Vol. 180, No. 3, 1991 November 14, 1991
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages ] 2 6 5 - ] 2 7 2
CLONING AND CHARACTERIZATION OF cDNA ENCODING H U M A N A-TYPE ENDOTHELIN RECEPTOR Miki Adachi, Yan-Yan Yang, Yasuhiro Furuichi* and Chikara Miyamoto Department of Molecular Genetics, Nippon Roche Research Center, Kamakura, 247, Japan Received July 17, 1991
Summary: A cDNA coding for the human A-type endothelin receptor (ETA) was cloned from a human placenta cDNA library. The c D N A contained the entire coding sequence for the 427 amino acid protein with a relative Mr of 48,722. The deduced amino acid sequence of the human ETA was, respectively, 94% and 93% homologous with the sequence of bovine ETA and rat ETA, but was only 64% homologous with that of the human ETB receptor. Upon expression in COS-1 cells, the human ETA receptor showed binding activity to ETA, with the highest selectivity to ET-1. Northern blot analysis showed that the mRNA of human placenta ETA consists of one species 5 kilo-nucleotides in length, and the same analysis for the uterus, testis, heart and adrenal gland of Cynomolgus monkey showed that the cognate mRNAs are widely distributed. ®~991Aoademic Press,
Inc.
I n t r o d u c t i o n : Endothelins (ETs) are a group of potent and longacting vasoconstrictor peptides consisting of 21 amino acids in length (1). Ligand binding-assays showed that the endothelin receptors are distributed widely in mammalian tissues (2). In addition, the presence of multiple subtypes of the endothelin (ET) receptor has been implicated by cross-linking experiments with 125I-labeled ETs (3,4). Indeed, the cDNAs coding for two different types of the ET receptor, ETA (specific for ET-1) and ETB (nonspecific for ET-1, 2, 3) have been respectively cloned from the lung tissue of bovine (5) and rat (6). More recently, cDNAs for the
* To whom correspondence should be addressed.
1265
0006-291X/91 $1.50 Copyright © 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.
Vol. 180, No. 3, 1991
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rat ETA (7) and human ETB (8) have also been cloned. The list of cloned cDNAs for ET receptor subtypes is constantly being added to and the structural features of these receptors appear r e m a r k a b l y similar; they contain seven m e m b r a n e - s p a n n i n g domains which are rich in c~-helix, N-linked glycosylation sites at the N - t e r m i n a l o u t e r m e m b r a n e d o m a i n and the p o t e n t i a l phosphorylation sites at the C-terminal cytoplasmic domains, consistent with a family of G protein-coupled receptors (5). To investigate the signal transduction of endothelin and its effects on vasoconstriction, we have cloned ETA cDNA from a human placenta cDNA library. In this paper, we characterize the human ETA receptor deduced from the cDNA sequence and describe its expression in COS ceils.
Materials
and
Methods
Materials: ET-I was purchased from Peptide Institute Inc. (Osaka, Japan) and the [125I]-labeled ET-1 (81.4 TBq/mmol) was from New England Nuclear. Oligotex-dT 30 was from Nippon Roche K.K. (Tokyo, Japan). Human placenta cDNA in lambda g t l l library was from Clontech Laboratories Inc. (Palo Alto, USA). Human placenta genomic DNA was prepared as described (9), P CR: After denaturation of human placenta genomic DNA by heating at 95 °C for 10 min, 0.5 unit of Taq polymerase was added and 30 cycles of polymerase chain reaction (PCR) were done (denaturation: 30 sec at 94 °C; annealing: 30 sec at 45 °C; elongation: 1 min at 72 °C) using the Perkin-Elmer Cetus automated thermal cycler (10). Cloning of Human ETA cDNA: The cDNA library was screened with the 32p-labeled PCR-amplified ETA DNA fragment as the probe. Positive clone was sequenced by the Sanger method (11) for the subcloned gene in pUC19. Northern Blot Analysis: Poly (A) + containing RNA (10 ~tg) was purified by Oligotex-dT 30 from human placenta and a variety of monkey organs. It was separated in agarose gel electrophoresis in the presence of formaldehyde and was hybridized with 32p_ labeled 1.2 Kb DNA of ETA cDNA as the probe. The membrane filters were washed in 0.1 x SSPE at 50 °C and autoradiographed overnight at -80 °C.
Cells and Transfection: COS-1 cells were cultured in Dulbecco Modified Eagle Medium (DMEM) (GIBCO Laboratories, New York, USA) supplemented with heat-inactivated 10% fetal calf serum. Transfections were performed as follows. The cells were grown at 1 x 106 to 1.5 x 106 cells per 90-mm-diameter dish 24 hr before 1266
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transfection. About 5 gg of pCDM8-ETA plasmid DNA which contained ETA cDNA was diluted in 0.4 ml of phosphate-buffered saline containing 1 mg of DEAE-dextran. The cells were washed once with warm PBS, the DNA solution was added to the cells, and the mixture was incubated at 37 °C for 30 rain. The medium containing 100 gM chloroquine was then added to the cells, and the mixture was incubated for 2 hr. After treatment of cells with 10% dimethyl sulfoxide for 2.5 min, the cells were fed again with fresh medium and incubated for 72 hr.
Binding Assay: COS-1 cells were removed from the monolayer plates and collected by centrifugation. After incubation of 3 x 105 cells with [125I]-ET-1 (0.47 KBq) in 100 gl of DMEM/25 mM HEPES buffer (pH 7.4)/0.1% bovine serum albumin for 1 hr at room temperature, the bound radioactivity was determined after removing free [125I]-ET-1 by centrifugation. Non-specific binding was determined in the presence of 1 gM nonradioactive ET-1.
Results Cloning of the cDNA Encoding Human ETA Receptor: The probe, specific for the human ETA gene, was prepared by PCR after testing various combinations of primers designed from the published sequence of bovine ETA (5). One set of primers corresponding to the bovine ETA coding sequence 911930 and 1,018-1,038 yielded a distinct 128 bp fragment by PCR from chromosomal DNA of human placenta used as template. The 128 bp fragment showed a 94% homology, upon sequence analysis, to the expected region of the bovine ETA gene. The 128 bp fragment was 32p-labeled and used for screening the ETAspecific cDNA clone from the phage library of human placental cDNAs. One positive clone, MA-1, was isolated from 4 x 105 phages. The sequence analysis revealed that the MA-1 contains 1,853 bp cDNA fragment which emcompasses the 67 nucleotides of 5'-noncoding region, the entire coding sequences of 1,281 nucleotides and the 3'-noncoding 505 nucleotide sequence (Fig. 1). The sequence predicts a protein of 427 amino acids that possesses 7 transmembrane domains. Its amino acid sequence is very homologous (94% and 93%) to those of the bovine and rat ETA receptors. The amino acid sequence homology between human and bovine/rat ETA diverges in N-terminal region (84%/73% homology) but is almost identical in seven transmembrane regions. Additional similarity between human, bovine and rat 1267
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GAATTCGGGAI~J~AGTGAAGGTGTAAAAGCAGCACAAGTGCAATAAGAGATATTrCCTCAAATTTGCCTCAAG • 120 A T G G A A A C C C T T T G CCTCAG G G C A T C C T T T T G G C T G G C A C T G G T T G G A T G T G T A A T C A G T G A T A A T C C T G A G A G A T A C A G C A C A A A T C T A A G C A A T C A T G T G G A T G A T T T C A C C A C T ~ M E T L C L R A S F W L A L V G C V I S D N P E R Y S T N L S N H V 0 D F T T F 40 • 240 C GT GGCACAGAGCT CA0 CTTC CT GGTTA C CAC T CAT CAAC CCACTAA'Iq'T 0 GT CCTAC C CAGCAAT 0 GCT CAATGCACAACTATTG C C CACAGCAGACTAAAATTA CTT CAGCTTTCAAA R G T E L S F L V T T H Q P T N L V L P S N 0 S M H N Y C P Q Q T K 1 T S A F K 80
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Figure 1. The eDNA sequence of the human ETA receptor and its deduced amino acid sequence. The predicted amino acid sequence is shown below the cloned eDNA sequence. Positions of the putative transmembrane segments I-VII are indicated by solid lines above the sequence. Arrow represents the predicted cleavage site of the secretory signal sequence. Triangles represent the potential N-glycosylation sites. Dots denotes the possible phosphorylation sites.
include conservation of (i) the potential asparagine-linked Nglycosylation sites in the predicted N-terminal outer cell domain (amino acid residues 29 and 62) and (ii) 3 serine-residues in the third cytoplasmic loop and the cytoplasmic C-terminal tail which may potentially be phosphorylated by serine/thereonine protein kinases. The ETA mRNA was detected in human placenta by Northern blot analysis, in which human ETA eDNA was used as probe. As shown in Fig. 2A (lane 1), human placenta contained a single class 1268
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BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
1 2
A
3
4
5
6
5 K~ 42K~ 3 K~
7
B
9 1011
~23S ~16S
B actin
Figure 2. A. Northern blot analysis of ETA mRNAs from human placenta and various monkey organs. Poly (A) + RNAs were isolated from human placenta (lane 1) and various monkey organs. Lane 2: testis, lane 3: uterus, lane 4: heart, lane 5: lung, lane 6: adrenal gland, lane 7: stomach, lane 8: large intestine, lane 9: cerebellum, lane 10: brain stem and lane 11: cerebral cortex. RNA blot-hybridization analysis (10 I~g of RNA each) was carried out as described in Materials and Methods. B. Simultaneous hybridizations for ~-actin mRNA were carried out as internal controls to evaluate the amount of ETA mRNAs.
of ETA mRNA, 5 kilo-nucleotides in size. To examine tissue distribution of ETA mRNA, the fresh mRNA was prepared from various organs of Cynomolgus monkey. The ETA mRNA is expressed in high levels in the testis, uterus, heart and adrenal gland and is also present in the lung and stomach at the low levels (Fig. 2A, lanes 2-7). However, we could not detect the ETAspecific sequence in cerebellum, brain stem and cerebral cortex of monkey (Fig. 2A, lanes 9-11). The size of monkey ETA m R N A s appears to be smaller (4.2 kilo-nucleotides) than that of human placenta. Monkey testis gave two hybridizing bands with mRNA sizes estimated at 3 and 4.2 kilo-nucleotides, while all the other monkey organs gave rise to a major ETA mRNA of 4.2 kilonucleotides.
Expression
of
Human
expression
plasmid
ETA
Gene in C 0 S - 1
Cells: An pCDM8-ETA was constructed by insertion of
human ETA cDNA into p C D M 8 v e c t o r d o w n s t r e a m of cytomegalovirus promoter (12). After transfection of pCDM8-ETA plasmid to C O S - 1 cells, the activity of the expressed receptor to bind to ET-1 was measured. Binding of 125I-labeled ET-1 to C O S - 1 1269
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B
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1 oo
15
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T 1 .0
[ ] 1 bound (fmol per 1X10 s cells) T T 2.0 3.0
0
['~2s I ] ET-1 (riM)
-5
Peptlde
(M)
Figure 3. Ligand-binding activity of ETA receptor expressed in COS cells. A: saturation isotherm, B: displacement of specific binding of [125I]-labeled ET-1 to COS-1 cells transfectd with pCDM8-ETA. A: the inset shows the Schatchard plot of [125I]-labeled ET-1 binding. B: binding of [125I]-labeled ET-1 was determined in the presence of the indicated concentrations of ET-1 (El), ET-2 (*) and ET-3 (A).
cells increased upon transfection with the plasmid DNA, and was saturable by non-radioactive ET-1 with a dissociation constant (Kd) of 1.26 nM (Fig. 3A). No or very low binding activity was detected for COS-1 cells transfected with the vector DNA alone or without transfection. Similar results were obtained when C O S - 7 cells were used as host (data not shown). Displacement experiments with 125I-labled ET-1 indicated that ET-1 is the most potent competitor of 125I-ET-1 binding. It is 4.5 and 350 times more e f f e c t i v e than ET-2 and ET-3, respectively. The values of the inhibition constant (Ki) for ET-1, ET-2 and ET-3 were calculated to be 2, 9 and 700 nM, respectively (Fig. 3B).
Discussion We have reported the presence of ET specific receptor in the membrane fraction of human placenta (13). Based on these results and others (5, 7), we have isolated an ETA-specific cDNA from a human placenta cDNA library. Expression of the cloned cDNA in COS-1 cells demonstrated that the MA-1 indeed encodes a functional ETA receptor, as determined by increased binding to 125I-ET-1. Nothern blot analysis of human placental mRNAs 1270
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revealed a single class of transcript with 5 kilo-nucleotides in length. The structure of the ETA receptor was found to be c o n s e r v e d among human, bovine and rat. The amino acid sequence of the human ETA receptor is 94% and 93% homologous, respectively, with those of bovine and rat (5, 6). Interestingly, the h o m o l o g y in the N-terminal region is relatively low as compared with that of the transmembrane regions. Diverged Nterminal sequences of ET receptors imply that the N-terminal region may be involved in ligand selection or other yet unknown regulation, but not ligand binding directly. Indeed, the natural endothelin receptor purified from the placenta extract is often devoid of the N-terminal region and yet represents the ET binding activity (our unpublished data). The data also imply that the three outer membrane-loop regions may constitute the site to which ET peptides binds. The tissue distribution of the ETA receptor in Cynomolgus monkey was also studied by Northern blot analysis under the high stringent conditions that allowed the detection of only ETAspecific mRNA. A significant similarity was found between bovine and monkey organs with respect to the distribution of the ETAspecific sequences and the high expression in heart (Fig. 2A). In addition, our data showed an extremely high expression of the E T A gene in uterus and testis, for which no comparable data was available in bovine (5). The relative content of ETA mRNA of human placenta was assumed to be less than those of monkey uterus, testis and heart but almost equivalent to that of adrenal gland. It should be noted that no detectable expression was observed in monkey brain. Much ETA was reported for lung and heart of rat and bovine (5, 6), however, there was only a low level of ETA mRNA in monkey lung in our data (Fig. 2A, lane 5). The cloned cDNA of human ETA receptor and its sequence information should provide valuable tools for further studies on the endothelin receptors which seemingly play important roles in vascular and reproductive systems.
Acknowledgments We thank Dr. I. Horii and Mr. A. Kuwahara in the Department of Toxicology in Nippon Roche Research Center for supplying us with Cynomolgus monkey organs, and Ms. A. Fujii for preparation of the manuscript. 1271
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References . Yanagisawa, M. and Masaki, T. (1989) Trends Pharmacol. Sci., (review), 10, 374-378. . Koseki, C., Imai, M., Hirata, Y., Yanagisawa, M. and Masaki, T. (1989) Am. J. Physiol. 256 (Regulatory Integrative Comp. Physiol. 25) R858-R866. . Watanabe, H., Miyazaki, H., Kondoh, M., Masuda, Y., Kimura, S., Yanagisawa, M., Masaki, T. and Murakami, K. (1989) Biochem. Biophys. Res. Commun., 161, 1252-1259. 4. Sugiura, M., Snajdar, R.M., Schwartzberg, M., Badr, K.F. and Inagami, T. (1989) Biochem. Biophys. Res. Commun., 162, 1396-1401. 5. Arai, H., Hori, S., Aramori, I., Ohkubo, H. and Nakanishi, S. (1990) Nature, 348, 730-732. 6. Sakurai, T., Yanagisawa, M., Takuwa, Y., Miyazaki, H., Kimura, S., Goto, K. and Masaki, T. (1990) Nature, 348, 732-735. 7.
Lin, H.Y., Kaji, E.H., Winkel, G.K., Ives, H.E. and Lodish, H.F. (1991) 88, 3185-3189.
. Nakamura, M., Takayanagi, R., Sakai, Y., Sakamoto, S., Hagiwara, H., Mizuno, T., Saitoh, Y., Hirose, S., Yamamoto, M. and Nawata, H. (1991) Biochem. Biophys. Res. Commun., 177, 34-39. .
Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989) Molecular Cloning, A laboratory manual (Second edition), 2, 9.22-9.23 (Cold Spring Harbor Laboratory)
10.
Saiki, R.K., Scharf, S., Faloona, F., Mullis, K.B., Horn, G.T., Erlich, H.A. and Arnheim, N. (1985) Science, 230, 1350-1354.
11.
Sanger, F., Nicklen, S. and Coulson, A.R. (1977) Proc. Natl. Acad. Sci. U.S.A., 74, 5463-5467.
12.
Seed, B. and Aruffo, A. (1987) Proc. Natl. Acad. Sci., U.S.A., 84, 3365-3369.
13.
Wada, K. Tabuchi, H. Ohba, R., Satoh, M., Tachibana, Y., Akiyama, N., Hiraoka, O., Asakura, A., Miyamoto, C. and Furuichi, Y. (1990) Biochem. Biophys. Res. Commun., 167, 251257.
1272
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CLONING AND CHARACTERIZATION OF cDNA ENCODING H U M A N A-TYPE ENDOTHELIN RECEPTOR Miki Adachi, Yan-Yan Yang, Yasuhiro Furuichi* and Chikara Miyamoto Department of Molecular Genetics, Nippon Roche Research Center, Kamakura, 247, Japan Received July 17, 1991
Summary: A cDNA coding for the human A-type endothelin receptor (ETA) was cloned from a human placenta cDNA library. The c D N A contained the entire coding sequence for the 427 amino acid protein with a relative Mr of 48,722. The deduced amino acid sequence of the human ETA was, respectively, 94% and 93% homologous with the sequence of bovine ETA and rat ETA, but was only 64% homologous with that of the human ETB receptor. Upon expression in COS-1 cells, the human ETA receptor showed binding activity to ETA, with the highest selectivity to ET-1. Northern blot analysis showed that the mRNA of human placenta ETA consists of one species 5 kilo-nucleotides in length, and the same analysis for the uterus, testis, heart and adrenal gland of Cynomolgus monkey showed that the cognate mRNAs are widely distributed. ®~991Aoademic Press,
Inc.
I n t r o d u c t i o n : Endothelins (ETs) are a group of potent and longacting vasoconstrictor peptides consisting of 21 amino acids in length (1). Ligand binding-assays showed that the endothelin receptors are distributed widely in mammalian tissues (2). In addition, the presence of multiple subtypes of the endothelin (ET) receptor has been implicated by cross-linking experiments with 125I-labeled ETs (3,4). Indeed, the cDNAs coding for two different types of the ET receptor, ETA (specific for ET-1) and ETB (nonspecific for ET-1, 2, 3) have been respectively cloned from the lung tissue of bovine (5) and rat (6). More recently, cDNAs for the
* To whom correspondence should be addressed.
1265
0006-291X/91 $1.50 Copyright © 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.
Vol. 180, No. 3, 1991
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
rat ETA (7) and human ETB (8) have also been cloned. The list of cloned cDNAs for ET receptor subtypes is constantly being added to and the structural features of these receptors appear r e m a r k a b l y similar; they contain seven m e m b r a n e - s p a n n i n g domains which are rich in c~-helix, N-linked glycosylation sites at the N - t e r m i n a l o u t e r m e m b r a n e d o m a i n and the p o t e n t i a l phosphorylation sites at the C-terminal cytoplasmic domains, consistent with a family of G protein-coupled receptors (5). To investigate the signal transduction of endothelin and its effects on vasoconstriction, we have cloned ETA cDNA from a human placenta cDNA library. In this paper, we characterize the human ETA receptor deduced from the cDNA sequence and describe its expression in COS ceils.
Materials
and
Methods
Materials: ET-I was purchased from Peptide Institute Inc. (Osaka, Japan) and the [125I]-labeled ET-1 (81.4 TBq/mmol) was from New England Nuclear. Oligotex-dT 30 was from Nippon Roche K.K. (Tokyo, Japan). Human placenta cDNA in lambda g t l l library was from Clontech Laboratories Inc. (Palo Alto, USA). Human placenta genomic DNA was prepared as described (9), P CR: After denaturation of human placenta genomic DNA by heating at 95 °C for 10 min, 0.5 unit of Taq polymerase was added and 30 cycles of polymerase chain reaction (PCR) were done (denaturation: 30 sec at 94 °C; annealing: 30 sec at 45 °C; elongation: 1 min at 72 °C) using the Perkin-Elmer Cetus automated thermal cycler (10). Cloning of Human ETA cDNA: The cDNA library was screened with the 32p-labeled PCR-amplified ETA DNA fragment as the probe. Positive clone was sequenced by the Sanger method (11) for the subcloned gene in pUC19. Northern Blot Analysis: Poly (A) + containing RNA (10 ~tg) was purified by Oligotex-dT 30 from human placenta and a variety of monkey organs. It was separated in agarose gel electrophoresis in the presence of formaldehyde and was hybridized with 32p_ labeled 1.2 Kb DNA of ETA cDNA as the probe. The membrane filters were washed in 0.1 x SSPE at 50 °C and autoradiographed overnight at -80 °C.
Cells and Transfection: COS-1 cells were cultured in Dulbecco Modified Eagle Medium (DMEM) (GIBCO Laboratories, New York, USA) supplemented with heat-inactivated 10% fetal calf serum. Transfections were performed as follows. The cells were grown at 1 x 106 to 1.5 x 106 cells per 90-mm-diameter dish 24 hr before 1266
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transfection. About 5 gg of pCDM8-ETA plasmid DNA which contained ETA cDNA was diluted in 0.4 ml of phosphate-buffered saline containing 1 mg of DEAE-dextran. The cells were washed once with warm PBS, the DNA solution was added to the cells, and the mixture was incubated at 37 °C for 30 rain. The medium containing 100 gM chloroquine was then added to the cells, and the mixture was incubated for 2 hr. After treatment of cells with 10% dimethyl sulfoxide for 2.5 min, the cells were fed again with fresh medium and incubated for 72 hr.
Binding Assay: COS-1 cells were removed from the monolayer plates and collected by centrifugation. After incubation of 3 x 105 cells with [125I]-ET-1 (0.47 KBq) in 100 gl of DMEM/25 mM HEPES buffer (pH 7.4)/0.1% bovine serum albumin for 1 hr at room temperature, the bound radioactivity was determined after removing free [125I]-ET-1 by centrifugation. Non-specific binding was determined in the presence of 1 gM nonradioactive ET-1.
Results Cloning of the cDNA Encoding Human ETA Receptor: The probe, specific for the human ETA gene, was prepared by PCR after testing various combinations of primers designed from the published sequence of bovine ETA (5). One set of primers corresponding to the bovine ETA coding sequence 911930 and 1,018-1,038 yielded a distinct 128 bp fragment by PCR from chromosomal DNA of human placenta used as template. The 128 bp fragment showed a 94% homology, upon sequence analysis, to the expected region of the bovine ETA gene. The 128 bp fragment was 32p-labeled and used for screening the ETAspecific cDNA clone from the phage library of human placental cDNAs. One positive clone, MA-1, was isolated from 4 x 105 phages. The sequence analysis revealed that the MA-1 contains 1,853 bp cDNA fragment which emcompasses the 67 nucleotides of 5'-noncoding region, the entire coding sequences of 1,281 nucleotides and the 3'-noncoding 505 nucleotide sequence (Fig. 1). The sequence predicts a protein of 427 amino acids that possesses 7 transmembrane domains. Its amino acid sequence is very homologous (94% and 93%) to those of the bovine and rat ETA receptors. The amino acid sequence homology between human and bovine/rat ETA diverges in N-terminal region (84%/73% homology) but is almost identical in seven transmembrane regions. Additional similarity between human, bovine and rat 1267
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GAATTCGGGAI~J~AGTGAAGGTGTAAAAGCAGCACAAGTGCAATAAGAGATATTrCCTCAAATTTGCCTCAAG • 120 A T G G A A A C C C T T T G CCTCAG G G C A T C C T T T T G G C T G G C A C T G G T T G G A T G T G T A A T C A G T G A T A A T C C T G A G A G A T A C A G C A C A A A T C T A A G C A A T C A T G T G G A T G A T T T C A C C A C T ~ M E T L C L R A S F W L A L V G C V I S D N P E R Y S T N L S N H V 0 D F T T F 40 • 240 C GT GGCACAGAGCT CA0 CTTC CT GGTTA C CAC T CAT CAAC CCACTAA'Iq'T 0 GT CCTAC C CAGCAAT 0 GCT CAATGCACAACTATTG C C CACAGCAGACTAAAATTA CTT CAGCTTTCAAA R G T E L S F L V T T H Q P T N L V L P S N 0 S M H N Y C P Q Q T K 1 T S A F K 80
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Figure 1. The eDNA sequence of the human ETA receptor and its deduced amino acid sequence. The predicted amino acid sequence is shown below the cloned eDNA sequence. Positions of the putative transmembrane segments I-VII are indicated by solid lines above the sequence. Arrow represents the predicted cleavage site of the secretory signal sequence. Triangles represent the potential N-glycosylation sites. Dots denotes the possible phosphorylation sites.
include conservation of (i) the potential asparagine-linked Nglycosylation sites in the predicted N-terminal outer cell domain (amino acid residues 29 and 62) and (ii) 3 serine-residues in the third cytoplasmic loop and the cytoplasmic C-terminal tail which may potentially be phosphorylated by serine/thereonine protein kinases. The ETA mRNA was detected in human placenta by Northern blot analysis, in which human ETA eDNA was used as probe. As shown in Fig. 2A (lane 1), human placenta contained a single class 1268
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1 2
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5 K~ 42K~ 3 K~
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Figure 2. A. Northern blot analysis of ETA mRNAs from human placenta and various monkey organs. Poly (A) + RNAs were isolated from human placenta (lane 1) and various monkey organs. Lane 2: testis, lane 3: uterus, lane 4: heart, lane 5: lung, lane 6: adrenal gland, lane 7: stomach, lane 8: large intestine, lane 9: cerebellum, lane 10: brain stem and lane 11: cerebral cortex. RNA blot-hybridization analysis (10 I~g of RNA each) was carried out as described in Materials and Methods. B. Simultaneous hybridizations for ~-actin mRNA were carried out as internal controls to evaluate the amount of ETA mRNAs.
of ETA mRNA, 5 kilo-nucleotides in size. To examine tissue distribution of ETA mRNA, the fresh mRNA was prepared from various organs of Cynomolgus monkey. The ETA mRNA is expressed in high levels in the testis, uterus, heart and adrenal gland and is also present in the lung and stomach at the low levels (Fig. 2A, lanes 2-7). However, we could not detect the ETAspecific sequence in cerebellum, brain stem and cerebral cortex of monkey (Fig. 2A, lanes 9-11). The size of monkey ETA m R N A s appears to be smaller (4.2 kilo-nucleotides) than that of human placenta. Monkey testis gave two hybridizing bands with mRNA sizes estimated at 3 and 4.2 kilo-nucleotides, while all the other monkey organs gave rise to a major ETA mRNA of 4.2 kilonucleotides.
Expression
of
Human
expression
plasmid
ETA
Gene in C 0 S - 1
Cells: An pCDM8-ETA was constructed by insertion of
human ETA cDNA into p C D M 8 v e c t o r d o w n s t r e a m of cytomegalovirus promoter (12). After transfection of pCDM8-ETA plasmid to C O S - 1 cells, the activity of the expressed receptor to bind to ET-1 was measured. Binding of 125I-labeled ET-1 to C O S - 1 1269
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B
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-5
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(M)
Figure 3. Ligand-binding activity of ETA receptor expressed in COS cells. A: saturation isotherm, B: displacement of specific binding of [125I]-labeled ET-1 to COS-1 cells transfectd with pCDM8-ETA. A: the inset shows the Schatchard plot of [125I]-labeled ET-1 binding. B: binding of [125I]-labeled ET-1 was determined in the presence of the indicated concentrations of ET-1 (El), ET-2 (*) and ET-3 (A).
cells increased upon transfection with the plasmid DNA, and was saturable by non-radioactive ET-1 with a dissociation constant (Kd) of 1.26 nM (Fig. 3A). No or very low binding activity was detected for COS-1 cells transfected with the vector DNA alone or without transfection. Similar results were obtained when C O S - 7 cells were used as host (data not shown). Displacement experiments with 125I-labled ET-1 indicated that ET-1 is the most potent competitor of 125I-ET-1 binding. It is 4.5 and 350 times more e f f e c t i v e than ET-2 and ET-3, respectively. The values of the inhibition constant (Ki) for ET-1, ET-2 and ET-3 were calculated to be 2, 9 and 700 nM, respectively (Fig. 3B).
Discussion We have reported the presence of ET specific receptor in the membrane fraction of human placenta (13). Based on these results and others (5, 7), we have isolated an ETA-specific cDNA from a human placenta cDNA library. Expression of the cloned cDNA in COS-1 cells demonstrated that the MA-1 indeed encodes a functional ETA receptor, as determined by increased binding to 125I-ET-1. Nothern blot analysis of human placental mRNAs 1270
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revealed a single class of transcript with 5 kilo-nucleotides in length. The structure of the ETA receptor was found to be c o n s e r v e d among human, bovine and rat. The amino acid sequence of the human ETA receptor is 94% and 93% homologous, respectively, with those of bovine and rat (5, 6). Interestingly, the h o m o l o g y in the N-terminal region is relatively low as compared with that of the transmembrane regions. Diverged Nterminal sequences of ET receptors imply that the N-terminal region may be involved in ligand selection or other yet unknown regulation, but not ligand binding directly. Indeed, the natural endothelin receptor purified from the placenta extract is often devoid of the N-terminal region and yet represents the ET binding activity (our unpublished data). The data also imply that the three outer membrane-loop regions may constitute the site to which ET peptides binds. The tissue distribution of the ETA receptor in Cynomolgus monkey was also studied by Northern blot analysis under the high stringent conditions that allowed the detection of only ETAspecific mRNA. A significant similarity was found between bovine and monkey organs with respect to the distribution of the ETAspecific sequences and the high expression in heart (Fig. 2A). In addition, our data showed an extremely high expression of the E T A gene in uterus and testis, for which no comparable data was available in bovine (5). The relative content of ETA mRNA of human placenta was assumed to be less than those of monkey uterus, testis and heart but almost equivalent to that of adrenal gland. It should be noted that no detectable expression was observed in monkey brain. Much ETA was reported for lung and heart of rat and bovine (5, 6), however, there was only a low level of ETA mRNA in monkey lung in our data (Fig. 2A, lane 5). The cloned cDNA of human ETA receptor and its sequence information should provide valuable tools for further studies on the endothelin receptors which seemingly play important roles in vascular and reproductive systems.
Acknowledgments We thank Dr. I. Horii and Mr. A. Kuwahara in the Department of Toxicology in Nippon Roche Research Center for supplying us with Cynomolgus monkey organs, and Ms. A. Fujii for preparation of the manuscript. 1271
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References . Yanagisawa, M. and Masaki, T. (1989) Trends Pharmacol. Sci., (review), 10, 374-378. . Koseki, C., Imai, M., Hirata, Y., Yanagisawa, M. and Masaki, T. (1989) Am. J. Physiol. 256 (Regulatory Integrative Comp. Physiol. 25) R858-R866. . Watanabe, H., Miyazaki, H., Kondoh, M., Masuda, Y., Kimura, S., Yanagisawa, M., Masaki, T. and Murakami, K. (1989) Biochem. Biophys. Res. Commun., 161, 1252-1259. 4. Sugiura, M., Snajdar, R.M., Schwartzberg, M., Badr, K.F. and Inagami, T. (1989) Biochem. Biophys. Res. Commun., 162, 1396-1401. 5. Arai, H., Hori, S., Aramori, I., Ohkubo, H. and Nakanishi, S. (1990) Nature, 348, 730-732. 6. Sakurai, T., Yanagisawa, M., Takuwa, Y., Miyazaki, H., Kimura, S., Goto, K. and Masaki, T. (1990) Nature, 348, 732-735. 7.
Lin, H.Y., Kaji, E.H., Winkel, G.K., Ives, H.E. and Lodish, H.F. (1991) 88, 3185-3189.
. Nakamura, M., Takayanagi, R., Sakai, Y., Sakamoto, S., Hagiwara, H., Mizuno, T., Saitoh, Y., Hirose, S., Yamamoto, M. and Nawata, H. (1991) Biochem. Biophys. Res. Commun., 177, 34-39. .
Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989) Molecular Cloning, A laboratory manual (Second edition), 2, 9.22-9.23 (Cold Spring Harbor Laboratory)
10.
Saiki, R.K., Scharf, S., Faloona, F., Mullis, K.B., Horn, G.T., Erlich, H.A. and Arnheim, N. (1985) Science, 230, 1350-1354.
11.
Sanger, F., Nicklen, S. and Coulson, A.R. (1977) Proc. Natl. Acad. Sci. U.S.A., 74, 5463-5467.
12.
Seed, B. and Aruffo, A. (1987) Proc. Natl. Acad. Sci., U.S.A., 84, 3365-3369.
13.
Wada, K. Tabuchi, H. Ohba, R., Satoh, M., Tachibana, Y., Akiyama, N., Hiraoka, O., Asakura, A., Miyamoto, C. and Furuichi, Y. (1990) Biochem. Biophys. Res. Commun., 167, 251257.
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