Vol. 181, No. 1, 1991 November 27, 1991
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 184-190
CLONING AND CHROMOSOMAL LOCALIZATION OF A H U M A N ENDOTHELIN ETA RECEPTOR Curt Cyr 1, Kay Huebner 2, Teresa Druck 2, and Richard Krisl, * 1Department of Pharmacology, New York University Medical Center, 550 First Avenue, New York, New York 10016 2Jefferson Cancer Institute, Thomas Jefferson University, 233 S. 10th Street, Philadelphia, Pennsylvania 19107 Received September 25, 1991
Summary A cDNA clone encoding a h u m a n endothelin receptor was isolated from a placenta cDNA library. The deduced amino acid sequence of the clone is 94% identical to the bovine endothelin ETA receptor and r e p r e s e n t s the h u m a n homologue. The h u m a n endothelin ETA receptor gene was localized to chromosome 4 by analysis of its segregation pattern in rodent-human hybrids. ®1991Aoad.... P..... Znc.
Endothelin is a twenty-one amino acid peptide originally isolated from porcine aortic endothelial cells and is a p o t e n t endogenous vasoconstrictor (1). Cloning and sequencing analysis showed that there are at least three genes encoding the endothelin family (2). One of these genes codes for endothelin 1 (ET1) and the other two genes code for peptides of the same length designated as endothelin 2 and 3 (ET2 and ET3). This family of proteins is remarkably similar to the sarafotoxins, a group of snake venoms derived from the Israeli burrowing asp (Atractaspis engaddensis) (3). The sarafotoxins are one of the most potent venoms known. Two ET receptor subtypes have recently been cloned and sequenced. The receptors are members of the rhodopsin-like superfamily of receptors that have seven putative t r a n s m e m b r a n e domains and are coupled with Gproteins for signal transduction. One ET receptor subtype was cloned from a r a t lung cDNA library and h u m a n placenta library (4,5). Displacement e x p e r i m e n t s performed on the expressed endothelin receptor revealed similar affinities for ET1, 2 a n d 3. This receptor s u b t y p e has been
* To whom correspondence should be addressed. Abbreviations u s e d in t b | . p a p e n Endothelin: ET; ETA: endothelin type ETA, 0006-291X/91 $1.50 Copyright © 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.
184
Vol. 181, No. 1, 1991
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
designated ETB. The endothelin receptor subtype cloned by Arai et al. (6) from a bovine lung cDNA library utilizing a Xenopus oocyte expression system has the highest affinity for ET1 with a Kd of 0.18 nM. This ET receptor subtype has been named ETA (6). More recently, a rat ETA receptor has been cloned from a rat heart cell line and is over 90% similar to the bovine sequence (7). The rat and bovine sequences differ most widely at the amino terminus. A cloned human endothelin receptor could make screening for specific h u m a n endothelin antagonists much easier. In addition, a human clone can be used to probe the role of endothelin receptor in disease states. Here we describe the cloning, sequencing and chromosomal localization of a human ETA receptor isolated from a human placenta cDNA library.
Materioh a n d Methods Cloning technique: A probe for the h u m a n endothelin receptor was produced using p o l y m e r a s e chain r e a c t i o n amplification. Two oligonucleotides (20 bases each) were produced from conserved regions corresponding to the bovine ETA sequence. The sense oligonucleotide was produced starting from base number 296 and the antisense was produced starting from base 830. The template DNA was cDNA produced from human placenta mRNA using a kit from Invitrogen. The resulting PCR product was 534 base pairs and was subcloned into pBluescript (Stratagene) (8). The insert (ET8) was sequenced for verification and removed from the plasmid using restriction enzymes BamHI and EcoRI. ET8 was labelled with a-32P-CTP by random priming and used to screen human brain and placenta libraries. Plaque hybridization was carried out at 55°C overnight in 6xSSPE (lxSSPE is 0.15M NaC1, 0.1M NaH2P04, 0.001M EDTA, pH 7.4) and blotto (0.2% nonfat dried milk) and the membranes washed once at room temperature with 6xSSC, 0.1% SDS and twice at 65°C with 0.1xSSC and 0.1% SDS (8). Positive clones were purified and inserts removed using EcoRI, subcloned into pBluescript and subjected to didioxy double stranded sequencing from both ends (9). Cells: Isolation, propagation and characterization of most somatic cell hybrids used in this study have been described elsewhere (10). Many of the hybrids were obtained from the NIGMS H u m a n Genetic M u t a n t Cell Repository (Coriell Institute, Camden, N.J.). Chromosome regions retained by each hybrid are diagrammed in Fig. 3. Southern Blot Analvsis: Hybrid and control DNAs were prepared by cell lysis, proteinase K digestion, phenol extraction, and ethanol precipitation. Cellular DNAs were digested with restriction enzyme EcoRI, fractionated on 0.8% agarose gels, transferred to nylon membrane and hybridized at 42°C for 16 hours in 50% formamide, 5x SSPE, 5X Denhardts solution, 0.1% SDS and 100 ug/ml salmon sperm DNA. ET8 probes (see above) for filter hybridizations were radiolabelled by nick translation with a-32P-CTP to a specific activity of 108 cpm/0.1 ug and 108 cpm were used for each filter hybridization. 185
Vol. 181, No. 1, 1991
BIOCHEMICAL AND BIOPHYSICAL RESEARCH C O M M U N I C A T I O N S
Results and Discussion 106 clones from each of three libraries (human brain stem, caudate, and placenta) were screened using a humanized P C R probe specificfor the endothelin E T A receptor (see methods). Positive clones were obtained from h u m a n brain stem, caudate, and placenta libraries. The positive clones from brain stem and caudate were not full length and repeated screenings did not produce a full length clone from these libraries (data not shown). All three clones had an internal EcoRI site and were subcloned into pBluescript for sequencing. The nucleotide sequence of the full length placenta clone is shown in figure 1. The c D N A consists of 1661 nucleotides with a coding region of 1263 nucleotides. The sequences of the partial clones were identical to the full length clone obtained from the placenta library. However, the 5' end of the caudate clone started at base 380 of the placental clone and the 5' end of the brain stem clone started at nucleotide 504 of the placental clone. The placenta c D N A sequence includes an open reading frame of 1263 bases which would encode a protein of 421 amino acids with a relative
TATAGGCGAATTGGGTA -121 CCGGGAAAAAAATCGAGGTCGACGGTAT•GGATGAAGCTTGATATCGAATTCGGGAAAAAGTGAAGGTGTAAAAGCAAGCACAAGTG•AATAAGAGATATTTCCTCAAATTTGCCTCAAG -i
MetG~uThrLeuCysLeuArgA~aSerPheTr~LeuA~aLeuVa~G~yCysVa~I~eSerAspAsnPr~G~uArgTyrSerThrAsnLeuSerAsnH~sVa~AspAspPheTh~ThrPhe 40 ATGGAAAC••TTTGCCTCAGGGCAT•CTTTTGGCTGGCACTGGTTGGATGTGTAATCAGTGATAATCCTGAGAGATACAGCACAAATCTAAGCAAT•ATGTGGATGATTTCA•CACTTTT 120 ArgG~yThrG~uLeuSerPheLeuVa~ThrThrHisG~nPr~ThrAsnLeuVa~LeuPr~SerAsnG~ySerMetHlsAsnTyrCysPr~G~nG~nThrLysI~eThrSerA~aPheLys 80 CGTGGCACAGAGCTCAGCTTCCTGGTTACCACTCATCAACCCACTAATTTGGTCCTACCCAGCAATGGCTCAATGCACAACTATTGCCCACAGCAGACTAAAATTACTTCAGCTTTCAAA 240 I T~rI~eA~nThrva~I1eSe~CYsThrI~ePheI~eVa~G~yMetVa~G~YAsnA~aThrLeuLeuAr~I~eI~eT~rG~nAsnLy~Cys~etArgAsnG~yPr~AsnA~aLeuI~eA~ 120 TACATTAACACTGTGATATCTTGTACTATTTTCATCGTGGGAATGGTGGGGAATGCAACTCTGCTCAGGATCATTTACCAGAACAAATGTATGAGGAATGGCCCCAACGCGCTGATAGCC 360 II ~erLeuA~aLeu$~As~L~uI~eTYrVa~Va~I~eAs~LeuPr~I~eAsnVa~heL~sLeuLeuA~aG~yArgTr~Pr~PheAs~HisAsnAs~PheG1yVa~PheLeuCysLysLeu 160 AGTCTTGCCCTTGGAGACCTTATCTATGTGGTCATTGATCTCCCTATCAATGTATTTAAGCTGCTGGCTGGGCGCTGGCCTTTTGATCACAATGACTTTGGCGTATTTCTTTGCAAGCTG 480 Ill PhePr~PheLeuG~nLvsSerSerVa~G~VI~eThrVa~LeuAsnLeuCvsA1aLeuSerVa~A~pArgTyrArgA~aVa~A~aSerTrp~erArgValG~nG~yI~eG~y~lePr~Leu 200 TTCCCCTTTTTGCAGAAGTCCTCGGTGGGGATCACCGTCCTCAACCTCTGCGCTCTTAGTGTTGACAGGTACAGAGCAGTTGCCTCCTGGAGTCGTGTTCAGGGAATTGGGATTCCTTTG 600 IV Va~ThrA~aI~eG~uI~eVa~SerI~eTrpI~eLeu~erPheI~eLeuA1aI~ePr~G~uA~aI~eG~yPh~Va~Me~a~Pr~PheG~uTyrArgG~yG~uG~n~isLysThrCy~Met 240 GTAACTGCCATTGAAATTGTCTCCATCTGGATCCTGTCCTTTATCCTGGCCATTCCTGAAGCGATTGGCTTCGTCATGGTACCCTTTGAATATAGGGGTGAACAGCATAAACCTGTATG 720 V LeuAsnA1aThrSerLysPheMetG~uPheTyrG1nAs~Va1LysA$pTrpTrpL~uPheG1YPheTYrPh~CYsMetPr~LeuVa1CYsThrA~aI1ePh~TyrThrLeuM~ThrCys 280 •TCAATGCCACATCAAAATTCATGGAGTTCTACCAAGATGTAAAGGACTGGTGGCT•TTCGGGTTCTATTTCTGTATGCCCTTGGTGTGCACTGCGATCTTCTACACCCTCATGACTTGT 840 # VI G~uMe~LeuAsnArgArgAsnG~ySerLeuArgI~eA~aLeuSerG~uHisLeuLysG~nArgArgG~uVa~A~aLysThrVa~phe~y~L~uVa1Val~epheA~aL~v~Trm~he 320 GAGATGTTGAACAGAAGGAATGGCAGCTTGAGAATTGCCCTCAGTGAACATCTTAAGCAGCGTCGAGAAGTGGCAAAAACAGTTTTCTGCTTGGTTGTAATTTTTGCTCTTTGCTGGTTC 960 VII Pr~LeuHisLeuSerAruI1eLeuL~sL~$ThrVa1~YrAsnG~uMetAspLysAsnArgCysG1uLeuLeuSerPh~Le~L~LeuH~AsDT~rI~eG~vI1~AsnLeuA~aThrMet 360 CCTCTTCATTTAAGCCGTATATTGAAGAAAACTGTGTATAACGAGATGGACAAGAACCGATGTGAATTACTTAGTTTCTTACTGCTCATGGATTACATCGGTATTAACTTGGCAACCATG 1080 # % ~ @ @ AsnSerCvsIleAsnPr~I~eA~aLeuT~rPheva~SerLysLysPheLysAsnCysPheG~nSerCysLeuCysCysCysCysTyrGlnSerLysSerLe~MetThrSerVa~Pr~Met 400 AATTCATGTATAAACCCCATAGCTCTGTATTTTGTGAGCAAGAAATTTAAAAATTGTTTCcAGTcATGCCTCTGCTGCTGCTGTTACCAGTCCAAAAGTCTGATGACCTCGGTCCCCATG 1200 # # AsnG•yThrSerI•eG•nTrpLysAsnHisAspG•nAsnAsnHisAsnThrAspArgSerSerHi•LysAspSerMetAsn 427 AACGGAACAAGCATCCAGTGGAAGAACCACGATCAAAACAACCACAACACAGACCGGAGCAGCCATAAGGACAGCATGAACTGACCACCCTTAGAAGCACTCCTCGGTACTCCCATAATC 1320 ~TcTCGGAGAAAAAAATCACAAGGCAACTGTC~GTCCGGGAATCTCTTCTcTGATCcTTCTTCCTTAATTCACTcCCACACcCAAGAAGAAATGCTTTCCAAAAC~GCAAGGTAGACTGG 1440 TTTATCCACCCACAACATCTACGAATCGTACTTCTTTTAATTGATCTAATTTACATATTCTGCGTGTTGTATTCAGCACTAAAA 1524
Fig. I. The nucleotide sequence and deduced peptide sequence of the human endothelin ETA receptor. The arrow denotes a potential cleavage
site of a hydrophobic signal sequence. Predicted hydrophobic transmembrane regions are underlined. Potential N-linked glycosylation
sitesare denoted by * and predictedproteinkinase C phosphorylationsites by #. Possible palmiteylation sites are marked by @. 186
V o l . 181, No. 1, 1991
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
6O Hetar Betar Retar
METLCLRASFWLALVGCVI SDNPERY STNLSNHVDDFTTFRGTE LSFLVTTHQPTNLVLP ---FW--L ........ G ....... S ...... I---SVA--H ...... V ......... A--GV--FL ......... GA-A--A .... ,---,--,---,-,---,,--,--,,-,--.-'I'IMT
:2:---G-:::::::--T--+::::::::
....
---:-
] ?(]
.......
+ _ i
......
~ ....
~ S LALGDL I YVVIDLP
INVFKL~AGRWPFDHNDFGVFLC~LFPFLQKS
1RD
SVGI TVLNLCAL
================================================ ........... 'I"M4 VDRYRAVASWSRVQGIG
1 240
IP LVT~I E IVS IWI LSF I LAI PEAI GFVM~PFEYRGEQHKTCM
1- . . . . . . . . . . . . . . . . . . . . . . .
.....................
1 ....
I-l- . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . .
K - ; , - - R - - -
-4. . . . K . . . . R - - -
~BML~
300
a,24s RREVAKI~VFCLW
I
IFALCWFP
a,w';
LHLSR ILKKTV~NEMDKNRCE m
LL ~F LLLMDY
36Q
IG INLATM
m
420 NSC INP I ALYFCKKFKNCFQSCLCCCCYQSKSLMTSVPNNGTS IQWKNHDQNNHNTDRS
::::::::::::::::::::::::::::::::::::::::::::::::::::::::::: 427 SHKDSMN . . . . .
I--
~ Alignment of deduced human, bovine and rat ETA receptor protein sequences. The seven transmembrane regions are denoted by boxes. Amino acids identical to the human sequence are denoted by a dashed line (-). The sequences of bovine and rat ETA receptor sequences are from ref 6 and 7, respectively.
molecular mass of 48,689 (Fig. 1). This protein appears to have seven t r a n s m e m b r a n e regions predicted by hydrophobicity analysis (11) and has a number of motifs found in the bovine and rat ETA receptors (6,7). There is a long N-terminal domain t h a t includes a potential signal sequence and two potential N-linked-glycosylation sites (11). A signal sequence has been detected in very few G-protein coupled receptors (14). The C-terminal domain has a group of four cysteine residues at least one of which m a y be p a l m i t o y l a t e d in a m a n n e r similar to rhodopsin a n d b e t a - a d r e n e r g i c r e c e p t o r (12,13). T h e r e are t h r e e p r e d i c t e d p r o t e i n k i n a s e C phosphlorylation sites in the C-terminus and one in the third cytoplasmic domain between transmembrane regions five and six (11). A comparison of the h u m a n , bovine and r a t ETA receptor protein sequences is shown in Fig. 2.
The h u m a n protein sequence is 94.6%
identical to the bovine sequence and 92.7% identical to the r a t sequence. The N - t e r m i n a l portion of the receptors r e p r e s e n t s the a r e a of l e a s t 187
V o l . 181, N o . 1, 1991
BIOCHEMICAL A N D BIOPHYSICAL RESEARCH COMMUNICATIONS
Human Chromosomes
Hybrid DE7 7297 10115 9142 7300 Nu9 G5 7301 10479 10449 8854 10888 734
| | iiii
lUll Bill
i|
!!
I
I
Fire 3. Presence of the ET8 gene in 13 rodent-human hybrids. A completely stippled box indicates that the hybrid named in the left column contains the chromosome indicated in the upper row; lower-right stippling indicates presence of the long arm (or part of the long arm, indicated by a smaller fraction of stippling) of the chromosome shown above the column; upperleft stippling indicates the presence of the short arm (or partial short arm) of the chromosome listed above the column; no stippling indicates the absence of the chromosome listed above the column. The column for chromosome 4 is boldly outlined and stippled to highlight correlation of presence of this chromosome (or region of chromosome) with presence of the probe. The pattern of retention of the ET8 gene in the panel is shown in the column to the right of the figure where presence of the probe in the hybrid is indicated by a stippled box with a plus sign and absence of the probe is indicated by an open box enclosing a minus sign.
homology b e t w e e n the different species, suggesting t h a t it m a y not be involved in ligand binding. The h u m a n and bovine sequences have the same n u m b e r of amino acids while t h e r a t sequence has one resi due deleted n e a r the C-terminus relative to the h u m a n and bovine sequences. More t h a n 20 r o d e n t - h u m a n hybrids were t est ed for the presence of the ET8 locus by hybridization of a radiolabelled h u m a n ET8 probe to EcoRI cleaved hybrid and control DNAs immobilized on nylon filters. Results of testing of the entire panel d e m o n s t r a t e d t h a t the endothelin receptor locus was p r e s e n t only in hybrids retaining chromosome 4 and was absent in all hybrids which did not contain chromosome 4, as sum m ari zed in Fig. 3. T he significance of t he l i nkage of t he e n d o t h e l i n A r e c e p t o r to c h r o m o s o m e 4 is di f f i cul t to g a u g e
without
a narrower
regional
localization. T h e r e are several chromosome 4-1inked disease loci such as H u n t i n g t o n ' s at 4p16, D e nt i o genesi s i m p e r f e c t a a t 4q12-23, Williams syndrome at 4q33-qter (15). Since one of our hybrids which was negative for the ETA receptor locus was positive for the craf-2 probe at 4p16 (K. Huebner, u n p u b l i s h e d ) , it is u n l i k e l y t h a t t he ETA r e c e p t o r locus is n e a r the Huntington's locus. Several other receptor genes (KIT and PDGFRA on 4q) an d gr ow t h factor genes (FGF5, EGF, FGFB, IL2) have been regionally 188
Vol. 181, No. 1, 1991
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
localized on 4q (15) and it will be of interest to determine if the endothelin A receptor is near any of these loci. In conclusion, a h u m a n endothelin ETA receptor has been cloned and mapped to h u m a n chromosome 4. The sequences of the partial clones obtained from h u m a n brain libraries suggest that the sequence of the ETA receptor expressed in the brain is identical to the ETA receptor expressed in the placenta. The h u m a n ETA receptor can be used to analyze the role of endothelin receptors in disease states and for the analysis of antagonists specific for the h u m a n endothelin ETA receptor.
A c k n o w l e d g m e n t s . We thank Vi Chu for her expert technical assistance and Millie Alvarez for secretarial assistance. We acknowledge helpful discussions with Drs. Joseph Schlessinger and S t e p h e n Felder. We acknowledge the help of Dr. Ross Smith, Dr. John Hill and Sara Kranczer for help with computer analysis. The work was supported in part by research grants from Irma T. Hirschl Charitable Trust/Monique WeillCaulier Trust (RK), Biomedical Research Support Grant (RK), NIH grants CA 21124 and CA 39860 (KH).
References
. Yanagisawa, M., Kurihara, H., Kimura, S., Tomobe, Y., Kobayashi, M., Mitsui, Y., Yazaki, Y., Gote, K. and Masaki, T. (1988) Nature 332.411415. Inoue, A.M., Yanagisawa, S., Kimura, Y., Miyauchi, T., Goto, K. and Masaki, T. (1989) Proc.Natl.Acad.Sci.USA 86.2863-2867.
.
. Wollberg, Z., Shabo-Shina, R., Intrator, N., Bdolah, A., Kochva, E., Shavit, G., Oron, Y., Vidne, B.A. and Gitter, S. (1991) Toxicon 26.525534. Sakurai, T., Yanagisawa, M., Takuwa, Y., Miyazaki, H., Kimura, S., Goto, I~ and Masaki, T. (1990) Nature 348.732-735.
.
. Ogawa, Y., Nakao, K., Arai, H., Nakagawa, O., Hosoda, K., Suga, Nakanishi, S. and Imura, H. (1991) Biochem.Biophys.Res.Commun. 178.248-255. S.,
.
Arai, H., Hori, S., Aramori, I., Ohkubo, H. and Nakanishi, S. (1990) Nature 348.730-732.
. Lin, H.Y., Kaji, E.H., Winkel, G.K., Ives, H.E. and Lodish, H.F. (1991) Proc.Natl.Acad.Sci.USA 88.3185-3189. .
Sambrook, J., Fritsh, E.F. and Maniatis, T. (1989). Moleular Cloning: A Laboratory Manual, New York:Cold Spring Harbor Press. 189
Vol. 181, No. 1, 1991
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
9. Sanger, F., Nicklen, S. and Coulson, A.R. (1977) Proc.Natl.Acad.Sci.USA 74.5463-5467. 10. Huebner, K., Druck, T., Croce, C.M. and Thiesen, H-J. (1991) Am.J.Hum.Genet. 48.726-740. 11. Devereux, J., Haeberli, P. and Smithies, O. (1984) Nucleic Acids Res. 12.387-395. 12. Yu, A., Ovchinnikov, N.G., Abdulaev, G. and Bogachuk, A.S. (1988) FEBS.Lett. 230.1-5. 13. O'Dowd, B.F., Hnatowich, M., Caron, M.G., Lefkowitz, R.J. and Bouvier, M. (1989) J.Biol.Chem. 264.7564-7569. 14. Sandou, F., Amlaiky, N., Plassat, J.L., Borrelli, E. and Hen, R. (1990) EMBO J. 9.3611-3617. 15. Cox, D.R., Murray, J.C. and Buetow, K.H. (1991) Cytogenet.Cell Genet. 51.121-136.
190
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 184-190
CLONING AND CHROMOSOMAL LOCALIZATION OF A H U M A N ENDOTHELIN ETA RECEPTOR Curt Cyr 1, Kay Huebner 2, Teresa Druck 2, and Richard Krisl, * 1Department of Pharmacology, New York University Medical Center, 550 First Avenue, New York, New York 10016 2Jefferson Cancer Institute, Thomas Jefferson University, 233 S. 10th Street, Philadelphia, Pennsylvania 19107 Received September 25, 1991
Summary A cDNA clone encoding a h u m a n endothelin receptor was isolated from a placenta cDNA library. The deduced amino acid sequence of the clone is 94% identical to the bovine endothelin ETA receptor and r e p r e s e n t s the h u m a n homologue. The h u m a n endothelin ETA receptor gene was localized to chromosome 4 by analysis of its segregation pattern in rodent-human hybrids. ®1991Aoad.... P..... Znc.
Endothelin is a twenty-one amino acid peptide originally isolated from porcine aortic endothelial cells and is a p o t e n t endogenous vasoconstrictor (1). Cloning and sequencing analysis showed that there are at least three genes encoding the endothelin family (2). One of these genes codes for endothelin 1 (ET1) and the other two genes code for peptides of the same length designated as endothelin 2 and 3 (ET2 and ET3). This family of proteins is remarkably similar to the sarafotoxins, a group of snake venoms derived from the Israeli burrowing asp (Atractaspis engaddensis) (3). The sarafotoxins are one of the most potent venoms known. Two ET receptor subtypes have recently been cloned and sequenced. The receptors are members of the rhodopsin-like superfamily of receptors that have seven putative t r a n s m e m b r a n e domains and are coupled with Gproteins for signal transduction. One ET receptor subtype was cloned from a r a t lung cDNA library and h u m a n placenta library (4,5). Displacement e x p e r i m e n t s performed on the expressed endothelin receptor revealed similar affinities for ET1, 2 a n d 3. This receptor s u b t y p e has been
* To whom correspondence should be addressed. Abbreviations u s e d in t b | . p a p e n Endothelin: ET; ETA: endothelin type ETA, 0006-291X/91 $1.50 Copyright © 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.
184
Vol. 181, No. 1, 1991
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
designated ETB. The endothelin receptor subtype cloned by Arai et al. (6) from a bovine lung cDNA library utilizing a Xenopus oocyte expression system has the highest affinity for ET1 with a Kd of 0.18 nM. This ET receptor subtype has been named ETA (6). More recently, a rat ETA receptor has been cloned from a rat heart cell line and is over 90% similar to the bovine sequence (7). The rat and bovine sequences differ most widely at the amino terminus. A cloned human endothelin receptor could make screening for specific h u m a n endothelin antagonists much easier. In addition, a human clone can be used to probe the role of endothelin receptor in disease states. Here we describe the cloning, sequencing and chromosomal localization of a human ETA receptor isolated from a human placenta cDNA library.
Materioh a n d Methods Cloning technique: A probe for the h u m a n endothelin receptor was produced using p o l y m e r a s e chain r e a c t i o n amplification. Two oligonucleotides (20 bases each) were produced from conserved regions corresponding to the bovine ETA sequence. The sense oligonucleotide was produced starting from base number 296 and the antisense was produced starting from base 830. The template DNA was cDNA produced from human placenta mRNA using a kit from Invitrogen. The resulting PCR product was 534 base pairs and was subcloned into pBluescript (Stratagene) (8). The insert (ET8) was sequenced for verification and removed from the plasmid using restriction enzymes BamHI and EcoRI. ET8 was labelled with a-32P-CTP by random priming and used to screen human brain and placenta libraries. Plaque hybridization was carried out at 55°C overnight in 6xSSPE (lxSSPE is 0.15M NaC1, 0.1M NaH2P04, 0.001M EDTA, pH 7.4) and blotto (0.2% nonfat dried milk) and the membranes washed once at room temperature with 6xSSC, 0.1% SDS and twice at 65°C with 0.1xSSC and 0.1% SDS (8). Positive clones were purified and inserts removed using EcoRI, subcloned into pBluescript and subjected to didioxy double stranded sequencing from both ends (9). Cells: Isolation, propagation and characterization of most somatic cell hybrids used in this study have been described elsewhere (10). Many of the hybrids were obtained from the NIGMS H u m a n Genetic M u t a n t Cell Repository (Coriell Institute, Camden, N.J.). Chromosome regions retained by each hybrid are diagrammed in Fig. 3. Southern Blot Analvsis: Hybrid and control DNAs were prepared by cell lysis, proteinase K digestion, phenol extraction, and ethanol precipitation. Cellular DNAs were digested with restriction enzyme EcoRI, fractionated on 0.8% agarose gels, transferred to nylon membrane and hybridized at 42°C for 16 hours in 50% formamide, 5x SSPE, 5X Denhardts solution, 0.1% SDS and 100 ug/ml salmon sperm DNA. ET8 probes (see above) for filter hybridizations were radiolabelled by nick translation with a-32P-CTP to a specific activity of 108 cpm/0.1 ug and 108 cpm were used for each filter hybridization. 185
Vol. 181, No. 1, 1991
BIOCHEMICAL AND BIOPHYSICAL RESEARCH C O M M U N I C A T I O N S
Results and Discussion 106 clones from each of three libraries (human brain stem, caudate, and placenta) were screened using a humanized P C R probe specificfor the endothelin E T A receptor (see methods). Positive clones were obtained from h u m a n brain stem, caudate, and placenta libraries. The positive clones from brain stem and caudate were not full length and repeated screenings did not produce a full length clone from these libraries (data not shown). All three clones had an internal EcoRI site and were subcloned into pBluescript for sequencing. The nucleotide sequence of the full length placenta clone is shown in figure 1. The c D N A consists of 1661 nucleotides with a coding region of 1263 nucleotides. The sequences of the partial clones were identical to the full length clone obtained from the placenta library. However, the 5' end of the caudate clone started at base 380 of the placental clone and the 5' end of the brain stem clone started at nucleotide 504 of the placental clone. The placenta c D N A sequence includes an open reading frame of 1263 bases which would encode a protein of 421 amino acids with a relative
TATAGGCGAATTGGGTA -121 CCGGGAAAAAAATCGAGGTCGACGGTAT•GGATGAAGCTTGATATCGAATTCGGGAAAAAGTGAAGGTGTAAAAGCAAGCACAAGTG•AATAAGAGATATTTCCTCAAATTTGCCTCAAG -i
MetG~uThrLeuCysLeuArgA~aSerPheTr~LeuA~aLeuVa~G~yCysVa~I~eSerAspAsnPr~G~uArgTyrSerThrAsnLeuSerAsnH~sVa~AspAspPheTh~ThrPhe 40 ATGGAAAC••TTTGCCTCAGGGCAT•CTTTTGGCTGGCACTGGTTGGATGTGTAATCAGTGATAATCCTGAGAGATACAGCACAAATCTAAGCAAT•ATGTGGATGATTTCA•CACTTTT 120 ArgG~yThrG~uLeuSerPheLeuVa~ThrThrHisG~nPr~ThrAsnLeuVa~LeuPr~SerAsnG~ySerMetHlsAsnTyrCysPr~G~nG~nThrLysI~eThrSerA~aPheLys 80 CGTGGCACAGAGCTCAGCTTCCTGGTTACCACTCATCAACCCACTAATTTGGTCCTACCCAGCAATGGCTCAATGCACAACTATTGCCCACAGCAGACTAAAATTACTTCAGCTTTCAAA 240 I T~rI~eA~nThrva~I1eSe~CYsThrI~ePheI~eVa~G~yMetVa~G~YAsnA~aThrLeuLeuAr~I~eI~eT~rG~nAsnLy~Cys~etArgAsnG~yPr~AsnA~aLeuI~eA~ 120 TACATTAACACTGTGATATCTTGTACTATTTTCATCGTGGGAATGGTGGGGAATGCAACTCTGCTCAGGATCATTTACCAGAACAAATGTATGAGGAATGGCCCCAACGCGCTGATAGCC 360 II ~erLeuA~aLeu$~As~L~uI~eTYrVa~Va~I~eAs~LeuPr~I~eAsnVa~heL~sLeuLeuA~aG~yArgTr~Pr~PheAs~HisAsnAs~PheG1yVa~PheLeuCysLysLeu 160 AGTCTTGCCCTTGGAGACCTTATCTATGTGGTCATTGATCTCCCTATCAATGTATTTAAGCTGCTGGCTGGGCGCTGGCCTTTTGATCACAATGACTTTGGCGTATTTCTTTGCAAGCTG 480 Ill PhePr~PheLeuG~nLvsSerSerVa~G~VI~eThrVa~LeuAsnLeuCvsA1aLeuSerVa~A~pArgTyrArgA~aVa~A~aSerTrp~erArgValG~nG~yI~eG~y~lePr~Leu 200 TTCCCCTTTTTGCAGAAGTCCTCGGTGGGGATCACCGTCCTCAACCTCTGCGCTCTTAGTGTTGACAGGTACAGAGCAGTTGCCTCCTGGAGTCGTGTTCAGGGAATTGGGATTCCTTTG 600 IV Va~ThrA~aI~eG~uI~eVa~SerI~eTrpI~eLeu~erPheI~eLeuA1aI~ePr~G~uA~aI~eG~yPh~Va~Me~a~Pr~PheG~uTyrArgG~yG~uG~n~isLysThrCy~Met 240 GTAACTGCCATTGAAATTGTCTCCATCTGGATCCTGTCCTTTATCCTGGCCATTCCTGAAGCGATTGGCTTCGTCATGGTACCCTTTGAATATAGGGGTGAACAGCATAAACCTGTATG 720 V LeuAsnA1aThrSerLysPheMetG~uPheTyrG1nAs~Va1LysA$pTrpTrpL~uPheG1YPheTYrPh~CYsMetPr~LeuVa1CYsThrA~aI1ePh~TyrThrLeuM~ThrCys 280 •TCAATGCCACATCAAAATTCATGGAGTTCTACCAAGATGTAAAGGACTGGTGGCT•TTCGGGTTCTATTTCTGTATGCCCTTGGTGTGCACTGCGATCTTCTACACCCTCATGACTTGT 840 # VI G~uMe~LeuAsnArgArgAsnG~ySerLeuArgI~eA~aLeuSerG~uHisLeuLysG~nArgArgG~uVa~A~aLysThrVa~phe~y~L~uVa1Val~epheA~aL~v~Trm~he 320 GAGATGTTGAACAGAAGGAATGGCAGCTTGAGAATTGCCCTCAGTGAACATCTTAAGCAGCGTCGAGAAGTGGCAAAAACAGTTTTCTGCTTGGTTGTAATTTTTGCTCTTTGCTGGTTC 960 VII Pr~LeuHisLeuSerAruI1eLeuL~sL~$ThrVa1~YrAsnG~uMetAspLysAsnArgCysG1uLeuLeuSerPh~Le~L~LeuH~AsDT~rI~eG~vI1~AsnLeuA~aThrMet 360 CCTCTTCATTTAAGCCGTATATTGAAGAAAACTGTGTATAACGAGATGGACAAGAACCGATGTGAATTACTTAGTTTCTTACTGCTCATGGATTACATCGGTATTAACTTGGCAACCATG 1080 # % ~ @ @ AsnSerCvsIleAsnPr~I~eA~aLeuT~rPheva~SerLysLysPheLysAsnCysPheG~nSerCysLeuCysCysCysCysTyrGlnSerLysSerLe~MetThrSerVa~Pr~Met 400 AATTCATGTATAAACCCCATAGCTCTGTATTTTGTGAGCAAGAAATTTAAAAATTGTTTCcAGTcATGCCTCTGCTGCTGCTGTTACCAGTCCAAAAGTCTGATGACCTCGGTCCCCATG 1200 # # AsnG•yThrSerI•eG•nTrpLysAsnHisAspG•nAsnAsnHisAsnThrAspArgSerSerHi•LysAspSerMetAsn 427 AACGGAACAAGCATCCAGTGGAAGAACCACGATCAAAACAACCACAACACAGACCGGAGCAGCCATAAGGACAGCATGAACTGACCACCCTTAGAAGCACTCCTCGGTACTCCCATAATC 1320 ~TcTCGGAGAAAAAAATCACAAGGCAACTGTC~GTCCGGGAATCTCTTCTcTGATCcTTCTTCCTTAATTCACTcCCACACcCAAGAAGAAATGCTTTCCAAAAC~GCAAGGTAGACTGG 1440 TTTATCCACCCACAACATCTACGAATCGTACTTCTTTTAATTGATCTAATTTACATATTCTGCGTGTTGTATTCAGCACTAAAA 1524
Fig. I. The nucleotide sequence and deduced peptide sequence of the human endothelin ETA receptor. The arrow denotes a potential cleavage
site of a hydrophobic signal sequence. Predicted hydrophobic transmembrane regions are underlined. Potential N-linked glycosylation
sitesare denoted by * and predictedproteinkinase C phosphorylationsites by #. Possible palmiteylation sites are marked by @. 186
V o l . 181, No. 1, 1991
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
6O Hetar Betar Retar
METLCLRASFWLALVGCVI SDNPERY STNLSNHVDDFTTFRGTE LSFLVTTHQPTNLVLP ---FW--L ........ G ....... S ...... I---SVA--H ...... V ......... A--GV--FL ......... GA-A--A .... ,---,--,---,-,---,,--,--,,-,--.-'I'IMT
:2:---G-:::::::--T--+::::::::
....
---:-
] ?(]
.......
+ _ i
......
~ ....
~ S LALGDL I YVVIDLP
INVFKL~AGRWPFDHNDFGVFLC~LFPFLQKS
1RD
SVGI TVLNLCAL
================================================ ........... 'I"M4 VDRYRAVASWSRVQGIG
1 240
IP LVT~I E IVS IWI LSF I LAI PEAI GFVM~PFEYRGEQHKTCM
1- . . . . . . . . . . . . . . . . . . . . . . .
.....................
1 ....
I-l- . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . .
K - ; , - - R - - -
-4. . . . K . . . . R - - -
~BML~
300
a,24s RREVAKI~VFCLW
I
IFALCWFP
a,w';
LHLSR ILKKTV~NEMDKNRCE m
LL ~F LLLMDY
36Q
IG INLATM
m
420 NSC INP I ALYFCKKFKNCFQSCLCCCCYQSKSLMTSVPNNGTS IQWKNHDQNNHNTDRS
::::::::::::::::::::::::::::::::::::::::::::::::::::::::::: 427 SHKDSMN . . . . .
I--
~ Alignment of deduced human, bovine and rat ETA receptor protein sequences. The seven transmembrane regions are denoted by boxes. Amino acids identical to the human sequence are denoted by a dashed line (-). The sequences of bovine and rat ETA receptor sequences are from ref 6 and 7, respectively.
molecular mass of 48,689 (Fig. 1). This protein appears to have seven t r a n s m e m b r a n e regions predicted by hydrophobicity analysis (11) and has a number of motifs found in the bovine and rat ETA receptors (6,7). There is a long N-terminal domain t h a t includes a potential signal sequence and two potential N-linked-glycosylation sites (11). A signal sequence has been detected in very few G-protein coupled receptors (14). The C-terminal domain has a group of four cysteine residues at least one of which m a y be p a l m i t o y l a t e d in a m a n n e r similar to rhodopsin a n d b e t a - a d r e n e r g i c r e c e p t o r (12,13). T h e r e are t h r e e p r e d i c t e d p r o t e i n k i n a s e C phosphlorylation sites in the C-terminus and one in the third cytoplasmic domain between transmembrane regions five and six (11). A comparison of the h u m a n , bovine and r a t ETA receptor protein sequences is shown in Fig. 2.
The h u m a n protein sequence is 94.6%
identical to the bovine sequence and 92.7% identical to the r a t sequence. The N - t e r m i n a l portion of the receptors r e p r e s e n t s the a r e a of l e a s t 187
V o l . 181, N o . 1, 1991
BIOCHEMICAL A N D BIOPHYSICAL RESEARCH COMMUNICATIONS
Human Chromosomes
Hybrid DE7 7297 10115 9142 7300 Nu9 G5 7301 10479 10449 8854 10888 734
| | iiii
lUll Bill
i|
!!
I
I
Fire 3. Presence of the ET8 gene in 13 rodent-human hybrids. A completely stippled box indicates that the hybrid named in the left column contains the chromosome indicated in the upper row; lower-right stippling indicates presence of the long arm (or part of the long arm, indicated by a smaller fraction of stippling) of the chromosome shown above the column; upperleft stippling indicates the presence of the short arm (or partial short arm) of the chromosome listed above the column; no stippling indicates the absence of the chromosome listed above the column. The column for chromosome 4 is boldly outlined and stippled to highlight correlation of presence of this chromosome (or region of chromosome) with presence of the probe. The pattern of retention of the ET8 gene in the panel is shown in the column to the right of the figure where presence of the probe in the hybrid is indicated by a stippled box with a plus sign and absence of the probe is indicated by an open box enclosing a minus sign.
homology b e t w e e n the different species, suggesting t h a t it m a y not be involved in ligand binding. The h u m a n and bovine sequences have the same n u m b e r of amino acids while t h e r a t sequence has one resi due deleted n e a r the C-terminus relative to the h u m a n and bovine sequences. More t h a n 20 r o d e n t - h u m a n hybrids were t est ed for the presence of the ET8 locus by hybridization of a radiolabelled h u m a n ET8 probe to EcoRI cleaved hybrid and control DNAs immobilized on nylon filters. Results of testing of the entire panel d e m o n s t r a t e d t h a t the endothelin receptor locus was p r e s e n t only in hybrids retaining chromosome 4 and was absent in all hybrids which did not contain chromosome 4, as sum m ari zed in Fig. 3. T he significance of t he l i nkage of t he e n d o t h e l i n A r e c e p t o r to c h r o m o s o m e 4 is di f f i cul t to g a u g e
without
a narrower
regional
localization. T h e r e are several chromosome 4-1inked disease loci such as H u n t i n g t o n ' s at 4p16, D e nt i o genesi s i m p e r f e c t a a t 4q12-23, Williams syndrome at 4q33-qter (15). Since one of our hybrids which was negative for the ETA receptor locus was positive for the craf-2 probe at 4p16 (K. Huebner, u n p u b l i s h e d ) , it is u n l i k e l y t h a t t he ETA r e c e p t o r locus is n e a r the Huntington's locus. Several other receptor genes (KIT and PDGFRA on 4q) an d gr ow t h factor genes (FGF5, EGF, FGFB, IL2) have been regionally 188
Vol. 181, No. 1, 1991
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
localized on 4q (15) and it will be of interest to determine if the endothelin A receptor is near any of these loci. In conclusion, a h u m a n endothelin ETA receptor has been cloned and mapped to h u m a n chromosome 4. The sequences of the partial clones obtained from h u m a n brain libraries suggest that the sequence of the ETA receptor expressed in the brain is identical to the ETA receptor expressed in the placenta. The h u m a n ETA receptor can be used to analyze the role of endothelin receptors in disease states and for the analysis of antagonists specific for the h u m a n endothelin ETA receptor.
A c k n o w l e d g m e n t s . We thank Vi Chu for her expert technical assistance and Millie Alvarez for secretarial assistance. We acknowledge helpful discussions with Drs. Joseph Schlessinger and S t e p h e n Felder. We acknowledge the help of Dr. Ross Smith, Dr. John Hill and Sara Kranczer for help with computer analysis. The work was supported in part by research grants from Irma T. Hirschl Charitable Trust/Monique WeillCaulier Trust (RK), Biomedical Research Support Grant (RK), NIH grants CA 21124 and CA 39860 (KH).
References
. Yanagisawa, M., Kurihara, H., Kimura, S., Tomobe, Y., Kobayashi, M., Mitsui, Y., Yazaki, Y., Gote, K. and Masaki, T. (1988) Nature 332.411415. Inoue, A.M., Yanagisawa, S., Kimura, Y., Miyauchi, T., Goto, K. and Masaki, T. (1989) Proc.Natl.Acad.Sci.USA 86.2863-2867.
.
. Wollberg, Z., Shabo-Shina, R., Intrator, N., Bdolah, A., Kochva, E., Shavit, G., Oron, Y., Vidne, B.A. and Gitter, S. (1991) Toxicon 26.525534. Sakurai, T., Yanagisawa, M., Takuwa, Y., Miyazaki, H., Kimura, S., Goto, I~ and Masaki, T. (1990) Nature 348.732-735.
.
. Ogawa, Y., Nakao, K., Arai, H., Nakagawa, O., Hosoda, K., Suga, Nakanishi, S. and Imura, H. (1991) Biochem.Biophys.Res.Commun. 178.248-255. S.,
.
Arai, H., Hori, S., Aramori, I., Ohkubo, H. and Nakanishi, S. (1990) Nature 348.730-732.
. Lin, H.Y., Kaji, E.H., Winkel, G.K., Ives, H.E. and Lodish, H.F. (1991) Proc.Natl.Acad.Sci.USA 88.3185-3189. .
Sambrook, J., Fritsh, E.F. and Maniatis, T. (1989). Moleular Cloning: A Laboratory Manual, New York:Cold Spring Harbor Press. 189
Vol. 181, No. 1, 1991
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
9. Sanger, F., Nicklen, S. and Coulson, A.R. (1977) Proc.Natl.Acad.Sci.USA 74.5463-5467. 10. Huebner, K., Druck, T., Croce, C.M. and Thiesen, H-J. (1991) Am.J.Hum.Genet. 48.726-740. 11. Devereux, J., Haeberli, P. and Smithies, O. (1984) Nucleic Acids Res. 12.387-395. 12. Yu, A., Ovchinnikov, N.G., Abdulaev, G. and Bogachuk, A.S. (1988) FEBS.Lett. 230.1-5. 13. O'Dowd, B.F., Hnatowich, M., Caron, M.G., Lefkowitz, R.J. and Bouvier, M. (1989) J.Biol.Chem. 264.7564-7569. 14. Sandou, F., Amlaiky, N., Plassat, J.L., Borrelli, E. and Hen, R. (1990) EMBO J. 9.3611-3617. 15. Cox, D.R., Murray, J.C. and Buetow, K.H. (1991) Cytogenet.Cell Genet. 51.121-136.
190
