL”ANGSGF”TREFLRSLR
300
I llllIlllllll l//l II I IllI LDLNDQVTLLKYGVYEEAIFTMLSSLMNKDGMLIAYGNGFITREFLKNLR
321 I
I
I
II
II/II
next best similarity with members of the retinoic acid/ thyroid receptors subfamily, including retinoic acid receptor-a, retinoid X receptor-a, thyroid hormone receptor, or vitamin D receptor (13, 15-l 8). The DNA-binding domains are 58-62% identical, and the ligand-binding domains exhibit a similarity in the range of 29-31%. Regarding the PCR procedure, it is of interest that sequence analysis of the amplified DNA products revealed that the primer ES12 was incorporated at both the 5’- and 3’-ends. At the 3’-end the primer only partially matched the actual cDNA sequence (Fig. 1 B). Thus, the amplification was the product of a single primer. Surprisingly, the nucleotide at the 3’-end of primer ES12 did not match the full-length cDNA sequence at all. None of the cloned PCR products contained the sequences of primer ESll. It is not clear whether primer ES12 was incorporated into the DNA fragments at the 3’-end, at the beginning of the amplification process, or only during late amplification cycles. of NUCI mFiNA
Northern analysis with a NUCI cDNA probe revealed that NUCI receptor mRNA is expressed as a 4.0-kilo-
Fig. 2. Nucleotide
Sequence and Deduced The cysteine residues at the predicted ligand-binding domains are boxed.
Activation
349
Fig. 3. Amino Acid Comparison between the NUCI Receptor and the Mouse PPAR (12) For maximum similarity, gaps were introduced, as indicated by dots. Identical amino acid residues are marked with vertical bars.
Expression
Receptor
II
Amino Acid Sequence DNA-binding domain
To search for a putative ligand for the NUCI receptor, we used the fact that the ligand-binding domains of the various receptors of the steroid superfamily can be interchanged to form chimeric receptors. These hybrid receptors are capable of exhibiting ligand-dependent transcription activation of a heterologous responsive DNA sequence (12, 19, 20). We prepared a chimeric receptor for NUCI (pJSGR/NUCI). The amino acid-terminus, which included the DNA-binding domain of the mouse glucocorticoid receptor (mGR) (21), was fused to the ligand-binding domain of NUCI in the manner described for mPPAR (12). For an additional control, we constructed a similar chimeric receptor in which the DNA-binding domain of the mouse estrogen receptor was fused to the ligand-binding domain of NUCI receptor (ER/NUCI). The plasmid pJA358 that contains the luciferase gene under the control of the modified mouse mammary tumor virus (MMTV) promoter was used as the reporter of the transcription unit. The screening assay was performed by transient transfection of COS cells, as previously described (12, 13, 22). Based on the similarity of NUCI receptor to the mPPAR, we tested if the Wy-14643 molecule could activate the NUCI hybrid receptor. In these experiments, WY-14643 stimulated luciferase activity after cotransfection with the GR/NUCI chimeric receptor (Fig. 6). Cotransfection of the reporter gene with wild-type glucocorticoid receptor or the ER/NUCI hybrid receptor did not result in similar stimulation (Fig. 6). We found that this compound, at a concentration of 100 PM, stimulated luciferase levels in GR/NUCI-transfected cells 4- to lo-fold. No activation was observed with other peroxisome proliferators, such as clofibrate, or other compounds that strongly stimulate liver fatty acid acyl coenzyme-A oxidase in mice at a concentration of 100 PM. No increase in luciferase activity was observed when WY-14643 was added to cells expressing either the unaltered mGR or NUCI receptors. The relatively low level of activation mediated
of the NUCI cDNA are circled. The amino
acid
residues
of the deduced
DNA-
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 03 February 2015. at 04:55 For personal use only. No other uses without permission. . All rights reserved.
and
MOL 1638
ENDO.
1992
72
Vol6
136
254
441
hNUC I 27%
102 m
166
1
23%
106 m
173
1
30
95
57%
244 m250 -4
474
60%
171
396
A
100
ER-NUC
No. 4
GR-NUC
466 . .
mPPAR
xPPARa xPPARf3 2
112 1
17%
m
177
257 41%
I-
477 '.
xPPARy
Fig. 4. Schematic Amino Acid Comparison between NUCI and Members of the PPAR Families The schematic amino acid alignment was prepared according to the functional domain structure of the steroid hormone receptors mPPAR (12) and the X. kevis xPPARLu, xPPARP, and xPPARr (14). The percentage of amino acid identity between the various receptors and NUCI is indicated for each domain.
0
GR-NUC
MMTV-LUC
A
Fig. 5; Northern Blot Analysis of NUCI Receptor mRNA RNA preparation and hybridization are described in Materials and Methods. A, Poly(A)-selected RNA (2 pg) of the osteosarcoma SAOSP/BlO cells was resolved on a formaldehyde-agarose gel. The RNA was transferred to a nylon membrane and hybridized with the cDNA probe for NUCI. The migration of the mol wt markers (kilobases) is indicated. B, Total RNA was isolated from the indicated rat tissues, and 20 and 4 fig of each sample (adjacent lanes) were treated and hybridized, as described in A.
by GR/NUCI may be attributed to the high baseline activation of the chimeric receptor in the absence of exogenous ligands. Cells transfected with the hybrid receptor had a 5fold higher level of luciferase activity than cells expressing the NUCI receptor itself or the native mGR. Similar findings were obtained in experiments in which the ligand-binding domain of NUCI was fused to the DNA-binding domain of the estrogen receptor or RXR (data not shown). Alternatively, the high basal activity observed with the chimeric receptor could be due to the presence of the natural ligand for NUCI receptor in the cell culture. It was recently shown that the rat PPAR was activated by various fatty acids, such as arachidonic (C18:4) and oleic (C18:l) acid (25). We found that the hybrid receptor GR/NUCI was also stim-
Fig. 6. Activation of Chimeric NUCI Receptor by Wy-14643 A, Cells were transfected with glucocorticoid response element-driven luciferase vector and either the wild-type GR, the hybrid estrogen-NUCI receptor (ER-NUC), or the chimeric glucocorticoid-NUCI receptor (GR-NUC). The transfected cells were treated with either Wy-14643 (WY) or dexamethasone (Dex) at the indicated concentrations. The luciferase assays were performed as described in Materials and Methods. The 100% value was the luciferase activity obtained by the addition of 0.1 M Dex to cells expressing the native GR. B, Experiments were performed as described in A. With MMTV-LUC, cells were transfected with the Luciferase reporter plasmid without steroid hormone receptor.
ulated by these agents. Both arachidonic and oleic acids, at concentrations of 100 and 60 PM, respectively, activated the reported gene that was cotransfected with the chimeric receptor (Fig. 7). No similar stimulation was observed when the reporter plasmid was transfected with the normal NUCI receptor or the normal mGR (Fig. 6). A large series of known ligands, including 1,25dihydroxyvitamin DS, 24,25-dihydroxyvitamin D3, Tq, estrogen, vitamin E, retinoic acid, progesterone, androgen, and other putative commercially available ligands, did not activate transcription via the hybrid receptor (at concentrations of l-10 PM; data not shown). Dexamethasone (0.1 PM) stimulated transcription mediated
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 03 February 2015. at 04:55 For personal use only. No other uses without permission. . All rights reserved.
New Member
of Steroid
Hormone
NUCI
Receptor
1639
Superfamily
GR-NUC
The experiments were performed as described in Fig. 6. The cells were treated with arachidonic acid (Ar.A.), oleic acid (OLA.), or dexamethasone (Dex.) at the indicated concentrations.
by the wild-type glucocortidoid receptor (Fig. 6). Low stimulation was also observed in cells transfected by GR/NUCI. This is probably a reflection of endogenous glucocorticoid receptors present in the cells, since the hybrid ER/NUCI receptor, which does not activate the reporter gene, was also responsive to dexamethasone (Fig. 6A). A similar response to dexamethasone was observed in cells that were transfected with only the MMTV-luciferase reporter gene (Fig. 68).
DISCUSSION
This report presents the cDNA cloning of a new member of the steroid hormone receptor superfamily. The amino acid sequence deduced from the DNA sequence has the characteristic features of both the DNA- and ligandbinding domains of this family of receptors. The putative DNA-binding domain of NUCI contains the conserved nine cysteine residues, which are found in all members of this receptor superfamily. Interestingly, two additional cysteine residues (Cys’” and CY@~) are located in the proximity of the nine conserved cysteine residues. The presence of additional cysteine residues in this region could lead to the formation of alternative structures for the DNA-binding domain. Sequence comparison with the different receptors of this superfamily indicates that NUCI is closely related to the mPPAR (12), with which it shares 86% of the amino
acids
in the DNA-binding
domain
and
72%
related, they may have regulatory roles in different target tissues. The structure of NUCI and that of its related receptor mPPAR suggest that they belong to a subgroup of the steroid receptor superfamily. Recently, three additional related receptors, XPPARa, XPPAPP, and XPPARr, were cloned from Xenopus (14). The DNA- and ligandbinding domains of NUCI receptor show 81-86% and 71-73% identity with the corresponding domains of the Xenopus receptors (14). However, in the less conserved regions of steroid receptors, there is a much lower degree of similarity. Thus, although it is clear that NUCI is a member of the PPAR receptor group, it remains to be determined whether NUCI is the human homolog of one of the Xenopus PPARs or a new member of this receptor family. These receptors were shown to activate the acyl coenzyme-A oxidase promoter. Whether NUCI receptor is also involved in peroxisome proliferation or P-oxidation of fatty acid is yet to be determined. Consistent with the sequence similarity, the chimeric NUCI receptor was activated by the peroxisome proliferator Wy-I 4643 at a concentration of 1 Om4 M. Lower concentrations ceptor. The
did not activate the chimeric NUCI relower sensitivity of NUCI receptor to the
peroxisome proliferator, in spite of the similarity in the ligand-binding domain, could be attributed either to differences in key amino acids in the ligand-binding domain or, possibly, to some unknown technical aspects of the assay. Recently, it has been shown that fatty acids, such as arachidonic and oleic acid, can stimulate transcription mediated by the rat PPAR (25). Similar results were obtained with the human NUCI receptor. Both arachidonic and oleic acids activated transcription of the reporter gene that was dependent on the expression of the chimeric NUCI receptor. The stimulation occurred at a concentration range of 60100 PM, similar to that observed with the rat PPAR. Since the fatty acids and the other synthetic peroxisome proliferators are structurally diverse molecules, it is not known if they are the actual ligands for these receptors or molecules structurally related to the real ligand(s). It is possible that the activation of the PPAR family by a variety of molecules could be attributed to pharmacological modulation of protein kinases and phosphatases, as shown for both the progesterone and chicken ovalbumin upstream promoter transcription factor receptors (23, 24). Since true ligand-receptor interactions are usually highly specific, we believe that the ultimate natural ligand for NUCI and the other members of this receptor family awaits isolation and identification.
in the
ligand-binding domain. Hybridization experiments revealed that the distribution of NUCI receptor is different from that of the mPPAR. NUCI is highly expressed in heart, kidney, lung, spleen, and ovaries and has very low expression in liver. In contrast, mPPAR was reported to show highest expression in liver (12). This suggests that although the two receptors seem to be
MATERIALS
AND METHODS
PCR Amplification Total RNA was isolated from human osteosarcoma BlO cells (26), and a random primed cDNA library pared from 2 pg RNA with the Moloney murine reverse transcriptase according to the manufacturer’s
SAOS2/ was preleukemia recom-
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 03 February 2015. at 04:55 For personal use only. No other uses without permission. . All rights reserved.
MOL 1640
ENDO.
1992
Vol6
mendations (Bethesda Research Laboratories, Gaithersburg, MD). The cDNA reaction (25 ~1) was diluted into 300 11 water, heat denatured at 95 C for 5 min, and quickly chilled on ice. The cDNA (2.5 ~1) and the first primer pair, ES1 1 and ES1 5 (0.5 FM each; Fig. 1 A), were employed in the amplification reaction with the Amplitaq kit and the DNA thermal cycler (Perkin-Elmer-Cetus, Norwalk, CT). We carried out the following amplification cycles; denaturation at 94 C, 1.5 min; annealing at 65 C, 3 min; extension at 72 C, 5 min for three cycles; denaturing at 94 C, 1 min; annealing at 60 C, 3 min; extension at 72 C, 5 min for 15 cycles; and denaturing at 94 C, 1 min; annealing at 57 C, 3 min; extension at 72 C, 5 min for 20 cycles. After completion of the first round of amplification, 5 ~1 of the reaction were added to an amplification reaction buffer containing a second set of primers: a partially nested oligomer ES12 and the same 3’-end primer ES15 (0.5 PM each; Fig. 1A). The second round of amplification was performed with the same program used for the first amplification cycles. The amplification products were separated on 5% polyacrylamide gel and stained by ethidium bromide. The DNA products were isolated from the gel, phosphorylated by T4 polynucleotide kinase, and cloned into the vector pUC 18 by blunt end ligation. Clones were identified by digestion of plasmid DNA with Pvull. The DNA inserts were analyzed by double stranded dideoxy DNA sequencing using Sequenase (U.S. Biochemical Corp., Cleveland, OH). Cloning
and
Sequencing
of cDNA
A human oligo(dT)-primed cDNA library was constructed from the human osteosarcoma SAOS-2/BlO cells in Xgtl 1 using the Lambda Librarian cloning kit (Invitrogen Corp., San Diego, CA). Several positive clones were identified by plaque screening using the 32P-labeled amplified DNA product (NUCI) as a probe. The hybridization conditions were previously described (27). The cDNA inserts were cloned into the EcoRl site of the cloning vector pUC 18. The complete DNA sequences of both strands were determined by dideoxy sequencing using Sequenase.
No. 4
of firefly luciferase is regulated by tandem repeats of the glucocorticoid hormone response element (Dr. M. Adam, Merck Frosst) linked to the MMTV promoter. Transient transfection of COS cells was performed as previously described (12, 13, 20). Cells were plated (1.5 x 1 O5 in 1 ml) into 12-well dishes in phenol red-free medium supplemented with activated charcoal-treated fetal calf serum. The next day, 0.12 ml DNA (10 pg/ml; 5 pg receptor DNA and 5 fig reporter plasmid), as a calcium phosphate precipitate, was added to the cells. Ligands were added to the cells 30 min after transfection. The next day (18 h), the cells were washed, and fresh ligands were added. Twenty-four hours later, cell extracts were prepared and assayed for luciferase enzyme activity, using the luciferase assay system (Promega, Madison, WI). Each transfection was performed in triplicate, and the fluorescence of each sample was measured using the AutoClinilumat (Berthold, Nashua, NH).
Acknowledgments We thank Dr. Carl Bennett for outstanding cooperation in the preparation of oligonucleotides. We thank Dr. J. Young for help in establishing the ligand screening assay and for the DNA-binding domain of the glucocorticoid receptor. We thank Dr. Ringold for the mouse glucocorticoid receptor cDNA (pSV2WREC). We thank Dr. Mohamad Adam for the reporter plasmid pJA358. Special thanks go to Ms. Mary Jo Zaboroski and Dr. Ellis Golub for help with the computer analysis, Dr. Larry Suva and Ms. Dianne McDonald for preparation of the manuscript, and Mr. Jeff Campbell for the art work.
Received April 15, 1992. Rerevision received August 3, 1992. Accepted August 3, 1992. Address requests for reprints to: Dr. Azriel Schmidt, Department of Bone Biology, Merck Research Laboratories, West Point, Pennsylvania 19486.
REFERENCES Northern
Blot Analysis
RNAs from various tissues or the listed cell lines were prepared by the guanidine isothiocyanate or guanidine hydrochloride method (28, 29). RNA samples were analyzed by formaldehyde agarose gel electrophoresis, as previously described (30). The RNA was transferred to N-Hybond (Amersham Corp., Arlington Heights, IL) and hybridized with 3’P-labeled NUCI, as described previously (22, 25). Ligand
Screening
Assay
The hybrid receptor GR-NUCI was prepared essentially as described for the construction of the GR-mPPAR chimeric protein (12). In the NUCI receptor, introducing the Xhol site resulted in a substitution of residues Leu13’ and GIu’~’ for Ser’39 and His14’. The DNA-binding domain of the mouse glucocorticoid receptor, which has a Xhol site at amino acid residues Leu495 and GIu~~~, was obtained from Dr. J. Young (Merck). The chimeric receptor pJ3GR\NUCI (GR/NUCI) was prepared by ligation of the DNA sequence coding for the DNAbinding domain of the glucocorticoid receptor to the DNA region of NUCI at the Xhol site. A hybrid receptor was prepared with the estrogen receptor similar to that described for the ER-mPPAR protein (12). The DNA-binding domain of the rat estrogen receptor was fused in frame to the above ligandbinding domain of NUCI (ER/NUCI). The cDNA of the human NUCI receptor (pJ3NUCI) and the native mouse glucocorticoid receptor (pSV2w~~c) were expressed under the control of simian virus-40-based expression vectors (21, 31). The reporter gene was the plasmid pJA358, in which the expression
5. 6.
7.
8.
9.
10.
Berg JM 1989 DNA binding specificity of steroid receptors. Cell 57:1065-l 068 Evans RM 1988 The steroid and thyroid hormone receptor super-family. Science 240:899-895 Beato M 1989 Gene regulation by steroid hormones. Cell 561335-344 Miller J, McLachlan AD, Klug A 1985 Repetitive zincbinding domains in the protein transcription factor IIIA from Xenopus oocytes. EMBO J 4:1609-l 614 Evans RM. Hollenbera SM 1988 Zinc finaers: ailt bv association. Cell 52:1-3 Carlstedt-Duke J, Okret S, Wrange 0, Gustafsson JA 1982 Immunological analysis of the glucocorticoid receptor identification of a third domain separate from the steroid-binding and DNA binding domains. Proc Natl Acad Sci USA 79:4260-4264 Carlstedt-Duke J, Stromstedt PE, Wrange 0, Bergman T, Gustafsson JA, Jornvall H 1987 Domain structure of the glucocorticoid receptor protein. Proc Natl Acad Sci USA 84~4437-4440 Scule R, Rangarajan P, Kliewer S, Ransone L, Bolodo J, Yang N, Verma IM, Evans RM 1990 Functional antagonism between oncoprotein c-iun and the glucocortiod receptor. Cell 62:1217-l 226 Yang HF, Chambard JC, Sun YL, Smeal T, Schmidt TJ, Bovin J, Karin M 1990 Transcriptional interference between c-iun and the glucocorticoid receptor: mutual inhibition of DNA binding due to direct protein-protein interaction. Cell 62:1205-l 215 Holloway JM, Glass CK, Adler S, Nelson CA, Rosenfeld I
-
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 03 February 2015. at 04:55 For personal use only. No other uses without permission. . All rights reserved.
I
New Member
11.
12.
13.
14.
15.
16.
17.
18.
19.
20.
of Steroid
Hormone
Receptor
Superfamily
MG 1990 The C-terminal interaction domain of the thyroid hormone receptor confers the ability of the DNA site to dictate positive or negative transcriptional activity. Proc Nat1 Acad Sci USA 87:8160-8164 Giquere V, Yang N, Segui P, Evans RM 1988 Identification of a new class of steroid hormone receptors. Nature 331:91-94 lssemann I, Green S 1990 Activation of the steroid receptor superfamily by peroxisome proliferators. Nature 3471645-650 Mangelsdorf DJ, Ong ES, Dyck JA, Evans RM 1990 Nuclear receptor that identifies a novel retinoic acid response pathway. Nature 345224-229 Dreyer C, Krey G, Keller H, Give1 F, Helftenbein G, Wahli W 1992 Control of the peroxisomal P-oxidation pathway by a novel family of nuclear hormone receptors. Ceil 68:879-887 Petkovich M, Brand NJ, Krust A, Chambon P 1987 A human retinoic acid receptor belongs to the family of nuclear receptors. Nature 330:444-450 Giguere V, Ong ES, Segui P, Evans RM 1987 Identification of a receptor for the morphogen retinoic acid. Nature 330:624-629 Weinberger C, Thompson CC, Ong ES, Lebo R, Gruol DJ, Evans RM 1986 The c-erbA gene encodes a thyroid receptor. Nature 324:642-646 Baker AR, McDonnell DP, Hughes M, Crisp TM, Mangelsdorf DJ, Haussler MR, Pike JW, Shine J, O’Malley BW 1988 Cloning and expression of full-length cDNA encoding human vitamin D receptor. Proc Natl Acad Sci USA 85:3294-3298 Greene S, Chambon P 1987 Oestradiol induction of a glucocorticoid-responsive gene by a chimeric receptor. Nature 32575-78 Webster NJG, Green S, Rui Jin J, Chambon P 1988 The hormone-binding domains of the estrogen and glucocorticoid receptors contain an inducible transcription activation function. Cell 54:199-207
1641
21.
Danielsen M, Northrop JP, Ringold GM 1986 The mouse glucocorticoid receptor: mapping of functional domains by cloning, sequencing, and expression of wild-type and mutant receptor proteins. EMBO J 5:2513-2525 22. Gorman CM, M&fat LF, Howard BH 1982 Recombinant genomes which express chloramphenicol acetyltransferase in mammalian cells. Mol Cell Biol 2:2044-l 051 23. Denner LA. Weiael NL. Maxwell BL. Schrader WT, O’Malley BW 1990 tiequlaiion of proaesterone receptor-mediated transcripfion by pho
Azriel Doron
Schmidt, Naoto Endo, Su Jane Shinar, and Gideon A. Rodan
Rutledge,
Robert
Vogel,
Department of Bone Biology and Osteoporosis Research Merck Research Laboratories West
Point,
Pennsylvania
19486
We have identified a novel member of the steroid hormone receptor superfamily by cDNA cloning from a human osteosarcoma SAOS-S/B10 cell library. Sequence analysis predicts a protein of 441 amino acids, which includes the conserved amino acid residues characteristic of the DNA- and ligand-binding domains of nuclear receptors. Amino acid sequence alignment and transcriptional activation experiments revealed that the new protein is closely related to the mouse peroxisome proliferator activated receptor. The overall homology is 62%, and the highest similarity is seen in the DNAand ligand-binding domains, 86% and 71%, respectively. Northern blot analysis showed that in mature rats, the receptor is highly expressed in heart, kidney, and lung as a transcript of approximately 3500 nucleotides. In human cells, the size of the mRNA is approximately 4000 nucleotides. Transcription assays using hybrid receptors consisting of the ligandbinding domain of the new protein and the DNAbinding domain of the glucocorticoid receptor showed weak stimulation by the peroxisome proliferator activator WY14643, suggesting a relationship to that receptor. Similar stimulation was observed with arachidonic and oleic acid (loo-250 PM). (Molecular Endocrinology 6: 1634-1641, 1992)
vealed two important regions of these receptors that exhibit a high degree of amino acid residue conservation. The highest level of similarity among the receptors is found in a region that contains nine cysteine residues that bind zinc atoms to form two “zinc fingers,” which interact with the cognate steroid response elements of DNA (4,5). The second region, which is less conserved, is the ligand-binding domain responsible for the interaction with the hormone (6, 7). Recent studies have attributed additional functions to other domains in these receptors, such as protein-protein interactions of domains that participate in transcriptional regulation (8IO). The amino acid conservation in the DNA-binding domain has led to the identification of new members of the steroid receptor superfamily. For example, hER1 and hER2 have been cloned by low stringency hybridization of cDNA libraries with a DNA probe coding for the DNA-binding domain of the estrogen receptor (11). Similar approaches have led to the discovery of multiple retinoic acid receptors and the mouse peroxisome proliferator activator receptor (mPPAR) (12, 13). We report here the use of polymerase chain reaction (PCR) to identify a new, as yet unreported member of this receptor family.
RESULTS INTRODUCTION Cloning Retinoids, steroid and thyroid hormones, and possibly other molecules produce their biological effects by binding to proteins of the steroid receptor superfamily. These receptors interact with specific DNA sequences and modulate gene expression (for reviews, see Refs. l-3). Sequence analysis and functional studies re0888-8809/92/1634-1641$0300/O Molecular Endocmology CopyrIght 1992 by The Endocrine
of NUCI cDNA
The PCR method was used to identify new members of the nuclear hormone receptor superfamily from bonederived cells. Degenerate oligonucleotides were synthesized according to the amino acid sequence of two conserved segments shared by members of the nuclear receptor superfamily (2). The 5’-end primers, ESI’I and ES12, were designed according to a segment of the DNA-binding domain (Fig. 1A). The primer at the 3’end, ES15 was prepared according to a conserved
Scmty
1634 The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 03 February 2015. at 04:55 For personal use only. No other uses without permission. . All rights reserved.
New
Member
of Steroid
Hormone
Receptor
Superfamily
1635
A SENSE ES11
CGAATTC
C E G C K TGT GAG GGC TGC AAD
A/G
5’
SENSE ES12
CGAATTC
CEGCK TGT GAG GGC TGC AAG
A/G
5’
ANTISENSE ES15
5’
GGMTTC
GCC A G
GCC A G
PGPXXKAF AAA ACC AGG IA1 G C C T T G G
III
1
P
F
TTC
TTC
2
3
MW
4 1078
CTT TGC lvuL T A G G C
-
603
-
310
4 234 - 194 4 118
B 917
950 TCACCGGCAAA II,, AACGTCGGGAGTGT~TTAAGC
5 ’ AAAAAGAAGGCCCGCAGCATCC 3’ IIIIIIIII III
CTTCTTCCGG
II
3 ’ 5’
ES12
Fig. 1. Amplification
of NUCI DNA by PCR The DNA sequences of the primers used in the PCR reaction are shown in A. The corresponding amino acid residues are indicated (upper line). On the right, the products of the second round of PCR amplification, separated on 5% polyacrylamide gel and stained by ethidium bromide, are shown. The alignment of primer ES12 to the actual cDNA sequence of NUCI is shown in B.
amino acid sequence in the ligand-binding domain of the retinoid receptor subfamily and the vitamin D receptor. Since thisconserved region contains two nonconserved amino acid residues, inosine nucleotides were used as part of this primer (Fig. 1A). Amplification of cDNA prepared from RNA of human osteosarcoma cells SAOS-2/BlO, amplified with the primers ES1 1 and ES15 yielded multiple DNA fragments with various sizes after the first round of amplification (data not shown). A portion of the reaction was subjected to a second round of amplification using the nested primer ES12 and the same 3’-end primer ES15. This amplification produced two DNA fragments of 270 and 320 basepairs (bp), respectively (Fig. 1 A), which were cloned into pUC 18 and sequenced (data not shown). The amino acid residues predicted by the DNA sequences indicated that each of these DNA fragments coded for a segment of a genuine and novel member of the steroid hormone receptor superfamily. The focus of this work is the cloning and characterization of one of these novel receptors, NUCI. To obtain the complete cDNA sequence we screened a human SAOS-2/BlO cell cDNA library with the amplified 320-bp fragment of NUCI. All the strongly hybridizing clones were identical and matched the sequence of the amplified 320-bp DNA fragment. Analysis of the deduced amino acid sequence revealed a long open reading frame, which starts at the putative initiation methionine codon at nucleotide 338 and codes for a protein of 441 amino acid residues (Fig. 2). The putative protein contains a cysteine-rich region at the amino-terminus, which probably represents the conserved DNA-binding domain of this receptor. The carboxyl-terminus of the protein contains conserved amino acid residues, which may be part of a ligandbinding domain (l-3). The DNA-binding region contains
11 cysteine residues, compared to 9 residues in other steroid receptors (Fig. 2) suggesting the potential formation of an alternative Zn’-dependent finger structure(s). As in the peroxisome proliferator-activated receptor, the conserved loop structure of the second Zn+ finger (Cys”’ to Cys”‘) contains only three amino acid residues, compared to five in other members of the receptor family (12). Comparison of the amino acid sequence of NUCI with those of other members of the nuclear receptor family revealed best overall similarity (62%) to the mouse peroxisome proliferator-activated receptor (Fig. 3) (12). The highest similarity is in the putative DNA-binding domain, where 86% of the 66 amino acid residues are identical (Fig. 4). The ligand-binding domain of NUCI also exhibited close similarity with the corresponding region of the mPPAR. These domains share 71% of the amino acid residues (Fig. 4). Outside these two regions, the similarity between NUCI and the mPPAR is lower. While 57% of the amino acid residues in the region between the DNA- and ligand-binding domains are identical, in the N-terminal portion only 27% are the same. Sequence comparisons between NUCI and the three X,enopus receptors, xPPARcu, xPPARP, and xPPARy, exhibited the same approximate similarities (14). The DNA- and ligand-binding domains of NUCI receptor show 81-86% and 71-73% similarity to the parralel domains of the Xenopus receptors, respectively (Fig. 4). None of the less conserved domains of these receptors showed closer sequence relationship to NUCI receptor. These observations support the idea that NUCI is a new member of this receptor family and not the human homolog of any of these receptors. Although the similarity between NUCI and PPAR is substantial, it is much lower than the similarity among members of the retinoic acid receptor family (2). NUCI shows the
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 03 February 2015. at 04:55 For personal use only. No other uses without permission. . All rights reserved.
MOL 1636
ENDO.
1992
Vol6
1 61 121 181 241 301 361 421 481 541 601 661 721 781 841 901 961 1021 1081 1141 1201 1261 1321 1381 1441 1501 1561 1621 1681 1741 1801 1861 1921 1981 2041 2101 2161 2221 2281 2341 2401 2461 2521 2581 2641 2701 2761 2821 2881 2941 3001 3061 3121 3241 3301
GAATTCTGCGGAGCCTGCGGGACGGCGGCGGGTTGGCCCGTAGGCAGCCGGGACAGTGTT GTACAGTGTTTTGGGCATGCACGTGATACTCACACAGTGGCTTCTGCTCACC~CAGATG AAGACAGATGCACCAACGAGGGTCTGGAATGGTCTGGAGTGGTCTGGAAAGCAGGGTCAG ATACCCCTGGAAAACTGAAGCCCGTGGAGCAATGATCTCTACAGGACTGCTTCAAGGCTG ATGGGAACCACCCTGTAGAGGTCCATCTGCGTTCAGACCCAGACGATGCCAGAGCTATGA CTGGGCCTGCAGGTGTGGCGCCGAGGGGAGATCAGCCATGGAGCAGCCACAGGAGG~GC MEQPQEEA CCCTGAGGTCCGGGAAGAGGAGGAGAAAGAGGAGGCAGGAGCCCCAGA PEVREEEEKEEVAEAE GAPE GCTCAATGGGGGACCACAGCATGCACTTCCTTCCAGCAGCTACACAGACCTCTCCCGGAG LNGGPQHALPSSSYT DLSRS CTCCTCGCCACCCTCACTGCTGGACCAACTGCAGATGCAGATGGGCTGTGACGGGGCCTCATGCGG s s P P SLLDQLQMGCDGASCG GTGCCGGGTGTGCGGGGACAAGGCATCGGGCTTCCACTACGGTGT S L N M @RV@G DKASGFHYGV TCATGCATGTGAGGGGTGCAGGGCTTCTTCCGTCGTACGATCCGCATG~GCTGGAGTA HA@EG@KGFFRRT I RMK L E Y CGAGAAGTGTGAGCGCAGCTGCAAGATTCAGAAGAAGAACCGCAACAGTGCCAGTACTG EK@ERS@K IQKKNRNK@QY@l28 CCGCTTCCAGAAGTGCCTGGACTGGGCATGTCACACAACT RF Q K CLALGMSHNAIRF G R M GCCGGAGGCTGAGAAGAGGAGCTGGTGGCAGGGCTGACTGC~CGAGGGGAGCCAGTA P EAEKRKLVAGLTANEGSQY CAACCCACAGGTGGCCGACCTGAAGGCCTTCTCCAAGCACATCTACAATGCCTACCTGAA S K H I YNAYLK N P Q VADLKAF AAACTTCAACATGACCAG~GGCCCGCAGCATCCTCACCGGC~GCCAGCCACAC NFNMTKKKARSI LTGKASHT GGCGCCCTTTGTGATCCACGACATCGAGACATTGTGGCAGGCAGAGIlAGGGGCTGGTGTG AP FV I H D I ET LWQAEK G LVW GAAGCAGTTGGTG~TGGCCTGCCTCCCTACAGGAGATCAGCGTGCACGTCTTCTACCG KQLVNGLPPYKEI SV HV F Y R CTGCCAGTGCACCAC GTGGAGACCGTGCGGGAGCTCACTGAGTTCGCCAAGAGCATCCC C QC T TVE TVRE LT EF AK S I P CAGCTTCAGCAGCCTCTTCCTCAACGACCAGGTTACCCTTCTCILAGTATGGCGTGCACGA S F S SLFLNDQVTLLK Y G V H E GGCCATCTTCCCGATGCTGGCCTCTATCGTCAACAAGGACGGGCTGCTGGTAGCCAACGG A IFPMLASIVNKDGLLVANG CAGTGGCTTTGTCACCCGTGAGTTCCTGCGCAGCCTCCGCAAACCCTTCAGTGATATCAT SGFVTREFLRS I L R K P F S D I TGAGCCTAAGTTTGAATTTGCTGTCAAGTTCAACGCCCTGGAACTTGATGACAGTGACCT EPKFEFAVKFNALELDD S D L GGCCCTATTCATTGCGGCCATCATTCTGTGTGG~~~~~~~~~~~~~~~~~~~~~~~~~C A L F I A A I ILCGDRPGLMNVP ACGGGTGGAGGCTATCCAGGACACCATCCTGCGTGCCCTCGAATTCCACCTGCAGGCCAA R V E A I Q D T I L RA L E F H L Q A N CCACCCTGATGCCCAGTACCTCTTCCCCAAGCTGCTGCAGGATGGCTGACCTGCGGCA HPDAQYLFPKL L Q K MA D L R Q ACTGGTCACCGAGCACGCCCAGATGATGCAGCGGATCAAGAAGACCGAAACCGAGACCTC LVTEHAQMMQRI KKTETETS GCTGCACCCTCTGCTCCAGGAGATCTACAAGGACATGTACTAACGGCGGCACCCAGGCCT ILHPLLQE I Y K D M Y/END CCCTGCAGACTCCAATGGGGCCAGCACTGGAGGGGCCCACCCACATGACTTTTCCATTGA CCAGCTCTCTTCCTGTCTTTGTTGTCTCCCTCTTTCTCAGTTCCTCTTTCTTTTCTAATT CCTGTTGCTCTGTTTCTTCCTTTCTGTAGGTTTCTCTCTTCCCTTCTCCCTTCTCCCTTG CCCTCCCTTTCTCTCTCCTATCCCCACGTCTGTCCTCCTTTCTTATTCTGTGAGATGTTT TGTATTATTTCACCAGCAGCATAGAACAGGACCTCTCTGCTTTTGCACACCTTTTCCCCAGG AGCAGAAGAGAGTGGGCCTGCCCTCTGCCCCATCATTGCACCTGCAGGCTTAGGTCCTCA CTTCTGTCTCCTGTCTTCAGAGCAAAAGACTTGAGCCATCCAAAGAAACACTAAGCTCTCTC TGGGCCTGGGTTCCAGGGAAGGCTAAGCATGGCCTGGACTGACTGCAGCCCCCTATAGTC ATGGGGTCCCTGCTGCAAAGGACAGTGGCAGACCCCGGCAGTAGAGCCGAGATGCCTCCC CAAGACTGTCATTGCCCCTCCGATCGTGAGGCCACCCACTGACCCAATGATCCTCTCCAG CAGCACACCTCAGCCCCACTGACACCCAGTGTCCTTCCATCTTCACACTGGTTTGCCAGG CCAATGTTGCTGATGGCCCCTCCAGCACACACACATAAGCTCACTTTACCTGC AGGCACCATGCACCTCCCTTCCCTCCCTGAGGCAGGTGAGAACCCAGAGAGAGGGGCCTG CAGGTGAGCAGGCAGGGCTGGGCCAGGTCTCCGGGGAGGCAGGGGTCCTGCAGGTCCTGG TGGGTCAGCCCAGCACCTCGCCCAGTGGGAGCTTCCCGGGAT~CTGAGCCTGTTCATT CTGATGTCCATTTGTCCCAATAGCTCTACTGCCCTCCCCTTCCCCTTTACTCAGCCCAGC TGGCCACCTAGAAGTCTCCCTGCACAGCCTCTAGTGTCCGGGGACCTTGTGGGACCAGTC CCACACCGCTGGTCCCTGCCCTCCCCTGCTCCCAGGTTGAGGTGCGCTCACCTCAGAGCA GGGCCAIlAGCACAGCTGGGCATGCCATGTCTGAGCGGCGCAGAGCCCTCCAGGCCTGCAG GGGCAAGGGGCTGGCTGGAGTCTCAGAGCACAGAGGTAGGAGAACTGGGGTTGCCCA GGCTTCCTGGGTCCTGCCTGGTCCTCCCTCCCAAGGAGCCATTCTATGTGACTCTGGGTG GAAGTGCCCAGCCCCTGCCTGACGGXXXXXXXGATCACTCTCTGCTGGCAGGATTCTTCC CGCTCCCCACCTACCCAGCTGATGGGGGTTGGGGTGCTTCTTTCAGCC~GGCTATG~G GGACAGCTGCTGGGACCCACCTCCCCCCTTCCCCGGCCACATGCCGCGTCCCTGCCCCCA CCCGGGTCTGGTGCTGAGGATACAGCTCTTCTCAGTGTCTG~C~TCTCC~TTG~ ACTGACGAAACTTTAAATAAATGGGAATTAAATATTAAATATTT WGCGGCCGCGAATT c 3301
8 28 48 68 88 108
148 168 188 208 228 248 268 288 308 328 348 368 388 408 428 441
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 03 February 2015. at 04:55 For personal use only. No other uses without permission. . All rights reserved.
No. 4
New Member
of Steroid
Hormone
Receptor
Superfamily
1637
base transcript in human SAOS-2/BlO cells (Fig. 5A). In mice and rats the transcript for NUCI is about 500 nucleotides shorter than that observed in human cell lines and baboon tissues. In mature rats we found high levels of NUCI expression in heart, lung, and kidney (Fig. 5B). About 5-fold lower levels were found in skin and bone RNA prepared from tibia. High expression levels were also observed in spleen and ovaries (data not shown). This result suggests that NUCI is expressed in many tissues and organs, and is most abundant in heart, kidneys, and lungs. Unlike mPPAR, which is highly expressed in liver, the concentration of NUCI RNA is lowest in adult liver.
273
LFLNDa”TLLKYG”.HEAIFPMLASIVNKDGLL”ANGSGF”TREFLRSLR
300
I llllIlllllll l//l II I IllI LDLNDQVTLLKYGVYEEAIFTMLSSLMNKDGMLIAYGNGFITREFLKNLR
321 I
I
I
II
II/II
next best similarity with members of the retinoic acid/ thyroid receptors subfamily, including retinoic acid receptor-a, retinoid X receptor-a, thyroid hormone receptor, or vitamin D receptor (13, 15-l 8). The DNA-binding domains are 58-62% identical, and the ligand-binding domains exhibit a similarity in the range of 29-31%. Regarding the PCR procedure, it is of interest that sequence analysis of the amplified DNA products revealed that the primer ES12 was incorporated at both the 5’- and 3’-ends. At the 3’-end the primer only partially matched the actual cDNA sequence (Fig. 1 B). Thus, the amplification was the product of a single primer. Surprisingly, the nucleotide at the 3’-end of primer ES12 did not match the full-length cDNA sequence at all. None of the cloned PCR products contained the sequences of primer ESll. It is not clear whether primer ES12 was incorporated into the DNA fragments at the 3’-end, at the beginning of the amplification process, or only during late amplification cycles. of NUCI mFiNA
Northern analysis with a NUCI cDNA probe revealed that NUCI receptor mRNA is expressed as a 4.0-kilo-
Fig. 2. Nucleotide
Sequence and Deduced The cysteine residues at the predicted ligand-binding domains are boxed.
Activation
349
Fig. 3. Amino Acid Comparison between the NUCI Receptor and the Mouse PPAR (12) For maximum similarity, gaps were introduced, as indicated by dots. Identical amino acid residues are marked with vertical bars.
Expression
Receptor
II
Amino Acid Sequence DNA-binding domain
To search for a putative ligand for the NUCI receptor, we used the fact that the ligand-binding domains of the various receptors of the steroid superfamily can be interchanged to form chimeric receptors. These hybrid receptors are capable of exhibiting ligand-dependent transcription activation of a heterologous responsive DNA sequence (12, 19, 20). We prepared a chimeric receptor for NUCI (pJSGR/NUCI). The amino acid-terminus, which included the DNA-binding domain of the mouse glucocorticoid receptor (mGR) (21), was fused to the ligand-binding domain of NUCI in the manner described for mPPAR (12). For an additional control, we constructed a similar chimeric receptor in which the DNA-binding domain of the mouse estrogen receptor was fused to the ligand-binding domain of NUCI receptor (ER/NUCI). The plasmid pJA358 that contains the luciferase gene under the control of the modified mouse mammary tumor virus (MMTV) promoter was used as the reporter of the transcription unit. The screening assay was performed by transient transfection of COS cells, as previously described (12, 13, 22). Based on the similarity of NUCI receptor to the mPPAR, we tested if the Wy-14643 molecule could activate the NUCI hybrid receptor. In these experiments, WY-14643 stimulated luciferase activity after cotransfection with the GR/NUCI chimeric receptor (Fig. 6). Cotransfection of the reporter gene with wild-type glucocorticoid receptor or the ER/NUCI hybrid receptor did not result in similar stimulation (Fig. 6). We found that this compound, at a concentration of 100 PM, stimulated luciferase levels in GR/NUCI-transfected cells 4- to lo-fold. No activation was observed with other peroxisome proliferators, such as clofibrate, or other compounds that strongly stimulate liver fatty acid acyl coenzyme-A oxidase in mice at a concentration of 100 PM. No increase in luciferase activity was observed when WY-14643 was added to cells expressing either the unaltered mGR or NUCI receptors. The relatively low level of activation mediated
of the NUCI cDNA are circled. The amino
acid
residues
of the deduced
DNA-
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 03 February 2015. at 04:55 For personal use only. No other uses without permission. . All rights reserved.
and
MOL 1638
ENDO.
1992
72
Vol6
136
254
441
hNUC I 27%
102 m
166
1
23%
106 m
173
1
30
95
57%
244 m250 -4
474
60%
171
396
A
100
ER-NUC
No. 4
GR-NUC
466 . .
mPPAR
xPPARa xPPARf3 2
112 1
17%
m
177
257 41%
I-
477 '.
xPPARy
Fig. 4. Schematic Amino Acid Comparison between NUCI and Members of the PPAR Families The schematic amino acid alignment was prepared according to the functional domain structure of the steroid hormone receptors mPPAR (12) and the X. kevis xPPARLu, xPPARP, and xPPARr (14). The percentage of amino acid identity between the various receptors and NUCI is indicated for each domain.
0
GR-NUC
MMTV-LUC
A
Fig. 5; Northern Blot Analysis of NUCI Receptor mRNA RNA preparation and hybridization are described in Materials and Methods. A, Poly(A)-selected RNA (2 pg) of the osteosarcoma SAOSP/BlO cells was resolved on a formaldehyde-agarose gel. The RNA was transferred to a nylon membrane and hybridized with the cDNA probe for NUCI. The migration of the mol wt markers (kilobases) is indicated. B, Total RNA was isolated from the indicated rat tissues, and 20 and 4 fig of each sample (adjacent lanes) were treated and hybridized, as described in A.
by GR/NUCI may be attributed to the high baseline activation of the chimeric receptor in the absence of exogenous ligands. Cells transfected with the hybrid receptor had a 5fold higher level of luciferase activity than cells expressing the NUCI receptor itself or the native mGR. Similar findings were obtained in experiments in which the ligand-binding domain of NUCI was fused to the DNA-binding domain of the estrogen receptor or RXR (data not shown). Alternatively, the high basal activity observed with the chimeric receptor could be due to the presence of the natural ligand for NUCI receptor in the cell culture. It was recently shown that the rat PPAR was activated by various fatty acids, such as arachidonic (C18:4) and oleic (C18:l) acid (25). We found that the hybrid receptor GR/NUCI was also stim-
Fig. 6. Activation of Chimeric NUCI Receptor by Wy-14643 A, Cells were transfected with glucocorticoid response element-driven luciferase vector and either the wild-type GR, the hybrid estrogen-NUCI receptor (ER-NUC), or the chimeric glucocorticoid-NUCI receptor (GR-NUC). The transfected cells were treated with either Wy-14643 (WY) or dexamethasone (Dex) at the indicated concentrations. The luciferase assays were performed as described in Materials and Methods. The 100% value was the luciferase activity obtained by the addition of 0.1 M Dex to cells expressing the native GR. B, Experiments were performed as described in A. With MMTV-LUC, cells were transfected with the Luciferase reporter plasmid without steroid hormone receptor.
ulated by these agents. Both arachidonic and oleic acids, at concentrations of 100 and 60 PM, respectively, activated the reported gene that was cotransfected with the chimeric receptor (Fig. 7). No similar stimulation was observed when the reporter plasmid was transfected with the normal NUCI receptor or the normal mGR (Fig. 6). A large series of known ligands, including 1,25dihydroxyvitamin DS, 24,25-dihydroxyvitamin D3, Tq, estrogen, vitamin E, retinoic acid, progesterone, androgen, and other putative commercially available ligands, did not activate transcription via the hybrid receptor (at concentrations of l-10 PM; data not shown). Dexamethasone (0.1 PM) stimulated transcription mediated
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 03 February 2015. at 04:55 For personal use only. No other uses without permission. . All rights reserved.
New Member
of Steroid
Hormone
NUCI
Receptor
1639
Superfamily
GR-NUC
The experiments were performed as described in Fig. 6. The cells were treated with arachidonic acid (Ar.A.), oleic acid (OLA.), or dexamethasone (Dex.) at the indicated concentrations.
by the wild-type glucocortidoid receptor (Fig. 6). Low stimulation was also observed in cells transfected by GR/NUCI. This is probably a reflection of endogenous glucocorticoid receptors present in the cells, since the hybrid ER/NUCI receptor, which does not activate the reporter gene, was also responsive to dexamethasone (Fig. 6A). A similar response to dexamethasone was observed in cells that were transfected with only the MMTV-luciferase reporter gene (Fig. 68).
DISCUSSION
This report presents the cDNA cloning of a new member of the steroid hormone receptor superfamily. The amino acid sequence deduced from the DNA sequence has the characteristic features of both the DNA- and ligandbinding domains of this family of receptors. The putative DNA-binding domain of NUCI contains the conserved nine cysteine residues, which are found in all members of this receptor superfamily. Interestingly, two additional cysteine residues (Cys’” and CY@~) are located in the proximity of the nine conserved cysteine residues. The presence of additional cysteine residues in this region could lead to the formation of alternative structures for the DNA-binding domain. Sequence comparison with the different receptors of this superfamily indicates that NUCI is closely related to the mPPAR (12), with which it shares 86% of the amino
acids
in the DNA-binding
domain
and
72%
related, they may have regulatory roles in different target tissues. The structure of NUCI and that of its related receptor mPPAR suggest that they belong to a subgroup of the steroid receptor superfamily. Recently, three additional related receptors, XPPARa, XPPAPP, and XPPARr, were cloned from Xenopus (14). The DNA- and ligandbinding domains of NUCI receptor show 81-86% and 71-73% identity with the corresponding domains of the Xenopus receptors (14). However, in the less conserved regions of steroid receptors, there is a much lower degree of similarity. Thus, although it is clear that NUCI is a member of the PPAR receptor group, it remains to be determined whether NUCI is the human homolog of one of the Xenopus PPARs or a new member of this receptor family. These receptors were shown to activate the acyl coenzyme-A oxidase promoter. Whether NUCI receptor is also involved in peroxisome proliferation or P-oxidation of fatty acid is yet to be determined. Consistent with the sequence similarity, the chimeric NUCI receptor was activated by the peroxisome proliferator Wy-I 4643 at a concentration of 1 Om4 M. Lower concentrations ceptor. The
did not activate the chimeric NUCI relower sensitivity of NUCI receptor to the
peroxisome proliferator, in spite of the similarity in the ligand-binding domain, could be attributed either to differences in key amino acids in the ligand-binding domain or, possibly, to some unknown technical aspects of the assay. Recently, it has been shown that fatty acids, such as arachidonic and oleic acid, can stimulate transcription mediated by the rat PPAR (25). Similar results were obtained with the human NUCI receptor. Both arachidonic and oleic acids activated transcription of the reporter gene that was dependent on the expression of the chimeric NUCI receptor. The stimulation occurred at a concentration range of 60100 PM, similar to that observed with the rat PPAR. Since the fatty acids and the other synthetic peroxisome proliferators are structurally diverse molecules, it is not known if they are the actual ligands for these receptors or molecules structurally related to the real ligand(s). It is possible that the activation of the PPAR family by a variety of molecules could be attributed to pharmacological modulation of protein kinases and phosphatases, as shown for both the progesterone and chicken ovalbumin upstream promoter transcription factor receptors (23, 24). Since true ligand-receptor interactions are usually highly specific, we believe that the ultimate natural ligand for NUCI and the other members of this receptor family awaits isolation and identification.
in the
ligand-binding domain. Hybridization experiments revealed that the distribution of NUCI receptor is different from that of the mPPAR. NUCI is highly expressed in heart, kidney, lung, spleen, and ovaries and has very low expression in liver. In contrast, mPPAR was reported to show highest expression in liver (12). This suggests that although the two receptors seem to be
MATERIALS
AND METHODS
PCR Amplification Total RNA was isolated from human osteosarcoma BlO cells (26), and a random primed cDNA library pared from 2 pg RNA with the Moloney murine reverse transcriptase according to the manufacturer’s
SAOS2/ was preleukemia recom-
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 03 February 2015. at 04:55 For personal use only. No other uses without permission. . All rights reserved.
MOL 1640
ENDO.
1992
Vol6
mendations (Bethesda Research Laboratories, Gaithersburg, MD). The cDNA reaction (25 ~1) was diluted into 300 11 water, heat denatured at 95 C for 5 min, and quickly chilled on ice. The cDNA (2.5 ~1) and the first primer pair, ES1 1 and ES1 5 (0.5 FM each; Fig. 1 A), were employed in the amplification reaction with the Amplitaq kit and the DNA thermal cycler (Perkin-Elmer-Cetus, Norwalk, CT). We carried out the following amplification cycles; denaturation at 94 C, 1.5 min; annealing at 65 C, 3 min; extension at 72 C, 5 min for three cycles; denaturing at 94 C, 1 min; annealing at 60 C, 3 min; extension at 72 C, 5 min for 15 cycles; and denaturing at 94 C, 1 min; annealing at 57 C, 3 min; extension at 72 C, 5 min for 20 cycles. After completion of the first round of amplification, 5 ~1 of the reaction were added to an amplification reaction buffer containing a second set of primers: a partially nested oligomer ES12 and the same 3’-end primer ES15 (0.5 PM each; Fig. 1A). The second round of amplification was performed with the same program used for the first amplification cycles. The amplification products were separated on 5% polyacrylamide gel and stained by ethidium bromide. The DNA products were isolated from the gel, phosphorylated by T4 polynucleotide kinase, and cloned into the vector pUC 18 by blunt end ligation. Clones were identified by digestion of plasmid DNA with Pvull. The DNA inserts were analyzed by double stranded dideoxy DNA sequencing using Sequenase (U.S. Biochemical Corp., Cleveland, OH). Cloning
and
Sequencing
of cDNA
A human oligo(dT)-primed cDNA library was constructed from the human osteosarcoma SAOS-2/BlO cells in Xgtl 1 using the Lambda Librarian cloning kit (Invitrogen Corp., San Diego, CA). Several positive clones were identified by plaque screening using the 32P-labeled amplified DNA product (NUCI) as a probe. The hybridization conditions were previously described (27). The cDNA inserts were cloned into the EcoRl site of the cloning vector pUC 18. The complete DNA sequences of both strands were determined by dideoxy sequencing using Sequenase.
No. 4
of firefly luciferase is regulated by tandem repeats of the glucocorticoid hormone response element (Dr. M. Adam, Merck Frosst) linked to the MMTV promoter. Transient transfection of COS cells was performed as previously described (12, 13, 20). Cells were plated (1.5 x 1 O5 in 1 ml) into 12-well dishes in phenol red-free medium supplemented with activated charcoal-treated fetal calf serum. The next day, 0.12 ml DNA (10 pg/ml; 5 pg receptor DNA and 5 fig reporter plasmid), as a calcium phosphate precipitate, was added to the cells. Ligands were added to the cells 30 min after transfection. The next day (18 h), the cells were washed, and fresh ligands were added. Twenty-four hours later, cell extracts were prepared and assayed for luciferase enzyme activity, using the luciferase assay system (Promega, Madison, WI). Each transfection was performed in triplicate, and the fluorescence of each sample was measured using the AutoClinilumat (Berthold, Nashua, NH).
Acknowledgments We thank Dr. Carl Bennett for outstanding cooperation in the preparation of oligonucleotides. We thank Dr. J. Young for help in establishing the ligand screening assay and for the DNA-binding domain of the glucocorticoid receptor. We thank Dr. Ringold for the mouse glucocorticoid receptor cDNA (pSV2WREC). We thank Dr. Mohamad Adam for the reporter plasmid pJA358. Special thanks go to Ms. Mary Jo Zaboroski and Dr. Ellis Golub for help with the computer analysis, Dr. Larry Suva and Ms. Dianne McDonald for preparation of the manuscript, and Mr. Jeff Campbell for the art work.
Received April 15, 1992. Rerevision received August 3, 1992. Accepted August 3, 1992. Address requests for reprints to: Dr. Azriel Schmidt, Department of Bone Biology, Merck Research Laboratories, West Point, Pennsylvania 19486.
REFERENCES Northern
Blot Analysis
RNAs from various tissues or the listed cell lines were prepared by the guanidine isothiocyanate or guanidine hydrochloride method (28, 29). RNA samples were analyzed by formaldehyde agarose gel electrophoresis, as previously described (30). The RNA was transferred to N-Hybond (Amersham Corp., Arlington Heights, IL) and hybridized with 3’P-labeled NUCI, as described previously (22, 25). Ligand
Screening
Assay
The hybrid receptor GR-NUCI was prepared essentially as described for the construction of the GR-mPPAR chimeric protein (12). In the NUCI receptor, introducing the Xhol site resulted in a substitution of residues Leu13’ and GIu’~’ for Ser’39 and His14’. The DNA-binding domain of the mouse glucocorticoid receptor, which has a Xhol site at amino acid residues Leu495 and GIu~~~, was obtained from Dr. J. Young (Merck). The chimeric receptor pJ3GR\NUCI (GR/NUCI) was prepared by ligation of the DNA sequence coding for the DNAbinding domain of the glucocorticoid receptor to the DNA region of NUCI at the Xhol site. A hybrid receptor was prepared with the estrogen receptor similar to that described for the ER-mPPAR protein (12). The DNA-binding domain of the rat estrogen receptor was fused in frame to the above ligandbinding domain of NUCI (ER/NUCI). The cDNA of the human NUCI receptor (pJ3NUCI) and the native mouse glucocorticoid receptor (pSV2w~~c) were expressed under the control of simian virus-40-based expression vectors (21, 31). The reporter gene was the plasmid pJA358, in which the expression
5. 6.
7.
8.
9.
10.
Berg JM 1989 DNA binding specificity of steroid receptors. Cell 57:1065-l 068 Evans RM 1988 The steroid and thyroid hormone receptor super-family. Science 240:899-895 Beato M 1989 Gene regulation by steroid hormones. Cell 561335-344 Miller J, McLachlan AD, Klug A 1985 Repetitive zincbinding domains in the protein transcription factor IIIA from Xenopus oocytes. EMBO J 4:1609-l 614 Evans RM. Hollenbera SM 1988 Zinc finaers: ailt bv association. Cell 52:1-3 Carlstedt-Duke J, Okret S, Wrange 0, Gustafsson JA 1982 Immunological analysis of the glucocorticoid receptor identification of a third domain separate from the steroid-binding and DNA binding domains. Proc Natl Acad Sci USA 79:4260-4264 Carlstedt-Duke J, Stromstedt PE, Wrange 0, Bergman T, Gustafsson JA, Jornvall H 1987 Domain structure of the glucocorticoid receptor protein. Proc Natl Acad Sci USA 84~4437-4440 Scule R, Rangarajan P, Kliewer S, Ransone L, Bolodo J, Yang N, Verma IM, Evans RM 1990 Functional antagonism between oncoprotein c-iun and the glucocortiod receptor. Cell 62:1217-l 226 Yang HF, Chambard JC, Sun YL, Smeal T, Schmidt TJ, Bovin J, Karin M 1990 Transcriptional interference between c-iun and the glucocorticoid receptor: mutual inhibition of DNA binding due to direct protein-protein interaction. Cell 62:1205-l 215 Holloway JM, Glass CK, Adler S, Nelson CA, Rosenfeld I
-
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 03 February 2015. at 04:55 For personal use only. No other uses without permission. . All rights reserved.
I
New Member
11.
12.
13.
14.
15.
16.
17.
18.
19.
20.
of Steroid
Hormone
Receptor
Superfamily
MG 1990 The C-terminal interaction domain of the thyroid hormone receptor confers the ability of the DNA site to dictate positive or negative transcriptional activity. Proc Nat1 Acad Sci USA 87:8160-8164 Giquere V, Yang N, Segui P, Evans RM 1988 Identification of a new class of steroid hormone receptors. Nature 331:91-94 lssemann I, Green S 1990 Activation of the steroid receptor superfamily by peroxisome proliferators. Nature 3471645-650 Mangelsdorf DJ, Ong ES, Dyck JA, Evans RM 1990 Nuclear receptor that identifies a novel retinoic acid response pathway. Nature 345224-229 Dreyer C, Krey G, Keller H, Give1 F, Helftenbein G, Wahli W 1992 Control of the peroxisomal P-oxidation pathway by a novel family of nuclear hormone receptors. Ceil 68:879-887 Petkovich M, Brand NJ, Krust A, Chambon P 1987 A human retinoic acid receptor belongs to the family of nuclear receptors. Nature 330:444-450 Giguere V, Ong ES, Segui P, Evans RM 1987 Identification of a receptor for the morphogen retinoic acid. Nature 330:624-629 Weinberger C, Thompson CC, Ong ES, Lebo R, Gruol DJ, Evans RM 1986 The c-erbA gene encodes a thyroid receptor. Nature 324:642-646 Baker AR, McDonnell DP, Hughes M, Crisp TM, Mangelsdorf DJ, Haussler MR, Pike JW, Shine J, O’Malley BW 1988 Cloning and expression of full-length cDNA encoding human vitamin D receptor. Proc Natl Acad Sci USA 85:3294-3298 Greene S, Chambon P 1987 Oestradiol induction of a glucocorticoid-responsive gene by a chimeric receptor. Nature 32575-78 Webster NJG, Green S, Rui Jin J, Chambon P 1988 The hormone-binding domains of the estrogen and glucocorticoid receptors contain an inducible transcription activation function. Cell 54:199-207
1641
21.
Danielsen M, Northrop JP, Ringold GM 1986 The mouse glucocorticoid receptor: mapping of functional domains by cloning, sequencing, and expression of wild-type and mutant receptor proteins. EMBO J 5:2513-2525 22. Gorman CM, M&fat LF, Howard BH 1982 Recombinant genomes which express chloramphenicol acetyltransferase in mammalian cells. Mol Cell Biol 2:2044-l 051 23. Denner LA. Weiael NL. Maxwell BL. Schrader WT, O’Malley BW 1990 tiequlaiion of proaesterone receptor-mediated transcripfion by pho
