THE OF CHEMISTRY Vol. 268, No. Issue 15, pp. 3463-3470 ... · PDF file0 1993 by The American Society for Biochemistry and Molecular Biology, ... The Human Endothelin-B Receptor Gene

THE J O U R N A L OF BIOLOGICAL CHEMISTRY 0 1993 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 268, No. 5, Issue of February 15, pp. 3463-3470, 1993 Printed in U. S. A.

The Human Endothelin-B Receptor Gene STRUCTURAL ORGANIZATION AND CHROMOSOMAL ASSIGNMENT*

(Received for publication, September 2, 1992)

Hiroshi Arai, Kazuwa NakaoS, Kazuhiko Takaya, Kiminori Hosoda, Yoshihiro Ogawa, Shigetada Nakanishi§, and Hiroo Imura From the Second Division, Department of Medicine, and §Institute for Immunology, Kyoto University School of Medicine, Kyoto 606, Japan

The gene encoding the human endothelin-B receptor (hET-BR) has been isolated, and its structural organi- zation and chromosomal assignment have been deter- mined. Southern blot analysis demonstrated a single copy of the hET-BR gene in the human genome. The hET-BR gene spans 24 kilobases and consists of seven exons and six introns. The size range for exons is 109- 2855 base pairs, although that for introns is 0.2-14.5 kilobases. Every intron occurs near the border of the putative transmembrane domain in the coding region. The major transcription initiation sites were mapped to the positions 258 and 229 base pairs upstream of the ATG initiation codon by primer extension and nu- clease S1 protection experiments. The 5”flanking re- gion of the hET-BR gene lacks conventional TATA and CCAAT boxes but contains a sequence of potential Spl binding sites upstream of the transcription initiation sites. There are some canonical consensus sequences of cis-elements including GATA motif, acute phase reac- tant regulatory element, and E box. Using human- rodent somatic hybrid cell lines, the hET-BR gene was assigned to human chromosome 13. The present study will lead to a better understanding of the mechanism for the transcriptional regulation of the hET-BR gene and will give a clue as to how to search for possible genetic disorders of hET-BR.

Multiple subtypes of endothelin (ET)’ receptor with dis- tinct ligand specificities have been defined by pharmacological and physiological studies (1). In 1990, a key step in charac-

*This work was supported in part by research grants from the Japanese Ministry of Education, Science, and Culture, the Japanese Ministry of Health and Welfare “Disorders of Adrenal Hormone” Research Committee, Japan, 1992, Japan Tabacco Inc., Yamanouchi Foundation for Research on Metabolic Disorders, Uehara Memorial Foundation, Inamori Foundation, and by research grants for cardio- vascular diseases (2A-3) from the Japanese Ministry of Health and Welfare. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in th ipaper has been submitted to the GenBankTM/EMBL Data Bunk with accession number(s)

4 To whom correspondence and reprint requests should be ad- dressed Second Division, Dept. of Medicine, Kyoto University School of Medicine, 54 Shogoin Kawahara-cho, Sakyo-ku, Kyoto 606, Japan. Tel.: 75-751-3170; Fax: 75-771-9452.

The abbreviations used are: ET, endothelin; ET-R, endothelin receptor; ET-BR, endothelin B receptor; ET-AR, endothelin A recep- tor; hET-AR, human ET-AR hET-BR, human ET-BR G protein, guanine nucleotide-binding protein; hp, base pair(s); kb, kilobase(s); APRRE, acute phase reactant regulatory element.

013162-013168.

terizing the molecular makeup of these receptors was achieved by two groups (2, 3) including ours (2). We succeeded in cloning the cDNA encoding an ET-1 selective E T receptor, designated ET-A receptor (ET-AR), from a bovine lung cDNA library using Xenopus oocyte expression system (2). Sakurai et al. (3) reported the cDNA cloning of a non-isopeptide- selective E T receptor, named ET-B receptor (ET-BR), from a rat lung cDNA library using the COS-7 cell. Both subtypes of ET-R have seven transmembrane domains, a common structure of the G protein-coupled receptor superfamily (4). Subsequently, to assess the clinical implication of ET family, we have isolated the cDNAs for human ET-AR (hET-AR) and human ET-BR (hET-BR) from a human placenta cDNA library (5,6). Human ET-AR and hET-BR consist of 427 and 442 amino acids, respectively, and the overall identity between the two proteins is 55%. The transmembrane domains and the cytoplasmic loops are highly conserved between hET-AR and hET-BR, but the N-terminal ertracellular domain ex- hibits a divergence in length (80 resic‘ues for hET-AR uersus 101 residues for hET-BR) and in amino acid sequence. The hET-AR mRNA distributes at the highest level in the aorta and at high levels in the lung, atrium, colon, and placenta (5). On the other hand, the hET-BR mRNA is expressed at the highest levels in the cerebral cortex and cerebellum, and at high levels in the placenta, lung, kidney, adrenal, colon, and duodenum (6). In the vascular wall, hET-AR is expressed mainly in smooth muscle cells, although hET-BR distributes in endothelial cells (5, 6).

To elucidate the transcriptional regulation of the ET-R gene family and to analyze possible genetic disorders of the ET-R family, we have tried to isolate and analyze the genes encoding the human ET-R family and have already reported on the hET-AR gene (7). The hET-AR gene consists of eight exons and seven introns spanning more than 40 kilobases (kb) and is assigned to human chromosome 4 ( 7 ) .

In the present study, we report the structural organization and chromosomal assignment of the hET-BR gene and com- pare them with those of the hET-AR gene and other G protein-coupled receptor genes.

MATERIALS AND METHODS

Isolation and Characterization of the hET-BR Gene-Two human genomic DNA libraries were screened in this study, a human placenta library in the cloning vector AEMBL3 SP6/T7 (Clontech Laborato- ries, Inc.) and a human leukocyte library in ADASH (8). Approxi- mately 3 X lo6 phage clones were screened as previously reported (7) with various restriction fragments of the hET-BR cDNA labeled with ”P by the random priming method (9). Positive clones were purified and characterized by restriction endonuclease mapping and Southern blot analysis. DNA sequencing of specific restriction fragments sub- cloned into pBluescript I1 vector (Stratagene) was performed by the dideoxy chain termination method using Sequenase version 2.0

3463

3464 Human Endothelin-B Receptor Gene (U. S. Biochemical Corp.). In addition to T3 and T7 primers, se- quence-specific oligonucleotides were synthesized with DNA synthe- sizer 381 A (Applied Biosystems, Inc.). Both strands of DNA were sequenced, and overlapping sequences from parental clones were obtained for all restriction sites used in subcloning. Nucleotides are numbered sequentially from the first nucleotide of the ATG initiation codon.

Polymerase Chain Reaction-Polymerase chain reaction was used to determine the sizes of introns and to generate the probes for Southern blot analysis (IO). They were carried out using the Gbne AMP DNA amplification reagent kit and Taq polymerase (Perkin-

cycle profile of 30 s at 94 “C, 30 s at 55 “C, and 1 min at 72 “C. Elmer Cetus Instruments). The reaction was cycled 30 times in a

Amplified DNA fragments were analyzed by agarose gel electropho- resis.

Primer Extension Analysis-Total RNA was extracted from the human cerebral cortex, liver, and placenta obtained at autopsy and childbirth, respectively. Informed consent was obtained from each family. This study was approved by the Ethical Committee on Human Research of Kyoto University (61-98). A synthetic oligonucleotide corresponding to nucleotides -136 to -165 in the 5”flanking region of the hET-BR gene was end-labeled with [T-~~PIATP, 3 X IO5 cpm of which was hybridized to 50 pg of total RNA for 4 h a t 42 “C. Primer extension analysis was carried out essentially according to the stand- ard method (7,11) using Superscript Moloney murine leukemia virus reverse transcriptase (Bethesda Research Laboratories).

Nuclease SI Protection Analysis-To prepare an antisense DNA probe for nuclease SI mapping, a synthetic oligonucleotide (-136 to -165) was end-labeled with [y3’P]ATP and hybridized to the ge- nomic clone XhETBR l (Fig. l). The probe was extended with the Klenow fragment of DNA polymerase. The DNA was then digested with restriction endonuclease SmaI, and the single-stranded probe (-136 to -875) was isolated by electrophoresis on a 1.2% alkaline- agarose gel. Total RNA (50 fig) was co-precipitated in ethanol with 1 X IO6 cpm of the end-labeled, single-stranded probe. Nuclease SI protection analysis was performed as described (11).

Genomic Southern Blot Analysis-Ten pg of human genomic DNA prepared from leukocytes were digested with restriction endonucle- ases, EcoRI, BglII, and BamHI, electrophoresed on a 0.7% agarose gel, and blotted to Genescreen Plus (Du Pont). The membrane was hybridized with the 32P-labeled hET-BR cDNA probe (+1642 to +2909) as previously described (9) and washed in 0.1 X SSC (1 X SSC is 0.15 M NaC1, 15 mM sodium citrate, pH 7.0), 0.1% SDS at 50 “C.

Chromosomal Assignment-EcoRI-digested DNA from 24 human- rodent somatic hybrid cell lines (Coriell Institute for Medical Re- search) and parental cell lines were analyzed by Southern blot hy- bridization method (7). The 32P-labeled hET-BR cDNA fragment (+599 to +2909) was used as a probe. Scoring was determined by the presence (+) or absence (-) of the human band in the hybrids on the blots.

RESULTS

Isolation and Characterization of the hET-BR Genomic Clones-Overall 3 x lo6 recombinants from two genomic DNA libraries were screened with various 32P-labeled hET-BR cDNA probes (6). Twelve positive clones were isolated, and three overlapping clones (XhETBR 1, XhETBR 2, and XhETBR 3) were used to construct a map of the hET-BR gene with restriction endonucleases EcoRI and BamHI (Fig. 1). A genomic clone XhETBR 1 was from the human leukocyte genomic library, and XhETBR 2 and XhETBR 3 were from the human placenta genomic library.

Exon-Zntron Structure of the hET-BR Gene-The nucleo- tide sequences of the exons, the exon-intron junctions, the 5’- flanking region, and the 3”flanking region were determined (Fig. 2). The positions of the exon-intron splice sites were deduced by comparing the nucleotide sequence of the hET- BR gene with that of the hET-BR cDNA, pHETBR11, which we cloned subsequent to our previous report (6). As shown in Fig. 1, the hET-BR gene spans 24 kb and consists of seven exons and six introns. The sizes of exon 1-7 are 741,113,205, 150, 134, 109, and 2855 base pairs (bp), respectively, while

1 23 156 1 - hET-BR GENE

hET-BR cDNA

FIG. 1. Schematic representation of the hET-BR gene. The gene locus is represented by a solid bar, and exons are shown as black rectangles. Isolated genomic clones (XhETBR 1, XhETBR 2, and XhETBR 3) are shown at the top along with the restriction sites for EcoRI and BamHI. The structure of the hET-BR cDNA is presented at the bottom. Encoded transmembrane domains (TM I-VU) and other domains are displayed as hatched boxes and open boxes, respec- tively. Exons in the genomic.DNA and their corresponding regions of the cDNA are connected by solid lines.

those of intron 1-6 are approximately 14.5, 0.2, 1.8, 0.4, 0.7, and 1.4 kb, respectively. Each exon encodes several structural units as follows: exon 1, the entire 5”noncoding region and the coding region through the second transmembrane domain; exon 2, the first extracellular loop and the third transmem- brane domain; exon 3, the second cytoplasmic loop, the fourth transmembrane domain, and the second extracellular loop; exon 4, the fifth transmembrane domain and the third cyto- plasmic loop; exon 5, the sixth transmembrane domain and the third extracellular loop; exon 6, the seventh transmem- brane domain; exon 7, the cytoplasmic carboxyl tail and the entire 3”noncoding region. Thus, every intron occurs near the border of the putative transmembrane domain. All splice sites conform to the GT-AG rule (12).

The 5’-Flanking. Region of the hET-BR Gene-The nucleo- tide sequence of the 1259-bp region upstream from the ATG initiation codon was determined (Figs. 2 and 3). As illustrated in Fig. 3, there is a sequence CCGCCC, consensus for Spl binding (13), at the position -301 to -306. Neither a “TATA box” (14) nor a “CCAAT box” (15) exists in the 5”flanking region.

The sites of transcription initiation were determined by primer extension and nuclease S1 protection experiments using the total RNA from the cerebral cortex, placenta, and liver (Fig. 4). Multiple positive bands were detected in both experiments, and the common bands between the two exper- iments were determined as specific ones. The nucleotide cor- responding to the most upstream of each cluster of specific bands was determined as a transcription initiation site. The rank order in the density of the specific bands was compatible with that in the tissue concentration of the hET-BR mRNA (6). The yeast tRNA, analyzed as a negative control, yielded no positive bands. The residual bands may be caused by premature termination of reverse transcription, nonspecific hybridization of the primer, incomplete digestion by nuclease S1, and degradation of mRNA. We have confirmed two major initiation sites a t adenine and guanine residues (positions -258 and -229), which are 43 and 72 bp, respectively, down- stream of the Spl binding site (Fig. 3). Another potentially minor initiation site occurred a t position -283.

A search of the 5”flanking region for canonical consensus sequences revealed the presence of a GATA motif (16) a t -1249 to -1244, a hexanucleotide sequence CTGGGA at -348 to -343, which is “the acute phase reactant regulatory element (APRRE)” (17), and inverted APRRE at -914 to -909. There are four hexanucleotide sequences of CANNTG, “E box” (181, at -1224 to -1219, -894 to -889, -847 to -842, and -250 to

Human Endothelin-B Receptor Gene 3465

L.US. T T O ~ D ~ ~ ..o...oO.............................................. I n l r o n i ( 0 , 7 K b ) ...................................... 11101

2 4 0 7 2 0

2 8 0 8 4 0

320 980

IO10 180

I200 4 0 0

I 4 4 0 1560 1180 1800

2 0 4 0 1 ¶ 2 0

21 10

2 4 0 0 2280

2520 2840

2880 2110

3 0 0 0 3 1 2 0 3240 3380 3 4 8 0 3100 3 7 2 0 3 8 4 0 3910 4 0 4 9

FIG. 2. The nucleotide sequence of the hET-BR gene. The exon sequences are shown in upper case letters. The intron and the flanking sequences are in lower cme letters. Nucleotides of the transcript are numbered sequentially from the first nucleotide of the ATG initiation codon. ATTTA motifs are underlined, and polyadenylation signals are doubly underlined. Polyadenylation sites of two short cDNA clones (6) are indicated by arrows. GT clusters downstream of the polyadenylation site are enclosed by boxes. The deduced amino acid sequence is shown ubooe the nucleotide sequence. Amino acids are numbered sequentially from the initial methionine. Positions of the putative transmembrane domains I-VI1 are indicated as solid lines above the sequence.

3466 Human Endothelin-B Receptor Gene

5 ' A C A T G G T G C G ~ C T T G C C C T T G A T T T G G G T T ~ A T T ~ A A G A G C G T A G A A C T C T A A OATA Ebmc

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C T A C T A C T G A T G C T G T C C A G G C A T C G C C C A A G G G G A A A G G T T G C A G C G G G G T C G G A A G G C

G C G G G A G G A G T C T G G C G G T G A T T G A T G G G A A G G G A T G A A T G A A T A A A A G T A C T T G T C T G A

T G G C A G C A G A G A C C C C G A G C A A A C G G T G G A G G C T A C A C T G T C T G G C A T T C T C G C A G C G T T

T C G T C A G A G C C G G A C C C G C C T G C A G C T C A A G G G A G G C G T G C T C C T C I T C C C lnvwbd

A Q ~ A G C A G G C T

G G A A C C [ ~ A G C T ~ J ~ G T T C C G C C T C C C G G G A A G G T G G T C T C C A T T C G T C G C T CTG-G Ebmc Ebm

T T T G T C A G A T C C G A G A G G T A A A C A T T C G G G C T T G G T G T T G A A T T A A A A T C A T T G A T T G A A

C C T T A T T C T G G G G C T T C G G T T T G G C T T A C T A G T T T G G G A T T T T A A A A A A A T A A A A A T T A A

G C C T A T A G A G A G G G C A A A T T A A A A T T A G G T T G G G T A A A G G A A G G A G C G C G A G T G T T T G A A

G C C G T T T G G A G G G A A C A G C G G T T T C C A A G T T C C T G C T G A C T T G A G A A G T C T C T G C G G G T T

T C C G A A T C T C C G G C G C A C T C C T G G G C G C G C T G C G G G A G C T G T A G C T C A G C C A G C C A G G G A

G T A G C G G C T T T C A T C C G C C G G G A G G A G T C T T T C G A G T T C A A T C G C G G G G T A T A G A G G T T C

C C C T G C G G G G C A A A A T G C A G A G C T T G A C A C A A G C C C T T G G C C T C T A G G T G C C T T A A T T C C

G C G G T T C C C A C G C A C G C T T A A C T A A G A C G T G T C T G T A T T C C T C C C G T T A C G T G A A A G A G T APm?

lnvrrbdSP1 T C G G A G C T T T GC-ICC C C C A T C A T T C C C T C C C T G G C A C A C C C C T T C C A G A A C G C C ~

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0 v v O a 0 0 0 was. G G C A T C A G G A A G G A G T T T C G A C C C G C G C T G G C G A G T C A T G A G C G C C A A G T T T C C C A C T G G

C G C G C A A A C T T G A G T T A C T T T T G A G C G T G G A T A C T G G C G A A G A G G C T G C G G G C G G T A T T A aaaaoo

G C G T T T G C A G C G A C T T G G C T C G G G C A G C T G A C C C A A G T G T C C T O T C T T C C T T C C T C T G C T

T G T C T C T A G G C T C T G A A A C T G C G G A G C G G C C A C C G G A C G C C T T C T G G A G C A G G T A G C A G C

- I 2 0 1

- 1 1 4 1

- I 0 8 I

- 1 0 2 1

- 9 6 I

- 9 0 1

- 8 4 1

- 7 8 1

- 1 2 1

- 6 6 1

- 6 0 1

- 5 4 I

- 4 8 I

- 4 2 1

- 3 6 I

- 3 0 I

- 2 4 1

- I 8 1

- 1 2 1

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FIG. 3. Structure of the 5"flanking region of the hET-BR gene. The first nucleotide of the ATG initiation codon is designated +1. The B'-end of the fragments generated by primer extension and nuclease S1 digestion are indicated by closed and open circles below the sequence, respectively. The common bands between the two experiments were determined as specific ones, and the nucleotide corresponding to the most upstream of each cluster of specific bands was determined as a transcription initiation site. The major transcription initiation sites and 5'-end of the cDNA pHETBRll are indicated by inverted closed triangles and an inverted open triangle aboue the sequence, respectively. A GATA motif, E boxes, APRRE, inverted APRRE, and a potential Spl binding site are enclosed by boxes. Twelve-bp repeated sequences are underlined, and a 18-bp palindromic sequence is doubly underlined.

-245, one of which is the core of the 18-bp palindromic sequence GGAACCCAGCTGGGTTCC a t -900 to -883. The DNA sequence search has revealed many repeated sequences in the 5"flanking region. The longest one is 12 bp long (CGGGAGGAGTCT) and is located at -1079 to -1068 and -522 to -511.

The 3"Flanking Region of the hET-BR Gene-As we pre- viously reported (6), Northern blot analysis identified two major mRNA species (4.3 and 1.7 kb in size) and a minor one (2.7 kb in size). We isolated three groups of cDNA clones, whose polyadenylation sites were roughly compatible with each size of the hET-BR mRNAs. As shown in Fig. 2, the 3'- noncoding region of the hET-BR gene contains multiple potential polyadenylation signals (AATAAA or ATTAAA), two of which are 32 and 29 bp upstream of the polyadenylation sites of the two major mRNA species. The presumed polyad- enylation site of the longest cDNA clone is followed by two GT clusters (TGTGTGTTT and TTGTTGTG) that have been conserved in the areas for transcription termination and 3"processing (19). A number of ATTTA motifs implicated in mRNA instability (20) exist in the 3"noncoding region as shown in Fig. 2.

Genomic Southern Blot Analysis of the hET-BR Gene-The copy number of the hET-BR gene in the human genome was determined by Southern blot hybridization analysis (Fig. 5). The hET-BR cDNA fragment containing a part of exon 7 (+1642 to +2909) was used to probe the human genomic DNA

digested with three restriction endonucleases, EcoRI, BglII, and BamHI. Each enzyme generated a single fragment that hybridized to the probe, indicating that the hET-BR locus exists as a single copy in the human genome. The length of the hybridized fragments, 6.5 kb for EcoRI and 25 kb for BamHI, was consistent with the restriction map of the hET- BR gene (Fig. 1).

Chromosomal Assignment of the hET-BR Gene-To deter- mine the chromosomal assignment of the hET-BR gene, DNA isolated from human-rodent somatic hybrid cell lines was examined for the presence or absence of the hET-BR gene by Southern blot techniques. EcoRI-digested DNA from 24 hy- brids were probed with a 32P-labeled hET-BR cDNA fragment (+599 to +2909). One hybrid (GM/NA10898) yielded positive bands with sizes of 6.5 and 12 kb, which were consistent with the length of EcoRI fragments of the hET-BR gene. No bands were detected in the lane of the rodent parental cell DNA. Thus, the hET-BR gene segregated with human chromosome 13 (Table I).

DISCUSSION

In the present study, we have cloned the gene encoding bET-BR and clarified its structural organization and chro- mosomal assignment. The hET-BR gene spans 24 kb and consists of seven exons divided by six introns, while the hET- AR gene spans more than 40 kb and contains eight exons and seven introns (7) (Figs. 6 and 7). The sizes of the ET-R genes

Human Endothelin-B Receptor Gene 3467

FIG. 4. Mapping the sites of transcription initiation by the primer extension ( A ) and nuclease S 1 protection ( B ) analyses. A, a synthetic primer (-136 to -165) was end-labeled and hybridized to 50 pg of total RNA from the human placenta (lane I ) , cerebral cortex (lane2),liver (laneS),andyeasttRNA (lane4).The hybridized primer/RNA was extended with reverse transcriptase and was sepa- rated on an 8 M urea/5% polyacrylamide gel. B, a synthetic primer (-136 to -165) was end-labeled and hybridized to the genomic clone XhETBR 1. The probe was extended with the Klenow fragment of DNA polymerase and digested with restriction endonuclease SrnaI. A 739-nucleotide single-stranded probe was isolated by alkaline-agarose gel electrophoresis. The probe was hybridized to 50 pg of total RNA from the human placenta (lane 1 ), cerebral cortex (lane 2), liver (lane 3), and yeast tRNA (lune 4 ) . The DNA/RNA hybrids were digested with nuclease S1 and analyzed on an 8 M urea/5% polyacrylamide gel. The probe was also electrophoresed (lane 5). Marker lanes C, T, A, and G indicate sequencing ladders of the hET-BR gene using the same primer (-136 to -165). Positions of the positive bands are indicated by solid arrows. The open arrow demonstrates the 5'-end of the cDNA clone, pHETBR11. The position of the putative Spl binding sequence is indicated on the left.

23 kb 9.4 kb

6.6kb

4.4 kb

2.0kb

FIG. 5. Southern blot hybridization analysis of the human genomic DNA with the hET-BR cDNA probe. Samples of human genomic DNA (10 pg) digested with restriction endonucleases EcoRI, BglII, and BarnHI were electrophoresed, blotted, and hybridized with the "P-labeled hET-BR cDNA probe (+1642 to +2909).

are consistent with those of other G protein-coupled receptor genes with introns that range from 3.5 kb (human blue opsin gene) (21) to more than 75 kb (rat-luteinizing hormone recep- tor gene) (22).

The hET-BR gene lacks an intron corresponding to the

intron 1 of the hET-AR gene that is located in the 5'- noncoding region. The intron 1-6 of the hET-BR gene is located a t equivalent positions to the intron 2-7 of the hET- AR gene. Furthermore, the intron phase (23) of every exon- intron splice site is completely conserved. Thus, the structural organization of the hET-BR gene is essentially similar to that of the hET-AR gene except for the absence of one intron in the 5"noncoding region. These findings indicate that the genes for hET-RR and hET-AR originated from the same ancestral intron-containing gene.

All introns in the coding region of the ET-R gene family occur near the border of the putative transmembrane domains as shown in Fig. 7. This finding suggests that each exon encodes a potentially functional unit of the receptor as pre- viously reported (24).

Many G protein-coupled receptors, like @-adrenergic recep- tor (25), lack introns in the coding region, but a number of exceptions are known today. As summarized in Fig. 6, the genes for opsins (21), dopamine receptors (Dr receptor, Da receptor, and Dq receptor) (26-29), tachykinin receptors (sub- stance K receptor, substance P receptor, and neuromedin K receptor) (8, 30, 31), and luteinizing hormone receptor (22) have been reported to contain introns. The gene for hET-BR, together with the hET-AR gene (7), belongs to the latter group. The number and the position of introns are not always conserved among the members of a given receptor gene family (21, 26-29). As for the opsin gene family (21) (Fig. 7), all members contain four introns a t equivalent positions but two of them, the green and red opsin genes, have an additional intron in the N-terminal extracellular domain (21). This finding is similar to that in the case of the ET-R family. Another interesting finding on the exon-intron structure is that the genes for ET-BR, ET-AR, tachykinin receptors, Dr receptor, and Dq receptor share an intron at the same position immediately after the third transmembrane domain. The intron phase (23) and the nucleotide sequence around the splice site are also highly conserved. These observations sug- gest that these genes originate from a common ancestral gene.

The 5"flanking region of the hET-BR gene lacks typical TATA box and CCAAT box in close proximity to the tran- scription initiation sites, but it contains a potential site for Sp l binding (Fig. 3). Downstream of the Spl binding &e, there are two major transcription initiation sites determined by primer extension and nuclease S1 protection experiments a t positions -258 and -229 (Fig. 4). Existence of multiple transcription initiation sites also has been reported on other genes lacking TATA and CCAAT boxes (32). The 5'-end of the cDNA clone pHETBRll was at the nucleotide -226, consistent with the position of the latter transcription initia- tion site. There are many consensus sequences for cis-ele- ments, a GATA motif, APRREs, and E boxes in the 5'- flanking region of the hET-BR gene. The GATA motif is recognized by DNA-binding proteins and is required for the expression of some genes in erythroid and non-erythroid cells (16). A nuclear factor with binding specificity for the GATA motif is reported to be necessary for efficient and cell-specific expression of the human prepro-ET-1 gene (33). APRRE is thought to mediate the induction of the mRNA under acute physical stress in uiuo (17). The E boxes interact with mem- bers of a family of transcription factors with common dimer- ization and DNA binding motifs called the basic helix-loop- helix family including MyoD (18). The 18-bp palindromic sequence GGAACCCAGCTGGGTTCC at -900 to -883, which has a core sequence CAGCTG that is an E box, may have relevance to transcription factor binding. The 12-bp- repeated sequence at -1079 to -1068 and -522 to -511 may

3468 Human Endothelin-B Receptor Gene

Human Endothelin-B Receptor Gene 3469

-1zw -1wo -8w -600 -400 -2w + 1 * ATG

I hn)

hET-AR GENE

FIG. 6. Structural organization of the hET-BR gene and the hET-AR gene. The gene locus is represented by a solid bar, and exons are shown as black rectangles at the top (the hET-BR gene) and the bottom (the hET-AR gene) (7). The structure of the hET-BR cDNA is represented in the middle panel. Transmembrane domains ( T M I -VII ) and other domains are indicated as hatched boxes and open boxes, respectively. Exons in the genomic DNA and their cor- responding regions of the cDNA are connected by solid lines.

ET-BR

ET-AR

T M I n m r v v V I W

1 2 3 4 5 6 1 v v 1 1 1

1 2 3 4 5 6 7 v q 1 1 v r 1

1 2 3 4 1 v 1 1 Tachykinin Rs

1 2 3 4 567 -~ D2R

D3R

D4R

Rhodopsin, Blue Op.

Green OP. Red Op.

LHR

~. I I 1 7 wr

1 2 3 4 5 1 1 1 1 v 1 2 34 1 1

1 2 3 4 1 1 1 1

1 2 3 4 5 1 1 v 1 v

1-10

FIG. 7. Exon-intron splice sites of G protein-coupled recep- tor genes. The location of exon-intron splice sites are compared among the genes for hET-BR, hET-AR (71, tachykinin receptors (Tachykinin Rs) (8, 27, 28), D2 dopamine receptor (DZR) (24), D3 dopamine receptor (D3R) (25), D, dopamine receptor (D4R) (261, rhodopsin (211, blue opsin (Blue Op.) (21), green opsin (Green Op.)

(LHR) (22). The upper panel depicts the cDNA for hET-BR sche- (21), red opsin (Red Op.) (21), and luteinizing hormone receptor

matically. The transmembrane domains ( 7 " I-VII) are represented by hatched boxes. Other parts of the coding region are indicated by open boxes. The cDNAs for G protein-coupled receptors are repre- sented by solid lines in the lower panel. The exon-intron splice sites are indicated by arrowheads.

also have significance in transcriptional regulation. Further study will be required to clarify that these elements are responsible for the transcriptional regulation of the hET-BR gene.

Fig. 8 shows the comparison of the consensus sequences in the 5"flanking region between the hET-BR gene and hET- AR gene (7). Both ET-R genes have a Sp l binding site and lack conventional TATA and CCAAT boxes upstream of the site(s) for transcription initiation. GATA motif, APRRE, and E box are also common to the ET-R genes. As mentioned above, the tissue distribution of the hET-BR mRNA is ap- parently different from that of the hET-AR mRNA, but the molecular mechanism for the tissue-specific expression of the ET-R gene family awaits further clarification.

There are two reports besides ours on the nucleotide se- quence of the hET-BR cDNA (34,35). One reported a nucleo- tide sequence identical to ours (34); however, the other re- vealed two different nucleotides in the coding region at posi- tions +29 and +30 (CG instead of GC), which result in the

FIG. 8. Comparison of 5"flanking regions of ET receptor genes. The region upstream of the ATG initiation codon in the hET- BR gene (upper) and that in the hET-AR gene (lower) (7) are represented by a solid line. The first base of the ATG initiation codon is designated +l. Positions of the consensus sequences for cis-ele- ments, potential Spl binding sites ( S P I ) , GATA motifs (GATA), APRRE, E boxes, and a CArG box (CArG) are demonstrated. One of the E boxes in the hET-AR gene is a consensus sequence for the MyoD-E2A binding site (MyoD-E2A). Major transcription initiation sites are indicated by inverted closed triangles.

replacement of Arg'" by Pro'' compared with ours (35). The nucleotide sequence at positions +29 and +30 of the hET-BR gene elucidated in the present study is GC, which is identical to that of the hET-BR cDNA in our previous report (6).

As for the 5"noncoding region of the hET-BR cDNA, there also have been two different sequences reported. The nucleo- tide sequence from -53 to -237 of our hET-BR cDNA clones (pHETBR31, pHETBR1, pHETBR20, and pHETBR34) (6) did not exist in the hET-BR gene, but that of the cDNA cloned by Sakamoto et al. (35) (-53 to -230) and pHETBRl1 that we subsequently isolated (-53 to -226) were present at the equivalent region of the hET-BR gene. To determine whether it is because of an alternative splicing or an aberrant cDNA synthesis, we examined Northern blot analysis on human placenta RNA from different sources and found that the region -53 to -237 of pHETBR31 is not transcribed. In addition, the DNA homology search disclosed a remarkably high homology (86%) between the region -53 to -237 of pHETBR31 and a part of the human endogenous retroviral DNA clone 4-1 (36). I t is concluded, therefore, that an endog- enous retroviral DNA fragment was integrated by a gene rearrangement into the ET-BR gene locus of the human placenta tissue from which we constructed the cDNA library (6).

Southern blot analysis on the DNA of human-rodent hybrid cells demonstrates that the hET-BR gene is assigned to human chromosome 13 (Table I). Because the hET-AR gene is assigned to chromosome 4 (71, the ET-R genes are mapped to different chromosomes, as is the case with the opsin genes (21), dopamine receptor genes (29), and a,-adrenergic receptor genes (37). Further analyses on the subchromosomal localiza- tion of the ET-R gene family may enable us to identify the closely linked genes or genetic disorders.

Knowledge of the structural organization and chromosomal assignment of the hET-BR gene elucidated in the present study, together with those of the hET-AR gene (7), will lead to a better understanding of the mechanism for the transcrip- tional regulation of the ET-R gene family and will give a clue in the search for possible genetic disorders of the ET-R family.

Acknowledgments-We thank Prof. Ryuichiro Kageyama for help- ful discussion, and Chisako Yamamoto, Hisayo Kito, and Ayumi Takakoshi for excellent secretarial work.

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