©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Structure of the Rat V1a Vasopressin Receptor Gene and Characterization of Its Promoter Region and Complete cDNA Sequence of the 3`-End (*)

(Received for publication, April 10, 1995; and in revised form, June 20, 1995)

Satoshi Murasawa Hiroaki Matsubara (§) Kazuhisa Kijima Katsuya Maruyama Yasukiyo Mori Mitsuo Inada

From the Second Department of Internal Medicine, Kansai Medical University, Fumizonocho 10-15, Moriguchi, Osaka 570, Japan

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The gene encoding the rat V1a arginine vasopressin (AVP) receptor was isolated, and its structural organization and 5`-flanking region were characterized. In addition, the complete cDNA sequence of the major transcript of the rat V1a receptor gene was determined. Southern blots demonstrated a single copy of the V1a receptor gene in the rat genome, spanning a region of 3.8 kilobases (kb) and consisting of two exons and one intron (1.8 kb). The location of the intron was unique among G protein-coupled receptor genes in that the first exon encodes six of the seven transmembrane regions, the seventh region being encoded by the second exon. Primer extension, RNase protection, and rapid amplification of the 5`-end of the cDNA identified three transcriptional initiation sites (-405, -243, and -237), the major transcription initiation sites being mapped to positions -243 and -237 base pairs (bp) upstream of the ATG initiation codon (+1 bp). This portion of the 5`-flanking region has neither a TATA nor a CCAAT box, is GC-rich but has no GC box motif, and has features of promoters seen in housekeeping genes. Chimeras containing 2.2 kb of the 5`-flanking region and deletion analyses using the chloramphenicol acetyltransferase gene indicated that a ``minimal'' region, exhibiting promoter activity and tissue specificity, is located between nucleotides -296 and -221, when transfected into vascular smooth muscle cells. Gel mobility shift assay and Southwestern blotting suggested that 30- and 28-kDa nuclear proteins specifically bind to this region. Rapid amplification of the 3`-end of the cDNA showed that the major transcript terminates 442 bp downstream of the stop codon, in agreement with the mRNA size (2.1 kb). This study demonstrated a distinctive feature in the structural organization of the AVP-oxytocin receptor family genes, and characterization of the 5`-flanking region reported here will lead to a better understanding of the mechanism of transcriptional regulation of the rat V1a AVP receptor gene.


INTRODUCTION

Arginine vasopressin (AVP) (^1)has diverse actions, including the contraction of smooth muscle, stimulation of liver glycogenolysis, modulation of adrenocorticotropic hormone release from the pituitary, and the inhibition of diuresis(1) . These physiological effects are mediated through binding to specific membrane receptors of target cells. AVP receptors are coupled to G proteins and have been divided into at least three types, V1a, V1b, and V2. The V1a (vascular/hepatic) and V1b (anterior pituitary) receptors act through phosphatidylinositol hydrolysis to mobilize intracellular Ca(2) , while the V1a receptor mediates physiological effects such as cell contraction and proliferation, platelet aggregation, coagulation factor release, and glycogenolysis. The V1b receptor is expressed in the anterior pituitary where it stimulates adrenocorticotropic hormone release. V2 receptors are found mainly in the kidney and are linked to adenylate cyclase and the production of cyclic adenosine monophosphate associated with antidiuresis(3) .

Morel et al.(4) recently reported the cloning of a cDNA encoding the rat V1a receptor; a 1354-nucleotide cDNA encodes a 394-amino-acid protein with seven membrane-spanning domains similar to bacterial rhodopsin and other G protein-coupled receptors. Moreover, Lolait et al.(5) and Birnbaumer et al.(6) described the molecular cloning of human and rat V2 receptors, respectively. Simultaneously, Kimura et al.(7) reported the structure of a cDNA encoding the human oxytocin receptor and showed that its amino acid sequence has a high degree of similarity to those of V1 and V2 receptors. Thibonnier et al.(8) demonstrated that the cDNA for human V1a receptor has 72% amino acid sequence identity to the rat V1a receptor. These findings suggested that the AVP-oxytocin receptors belong to a subgroup of the family of G protein-linked receptors. With respect to genomic cloning of AVP-oxytocin receptor family genes, Seibold et al.(9) have described the structure of the gene encoding the human V2 receptor, which showed two very small intervening sequences in the coding region. The sizes and location of these introns are unique among the G protein-coupled receptor genes in that the second exon encodes only six of the seven transmembrane regions, the seventh such region being encoded by exon 3. Very recently, Rosen et al.(10) reported that the rat oxytocin receptor gene contains two introns, the second of which interrupts the coding region between the sixth and seventh transmembrane domains. However, the structures of the V1 type receptor genes and analyses of the promoter regions of these genes remain to be determined. In addition, the cDNA sequence of the rat V1a receptor reported by Morel et al.(4) lacked a 3`-noncoding region. In the present report, we describe the structural organization of the rat V1a receptor gene, the complete cDNA sequence, and also the DNA sequence of the promoter region. The V1a receptor gene spans 3.8 kb and consists of two exons and one intron. The 3`-end of the cDNA showed that the major transcript terminates 442 bp downstream of the stop codon, in agreement with the reported mRNA size. The promoter region has features common to promoters of housekeeping genes, and the promoter activity is specifically expressed in vascular smooth muscle cells, but not in NIH3T3 or skeletal muscle cells. We characterized the minimal region from nucleotides -296 and -222 which confers promoter activity and tissue specificity on the V1a receptor gene and found that 30- and 28-kDa nuclear proteins bind to this region.


EXPERIMENTAL PROCEDURES

Library Screening and DNA Sequencing

Methods of restriction analysis, screening recombinant genomic libraries, Southern blot transfer, hybridization to DNA on filters, subcloning into plasmid vectors, agarose, and polyacrylamide electrophoresis were performed as described(11) . A Lambda DASH II rat genomic library prepared from Sprague-Dawley male testes (Stratagene) was screened using a rat V1a receptor cDNA fragment corresponding to nucleotides -91 to +1343 of a V1a receptor cDNA clone (4) relative to the ATG initiation codon as a probe. The cDNA fragment was obtained by reverse transcriptase and polymerase chain reaction (PCR) as follows. Adult rat liver total RNA (1 µg) was transcribed with random hexamers (2.5 µM), dNTPs (1 mM), and RNase inhibitors (1 unit/µl) using murine Moloney leukemia virus reverse transcriptase (2.5 units/µl) for 45 min at 42 °C as previously reported(12, 13) . The resultant single strand cDNA was amplified with the forward primer in the 5`-noncoding region (5`-GCGCAGAGCTTAGAACTCGGATCCTCCGGT-3`) and reverse primer in the 3`-noncoding region (5`-CTTTGGACGCAGTCTTGCAGGAGATGGCC-3`) using Taq polymerase (Takara Shuzo, Kyoto, Japan). The resultant PCR product was radiolabeled by random oligonucleotide primer extension using [alpha-P]dCTP and used as a probe(14) . Positive clones were purified and characterized by restriction endonuclease mapping and Southern blotting. The specific restriction fragments subcloned into the pBluescript II vector (Stratagene) were sequenced by dideoxy chain termination. In addition to T3 and T7 primers, sequence-specific oligonucleotides were synthesized using a DNA synthesizer 381A (Applied Biosystems, Inc.).

RNA Analysis

Primer extension reactions and RNase protection were carried out with 40 µg and 10 µg of total RNA, respectively, as described(11) . For analysis of V1a receptor mRNA, 30-nucleotide (nt)-long primers (nt -68 to -98 and +21 to +51 relative to the first ATG codon) were synthesized. For analysis of chloramphenicol acetyltransferase (CAT) mRNA, a 30-nt-long primer corresponding to nt +2 to +32 downstream from the HindIII site of the CAT gene was used(15) . For RNase protection analysis, a HincII-digested EcoRI and BamHI genomic sequence (-480 to -72 relative to the ATG codon) was used for the probe. A RNA probe was generated using [alpha-P]CTP and T7 RNA polymerase (Takara Shuzo, Kyoto, Japan). After the probe was hybridized with RNA at 52 °C overnight, it was digested with RNase T1 (0.21 unit/ml, U. S. Biochemical Corp.) and RNase A (2.1 units/ml, U. S. Biochemical Corp.) and analyzed on a 6% sequence gel, as described previously(18) .

The Sequence of the Rapid Amplification Product of the 5`-End of Rat V1a Receptor cDNA

Two micrograms of total cellular RNA from liver and A10 cells were reverse-transcribed for 45 min at 42 °C using murine Moloney leukemia virus reverse transcriptase (2.5 units/µl, Life Technologies, Inc.). The primer was -264 to -294 relative to the ATG codon (Fig. 2). After removing excess primer with a spin column (Chroma spin-100, Clontech), the first strand reaction products were tailed with dATP using 10 units of terminal deoxytransferase as reported(18) . Amplification proceeded using a primer consisting of a T7 primer sequence and (dT) (TAATACGACTCACCTATAGGGCGAATTGAAGCTTTTTTTTTTTTTTTTTTTT) and a nested primer complementary to the nucleotides from position -316 to -336 relative to the ATG codon. The PCR product was subcloned into pGEM-T vector (Promega) and sequenced using the nested primer(16) . The product was analyzed on a 6% denaturing polyacrylamide gel. After fixing and drying, the gel was autoradiographed at -70 °C for 4 days.


Figure 2: Structure of the 5`-flanking region of the rat V1a receptor gene. The first base of the ATG codon is presented as position +1. Major (-243 and -236) and minor(-405) transcriptional initiation sites are indicated by thick and thin arrows, respectively. GATA motif, a potential AP-1, AP-2, SP-1, NF-kappaB, and PEA3 binding site, TATA box, and CAAT box are underlined. The primers used for 5`-RACE and primer extension analyses are indicated by the double underline and the dotted line, respectively.



The Sequence of the Rapid Amplification Product of the 3`-End of Rat V1a Receptor cDNA

Total cellular RNA (2 µg) from liver and A10 cells was reverse-transcribed for 45 min at 42 °C using murine Moloney leukemia virus reverse transcriptase. The primer was the same (dT) sequence as that used to determine the 5`-end of the cDNA. After removing excess primer, amplification proceeded with the (dT) primer and oligonucleotides designed from the 3`-noncoding region of the rat V1a cDNA sequences reported by Morel et al.(4) (+1280 to +1300 relative to the ATG initiation codon). The PCR product was subcloned into the pGEM-T vector (Promega) and sequenced using M13 primers. The product was analyzed on a 6% denaturing polyacrylamide gel, which was autoradiographed at -70 °C for 4 days.

Construction of V1a Receptor CAT Expression Vectors

The 2.2-kb EcoRI-ApaI fragment was fused 5` to the chloramphenicol acetyltransferase (CAT)-pBluescript KS(-) construct (V1a 2208, Fig. 5). Sequential deletion mutants of the 5`-flanking region (Fig. 5) were prepared by using restriction enzyme sites of XhoI (V1a 1605) and SacI (V1a 221), or Exonuclease III (double-stranded Nested Deletion Kit, Pharmacia). In brief, the V1a 2208-CAT construct was digested with KpnI and EcoRV (linker sites of pBs vector). The linearized DNA was incubated with exonuclease III, and samples were removed at timed intervals, then treated with S1 nuclease to remove single-stranded regions generated by the exonuclease III. Deletion mutants of desired sizes were recircularized with T4 DNA ligase. The 5`-end of deletion mutant was determined by reading the sequence. The DeltaV1a 2208-CAT that lacks the sequence between -348 and -222 from V1a 2208-CAT construct was constructed by digestion with PstI and SacI followed by treatment with S1 nuclease and T4 DNA ligase.


Figure 5: Expression of CAT fusion genes containing 5`-deleted V1a receptor gene sequences. The 5`-deletion mutants were transiently expressed in A10 and NIH3T3 cells. The cell extracts were prepared 48 h after transfection and assayed. The names of these deletion mutants indicate the 5`-end points of the promoter sequence. DeltaV1a 2208 indicates the CAT construct lacking the sequence between -348 and -222 from V1a 2208. The CAT activity levels, normalized with beta-galactosidase activities and protein contents, are expressed relative to those in promoterless pBs-CAT, assigned a value of 1.0. The mean activities and S.E. from four separate assays are presented. *, p < 0.05;**, p < 0.01 compared with the V1a 1006- and V1a 296-CAT construct, respectively.



DNA Transfection and CAT Assay

Plasmids were banded in CsCl before transfection. Transfections were performed by the calcium phosphate/DNA precipitate method(15) . Cells were incubated for 4 h with 30 µg of plasmid DNA, treated with 15% glycerol for 1 min (glycerol shock), and supplemented with the growth medium. After a 48-h incubation, cells were harvested and extracted in 0.25 M Tris-HCl, pH 7.8, by freezing-thawing. Using cell extracts, CAT activity was determined by a dual-phase diffusion assay that relies on the direct diffusion of the ^14C-labeled acetylchloramphenicol into liquid scintillation counting fluid, as previously reported(15, 18) . During this period, each vial was counted at 30-min intervals (at least six times). The relationship between incubation time and radioactivity was linear, and the slope (calculated by linear regression) was proportional to CAT activity. In each transfection, 5 µg of Rous sarcoma virus beta-galactosidase was co-transfected. CAT activity was normalized for transfection efficiency by beta-galactosidase activity and for cell density by protein concentration. The beta-galactosidase assay was performed as previously reported(15, 18) . Protein assay was performed by the Bradford method (Bio-Rad). CAT activity was expressed relative to the activity of a promoterless CAT construct. Most transfection experiments were repeated a minimum of four separate times.

Gel Retardation Assays

The gel retardation assay was performed and nuclear extracts from culture cells were prepared as described(11) . The DNA fragment between -296 and -221 bp relative to the ATG codon was obtained from V1a 296-CAT construct (Fig. 5) by BssHII (linker site of pBs vector) and SacI digestion and labeled with [alpha-P]dCTP using Klenow fragment (Takara Shuzo) and purified as reported(11) . Nuclear extracts were incubated for 15 min on ice in a 30-µl reaction mixture containing 12 mM Hepes, pH 7.9, 60 mM KCl, 0.1 mM EDTA, 0.5 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride, 12% glycerol, and 2 µg of double-stranded poly(dI-dC) in the presence or absence of excess competitor DNA. A radiolabeled DNA probe was added (0.1 0.5 ng; 15,000 cpm), and the incubation was continued for 30 min at room temperature. Thereafter, the mixture was loaded on a 6% polyacrylamide gel in 1 TBE (90 mM Tris-HCl, pH 8.0, 89 mM boric acid, 2 mM EDTA) and electrophoresed at 140 V for 3 h followed by autoradiography.

Southwestern (DNA-Protein) Blotting

Southwestern blotting proceeded as follows. Crude nuclear extracts (80 µg of protein each) mixed with 2.5% SDS, 2.5 mM Tris-HCl, pH 6.8, 100 mM dithiothreitol, 10% glycerol, 0.025% pyronin for 5 min at room temperature were separated by SDS-PAGE (10% polyacrylamide) and blotted onto a nitrocellulose membrane. The membrane was incubated in binding buffer (10 mM Hepes, pH 8.0, 100 mM KCl, 10 mM MgCl(2), 0.1 mM EDTA) containing 5% nonfat dried milk for 30 min at room temperature, and then in the same buffer containing salmon sperm DNA (100 µg/ml), 0.25% nonfat dried milk, and P-labeled probe (nt -296 and -221 relative to the ATG codon) for 1 h at room temperature. After washing three times for 10 min at room temperature in the binding buffer, the membrane was exposed to x-ray film.

Stable Transfectants of the Promoter-CAT Chimeric Plasmid and Cell Culture

Fifty micrograms of the V1a 2208-CAT construct were cotransfected with 5 µg of pcDNAneo (Invitrogen) into A10 cells. Two days after transfection, 400 µg/ml G418 (Life Technologies, Inc.) was added to the medium. Three weeks after transfection, G418-resistant colonies were pooled and used for the experiments. The rat vascular smooth muscle (A10) cells (Dainippon Pharmaceutical, Osaka, Japan), NIH3T3 (RIKEN Cell Bank, Tsukuba, Japan), and rat skeletal muscle (Sol 8) cells (a kind gift of Dr. Koren, Harvard University) were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum. All culture medium contained 100 units/ml penicillin G and 100 µg/ml streptomycin.

Genomic Southern Blots

Ten micrograms of rat genomic DNA prepared from the spleen was digested with the restriction endonucleases, EcoRI, BamHI, and XbaI, resolved by electrophoresis on a 0.9% agarose gel, and blotted onto a nylon membrane. The membrane was hybridized with the P-labeled 708-bp BamHI-KpnI cDNA fragment as described (14) and washed in 0.1 SSC + 0.1% SDS at 60 °C.

Reagents and Statistical Methods

All reagents were purchased from Sigma unless otherwise indicated below. Results are expressed as mean ± S.E. Analyses of variance and the Newmann-Keuls test were used for multigroup comparisons. Values of p < 0.05 were considered statistically significant.


RESULTS

Isolation and Characterization of Rat V1a Receptor Genomic Clones

A total of 10^6 clones from the genomic DNA library were screened with a P-labeled rat V1a cDNA probe. Four positive clones were isolated and analyzed further by means of restriction enzyme mapping and Southern blotting. The results indicated that these could be segregated into two overlapping clones, of which two (RGV1 and RGV2) harbored the entire rat V1a receptor cDNA sequence as reported by Morel et al.(4) . Fig. 1shows a map of these clones with recognition sites for the restriction endonucleases EcoRI and BamHI.


Figure 1: Schematic representation of the rat V1a receptor gene. A, the gene locus is shown with the recognition sites for EcoRI (R) and BamHI (B) marked. Open and hatched boxes indicate nonexpressed and expressed exons, respectively. B, the structure of the rat V1a cDNA is presented at the bottom. Encoded transmembrane domains (I-VII) and other domains are displayed as hatched and open boxes, respectively. Exons in the genomic DNA and their corresponding regions of the cDNA are connected by solid lines.



Exon-Intron Structure of Rat V1a Receptor Gene

A 3.7-kb BamHI-BamHI DNA fragment (Fig. 1) was subcloned into pBs KS(-) and sequenced bidirectionally with oligonucleotide primers designed from the V1a cDNA sequences(4) . A comparison of the V1a receptor genomic sequence with that of its cDNA indicated that one intron (1.8 kb) was located between the sixth and seventh putative transmembrane domains. The sequence of the splice site conformed to the GT-AG rule (Table 1)(17) . PCR using the primers designed from the intron sequence showed that the intron is about 1.8 kb in length (data not shown).



Sequence Analysis of Upstream 5`-Flanking Region

To identify the DNA elements that control and direct rat V1a receptor gene transcription, we sequenced the 2208-bp region upstream from the ATG initiation codon. As shown in Fig. 2, neither TATA nor CCAAT promoter elements were found within the region containing the proximal major transcriptional initiation sites and the promoter activity, although sequences homologous to these motifs could be identified more than 800 bp upstream of the translational initiation site. It was, however, noted that the G + C content was 62% in the 400 bp immediately 5` to the ATG initiation codon. This is higher than that of the average mammalian genome (40%); however, a GC box motif was not found in this region, suggesting that the promoter of the V1a receptor gene has features typical of a housekeeping gene(19) .

Further analysis of the 5`-flanking region using a computer program (TFD 7.2) revealed potential TATA-like (-616 and -447)(20) , Sp1 binding (-557 and -1449)(21) , CCAAT(-1530)(22) , and AP-1(-1705) and AP-2 binding (-1688 and -605) (23) sequences (Fig. 2). The TATA-like sequence(-447) is located at the position more than 200 bp upstream of the major transcriptional initiation sites and 48 bp upstream of the minor initiation site. The rat oxytocin receptor gene also has a promoter region and transcriptional initiation sites at positions more than 500 bp downstream of the TATA sequence(10) . Although NF-kappaB (-716 and -677), PEA3 (-1122 and -683), and GATA (-1713) binding sites (23) are located in the 5`-flanking region, the regulation of the rat V1a receptor expression by these factors has not been reported.

Determination of Transcriptional Initiation Sites by Primer Extension, RNase Protection, and 5`-RACE

Results of primer extension experiments using total RNA isolated from rat liver and A10 (rat vascular smooth muscle) cells are shown in Fig. 3A. Two primers were designed from the sequences from nt -68 to -98 and +21 to +51 relative to the ATG initiation codon. Three extended bands were detected by both primer extension analyses, corresponding to positions -405, -243, and -236 relative to the ATG codon (the results obtained using nt -68 to -98 primer are shown in Fig. 3A). No extended signals were observed when yeast tRNA or total RNA prepared from NIH3T3 or Sol 8 skeletal muscle cells, in which no V1a receptor mRNA was detected by reverse transcription-PCR analyses, was used as a control. To confirm the results obtained by primer extension analyses, we performed RNase protection analyses. The riboprobe used in this assay was an antisense RNA runoff from a HincII-digested EcoRI-BamHI genomic DNA fragment subcloned into pBs KS(-). This HincII site is located at position -480 from the first ATG codon. If transcriptions begin at position -405, -243, and -236, as the result of primer extension analyses suggested, this probe would protect fragments 334, 171, and 164 nucleotides in length, respectively. Fig. 3B shows the results of RNase protection assays showing two major bands migrating at positions corresponding to approximately 171 nt and 164 nt, and one faint band of approximately 334 nt following RNase protection of total RNA prepared from liver and A10 cells. Protected bands were not evident when yeast tRNA or total RNA from NIH3T3 or Sol 8 skeletal muscle cells were used as controls. Thus, RNase protection analyses confirmed the presence of the three transcriptional start sites indicated by primer extension analyses.


Figure 3: Primer extension (A) and RNase protection (B) analyses of the transcriptional initiation sites of the rat V1a receptor gene. In A, the end-labeled 30-nt primer (see Fig. 2) was hybridized at 42 °C to 40 µg of yeast tRNA and total RNA (40 µg) from liver, A10, NIH3T3, and Sol 8 cells and then extended with reverse transcriptase at 42 °C for 60 min. A sequencing ladder, generated using the same primer, was run in parallel. In B, HincII-digested EcoRI-BamHI genomic sequence (nt -480 to -72 bp relative to the ATG codon) was used as a probe. The riboprobe (5 10^5 cpm) was hybridized with 10 µg of yeast tRNA and total RNAs from liver, A10, NIH3T3, and sol 8 cells before being digested with RNase T1 and RNase A. A known DNA sequence was run in parallel, and the sizes of nucleotides were determined. Exposure time was three weeks with an intensifying screen in both A and B.



The most upstream start site was located 42 bp downstream from the putative TATA box and other proximal start sites were 205 and 212 bp downstream from the putative TATA box (Fig. 2). The transcriptional activity at the most upstream start site was considered to be much weaker than those at the proximal start sites, as suggested by the results of primer extension and RNase protection analyses (Fig. 3). To further elucidate whether the faint signal at position -405 observed in primer extension and RNase protection analyses reflects true transcriptional initiation of the V1a receptor gene, we attempted rapid amplification of the 5`-cDNA end (RACE) by means of PCR(18) . Total RNA from A10 cells or adult rat liver was reverse-transcribed with oligonucleotides corresponding in sequence to that of nt -254 to -224 relative to the ATG codon (Fig. 4) at 42 °C. The single-strand reaction products were tailed with dATP, then amplified with a (dT) primer and a nested primer and sequenced. As shown in Fig. 4, the ``G'' band subsequent to the poly(dT) tail was identified, which corresponded to the most upstream start site determined by primer extension analyses. The DNA sequences following the G band completely matched the genomic sequence of the rat V1a receptor gene. The same band was detected in total RNAs from A10 cells as well as rat liver. No detectable bands were observed, when yeast tRNA or total RNA from NIH3T3 or skeletal muscle cells was used as a template. Reverse transcription at other incubation temperatures (37 °C, 47 °C) also yielded the same band in the RACE experiments. From the results of the 5`-RACE experiment, primer extension, and RNase protection analyses, we concluded that there are three transcriptional start sites in the rat V1a receptor gene, of which the major start sites are at positions -243 and -236 relative to the ATG codon. This conclusion is supported below by the observation that these nucleotides are included in a region with promoter activity.


Figure 4: The nucleotide sequence of the product of rapid amplification of the 5`-end of the rat V1a receptor cDNA (RACE). Left panel, 2 µg of total RNA from liver was reverse-transcribed with primer (nt -264 to -294 relative to the ATG codon). After tailing the first strand reaction products with dATP, the cDNA was amplified using a T7 oligonucleotide (dT) primer and a nested primer (nt -260 to -280). The PCR product was then subcloned and sequenced using the nested primer. The arrow indicates the site of transcriptional initiation. Right panel, the nucleotide sequence of rat V1a receptor gene was determined with the same primer as that used for reverse transcription in RACE.



Promoter Activity of 5`-Flanking Region of Rat V1a Receptor Gene

To examine the promoter activity of the 5`-flanking region of the V1a receptor gene, we constructed a series of CAT expression plasmids with various 5` deletions generated using restriction enzymes and exonuclease III. A portion of the V1a receptor gene from positions -2208 to -221 relative to the ATG codon was inserted into the plasmid pBsKS(-) immediately upstream of the CAT gene coding sequence (V1a 2208, Fig. 5). A series of deletion mutants extending from -2208 toward the most downstream start site of transcription were then constructed; the deletion mutant end points were -1605, -1214, -1006, -824, -526, -404, -296, and -221. The promoter activities of the 5`-flanking sequences were then examined in terms of CAT activity using A10 vascular smooth muscle cells and, as negative controls, NIH3T3 and skeletal muscle (Sol 8) cells.

The CAT activities normalized with beta-galactosidase activity and protein concentration are shown in Fig. 5as values relative to those of the promoterless pBs-CAT construct. Transfection of V1a 2208-CAT construct into A10 cells resulted in an approximately 7.4-fold increase in CAT activity relative to the promoterless CAT construct, whereas no significant increases in CAT activities were observed in NIH3T3 or Sol 8 cells (data for NIH3T3 are shown in Fig. 5). Deletions from -2208 to -1606, -1605 to -1215, and -1214 to -1007 did not significantly affect CAT activity of the V1a-CAT construct. Successive deletion from -1006 to -825 resulted in a slight, but significant increase in CAT expression (1.4-fold, p < 0.05), suggesting the presence of a negatively acting element. Comparison of V1a 824 and V1a 526 showed that the sequence from -824 to -526 had no effect on CAT expression. Progressive deletion from -526 to -405, which lacks a putative TATA sequence, resulted in a modest, but not significant decrease in CAT activity. Whereas the sequence from -404 to -297 also had no effect on CAT expression, further deletion from -296 to -222 caused a marked reduction (p < 0.01) to basal levels of CAT activity. Cells transfected with the DeltaV1a 2208-CAT construct lacking the sequences between -348 and -222 also showed CAT activity similar to the basal level. Thus, cell type-specific promoter activity was detected in the 2.2-kb 5`-flanking region, and the smallest region necessary for this activity, from nt -296 to -222 relative to the ATG codon, is encompassed by the V1a 296-CAT construct and includes the major transcriptional start sites identified above.

Initiation of V1a CAT Transcripts at the Correct Start Sites

To verify that the products of the CAT gene reflect correctly initiated mRNA transcripts and that CAT activity measurements in this system do indeed reflect events at the RNA level, total cellular RNA from A10 cells that were stably transfected with the V1a 2208-CAT gene construct was examined by primer extension analysis. As shown in Fig. 6B, total RNA from the stably transfected cells produced two prominent bands and one faint band. These extended bands corresponded to the correct transcriptional initiation sites determined by primer extension using the native A10 cells.


Figure 6: Gel mobility shift assays (A) and primer extension analysis of V1a receptor CAT fusion gene transcript in stably transfected A10 cells (B). A, competition experiments with the 5`-end-labeled promoter fragment (position -296 to -222) and nuclear extract from A10, NIH3T3, and Sol 8 cells. The proposed promoter region between -296 and -222 bp relative to the ATG codon was labeled and incubated with nuclear extracts (5 µg) from A10, NIH3T3, and Sol 8 cells. The unlabeled DNA fragment at 10, 30 and 100 molar excess was used as the competitor. B, primer extension analysis was performed with a 5`-end-labeled 30-bp oligonucleotide designed in the CAT coding sequence. Forty µg of total RNA from A10 cells stably transfected with V1a 2208-CAT and yeast tRNA were analyzed. The sequencing reaction was performed with the V1a 2208-CAT construct and the oligonucleotide used for primer extension. Exposure time was 3 weeks with an intensifying screen.



Specific Binding of a Proximal Promoter Fragment to a Nuclear Factor

Gel mobility shift assays were performed to detect nuclear proteins that bind to the proximal positive regulatory region between nt -296 and -222. In these experiments, a radiolabeled DNA fragment corresponding to this region was incubated with nuclear extracts from A10, NIH3T3, and Sol 8 cells. As shown in Fig. 6A, we found three retarded bands (indicated by arrows A, B, and C) in A10 cells and one retarded band (arrow C) in NIH3T3 and Sol 8 cells. The arrow A and B bands were completely competed out with a molar excess of unlabeled probe fragment, whereas the arrow C band showed no such inhibition. These results indicate that specific trans-acting nuclear proteins bind to this region and may confer the promoter activity and tissue-specific expression pattern on the V1a receptor gene.

Southwestern Blots of the trans-Acting Factor

To characterize the binding factor, the nuclear extract from A10 cells was resolved by SDS-PAGE and Southwestern blotted with the P-labeled promoter region between nt -296 and -222 relative to the ATG codon. The probe bound to two proteins (30 and 28 kDa) with the same intensity, the binding of which disappeared in the presence of excess cold probe (Fig. 7). The nuclear extract from NIH3T3 or Sol 8 cells did not contain the protein which bound to the probe. Although the conditions of protein-DNA binding differ between gel shift assays and Southwestern blotting, this result confirms the finding obtained by the gel shift analysis showing that two nuclear proteins bind to the promoter region.


Figure 7: Southwestern blots of nuclear protein prepared from A10, NIH3T3, and Sol 8 cells. Nuclear extracts (80 µg of protein per lane) were separated by SDS-PAGE, blotted onto a nitrocellulose membrane, and then detected with the P-labeled fragment of rat V1a receptor gene (nt -296 to -222 relative to the ATG codon). The unlabeled probe (50 molar excess) was used as a competitor. The film was exposed for 4 weeks at -80 °C with an intensifying screen. Size markers are indicated in kilodaltons on the left side of the gel.



Determination of the 3`-End of the Rat V1a Receptor Transcript and Its 3`-Flanking Sequence

The cDNA sequence for the rat V1a receptor gene, reported by Morel et al.(4) , showed only 169 nucleotides following the stop codon, and neither polyadenylation signals nor the poly(A) tail were characterized. We determined the 3`-end of rat V1a receptor gene transcript by means of RACE. Total RNA from rat liver was reverse-transcribed with a (dT) primer, and the resultant single-strand cDNAs were amplified with the (dT) primer and oligonucleotides designed from the 3`-noncoding region of the rat V1a cDNA sequences reported by Morel et al.(4) (nucleotides +1280 to +1300 relative to the ATG initiation codon). As shown in Fig. 8, the RACE procedure allowed characterization of the poly(A) tail and adjacent sequences in the 3`-noncoding region of the rat V1a receptor gene transcript, which was 449 bp downstream from the stop codon. The poly(A) tail was not detected in the genomic sequence, and the upstream sequence of the poly(A) tail completely matched the genomic sequence that we determined. (Fig. 8, A and B).


Figure 8: The nucleotide sequence of 3`-flanking region and 3`-cDNA end of rat V1a receptor gene determined by rapid amplification of the 3`-end of the cDNA (RACE). A, the exon sequences are shown in uppercase letters, and the 3`-flanking sequences are shown in lowercase letters. The putative polyadenylation signal is doubly underlined. GT clusters downstream of the polyadenylation and ATTTA motif are underlined. The arrow indicates the 3`-end of the mRNA determined by RACE. B, 2 µg of total RNA from A10 cells was reverse-transcribed with (dT) primer. The resultant single-stranded cDNA pool was amplified with (dT) and a primer designed from the known cDNA sequence reported by Morel et al.(4) . The PCR product was subcloned into pGEM-T vector and sequenced with the M13 reverse primer (right panel). The left panel shows the genomic sequence around the cleavage site.



In the 3`-noncoding region, there was no consensus polyadenylation signal (AAUAAA); however, a polyadenylation-like signal (AACAAA) was found 23 nucleotides upstream of the polyadenylation site. The polyadenylation site was followed by two GT clusters (GTTTGT and TTGGGTGG). An ATTTA motif, which has been implicated in mRNA instability(24) , was found in the 3`-noncoding region. These results indicated that the sizes of the major transcripts of the rat V1a receptor gene are 1974 bp and 1967 bp followed by a poly(A) tail, which is in agreement with the size of the mRNA (2.1 kb) postulated by Morel et al.(4) .

Genomic Southern Blot Analysis of the Rat V1a Receptor Gene

The copy number of the rat V1a receptor gene in the rat genome was determined by Southern blot hybridization (Fig. 9). The rat V1a receptor cDNA fragment containing a part of exon 1 (BamHI-KpnI, 709 bp) was used to probe human genomic DNA digested with four restriction endonucleases: EcoRI, HindIII, BamHI, and XbaI. Each enzyme generated a single fragment which hybridized to the probe, indicating for the first time that the rat V1a receptor locus exists in a single copy in the rat genome. The length of the hybridized bands in the genomic DNA digested with restriction enzymes was consistent with those in the genomic clone RGV1 (Fig. 9).


Figure 9: Southern hybridization analysis of rat genomic DNA and DNA prepared from the genomic clone RGV1. Either 10 µg of genomic DNA (left panel) prepared from the rat spleen or 4 µg of DNA of RGV1 (right panel) was digested to completion with EcoRI (R), HindIII (H), BamHI (B), or XbaI (X). The digests were electrophoretically resolved and transferred on a nylon membrane, followed by hybridization with the P-labeled 708-bp BamHI-KpnI fragment prepared from exon 1 of RGV1.




DISCUSSION

In this study, we cloned the gene encoding the rat V1a receptor to characterize its structural organization and promoter region and determined the entire cDNA sequence of its transcript. The rat V1a receptor gene spans 3.8 kb and consists of two exons divided by one intron (approximately 1.8 kb). Among the AVP-oxytocin receptor family, the gene structures for the human V2 AVP receptor and the rat oxytocin receptor have been reported previously(9, 10) , whereas that of the V1 type AVP receptor gene had not been determined. The human V2 AVP gene harbors three exons framing two intervening sequences; the first intron, 360 bp, interrupts the codon corresponding to the ninth amino acid of the receptor sequence, and the second intron begins between the sixth and seventh transmembrane regions(9) . The rat oxytocin receptor gene contains two introns; the first of which is in the 5`-untranslated region and the second interrupts the coding region between the sixth and seventh transmembrane domains(10) . The present study demonstrated that the intron of the rat V1a receptor gene is also located between the sixth and seventh transmembrane regions. These exon/intron boundaries completely matched the consensus donor/acceptor splice sequences.

Cloning of the genes encoding G protein-coupled receptors has revealed that they are a heterogeneous family with regard to their exon/intron structure. Whereas the first genes cloned were intronless (beta-adrenergic receptor)(25) , a number of exceptions are known. As summarized in Fig. 10, the genes for human endothelin receptors (A and B receptors)(26, 27) , opsins(28) , dopamine receptors (D(2), D(3), and D(4) receptors) (29, 30, 31) , tachykinin receptors (substance K, substance P, and neuromedin K receptors)(32, 33, 34) , and luteinizing hormone receptor (35) had been reported to contain introns. The genes in the AVP-oxytocin receptor family, like the rat V1a receptor in this study and the human V2 and rat oxytocin receptors(9, 10) , belong to the latter group. The number and positions of introns are not always conserved among the members of a given receptor gene family, and multiple large introns often interrupt the open reading frame (Fig. 10). The location of the introns of the rat V1a, human V2, and rat oxytocin receptor genes is unique in that the first (rat V1a) and second (human V2 and rat oxytocin receptors) exons encode only six of the seven transmembrane regions, the seventh being encoded by the following exon. These findings suggest that the genes belonging to the AVP-oxytocin receptor family may originate from a common ancestral gene and comprise a particular group in the G protein-coupled receptor genes.


Figure 10: Exon-intron splice sites of G protein-coupled receptor genes. The location of exon-intron splice sites are compared among the genes for rat V1a (4) and rat V2 receptors (9) , rat oxytocin receptor(10) , human endothelin A (26) and B (27) receptors, tachykinin receptor(26, 27, 28) , D(2), D(3), and D(4) dopamine receptors(29, 30, 31) , rhodopsin(28) , blue, green, and red opsins(28) , and luteinizing hormone receptor (LHR)(35) . The upper panel depicts the cDNA for the rat V1a receptor schematically. The transmembrane domains (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 represented by solid lines in the lower panel. The exon-intron splice sites are indicated by arrowheads.



Analyses of the 5`-flanking region revealed the existence of three transcriptional initiation sites (-405, -243, and -236), the major ones being mapped at nt -243 and -236 relative to the ATG initiation codon. The existence of transcriptional initiation sites at -405 and -243 bp is consistent with the observation that the 5`-end of the rat V1a receptor cDNA cloned from the rat liver (4) was at -241 bp. The initiation of transcription at position -236 suggests the existence of an as yet undefined transcript lacking 7 nt from the 5`-end of the previously reported cDNA(4) . Sequential deletion mutants of the rat V1a-CAT chimeras have been used to delineate several functional regions of the 5`-flanking sequence. These revealed that the promoter activity is encompassed within the sequence between positions -296 and -222 relative to the ATG codon. This promoter region exhibits features typical of housekeeping genes(19) : the absence of TATA or CCAAT promoter elements and a high G + C content. It should be noted that the promoter region of the rat oxytocin receptor gene also lacks an apparent TATA or CCAAT box(10) . Promoters for thyrotropin receptor(36) , some growth factor receptors, and oncogenes also have these features; e.g. the EGF receptor(37) , insulin receptor (38) , nerve growth factor receptor(39) , Ha-ras(40) , and N-myc(41) .

Next, we showed that expression of the V1a receptor appears to be tissue-specific, since the promoter activity of the 2.2-kb 5`-flanking region was detected in vascular smooth muscle cells, but not in NIH3T3 or skeletal muscle cells. Sequential deletion analyses using the V1a-CAT chimeras indicated that a ``minimal'' region exhibiting promoter activity and tissue specificity is located between nt -296 and -222. Gel mobility shift assay and the Southwestern blot analyses demonstrated that 30- and 28-kDa nuclear proteins specifically bind to this region. A computer-assisted search indicated that there are no binding sites for known transcriptional factors in this region. In the present study, we observed no significant promoter activity in the 5`-flanking region encompassing the most upstream site of the gene transcription (position -405). This finding was consistent with the results of primer extension and RNase protection analyses showing that the level of transcriptional product initiated at this site is much less than those at proximal start sites and with Northern blot analyses showing that there is a single major transcript in rat tissues including liver and vascular smooth muscle cells(4) .

The 3`-ends of mRNA are generated in the nucleus in two steps. The precursor RNA is cleaved endonucleolytically at a specific phosphodiester bond, and the 3`-OH group of the upstream fragment then receives the poly(A) tail by polymerization from ATP. The cleavage reaction depends upon two RNA sequence elements, the highly conserved sequence AAUAAA 10-30 nucleotides upstream of the cleavage site and poorly defined GU- or U-rich sequences located approximately the same distance downstream(42) . Here, we determined the cleavage site and the 3`-noncoding sequences for the rat V1a receptor gene, in which there was no consensus poly(A) signal, and, instead, a poly(A)-like sequence (AACAAA) was found 23 bp upstream from the cleavage site. In addition, a GT-rich sequence was located 26 bp downstream from the cleavage site. Wickens et al.(43) reported that variants of the poly(A) signal are present in natural genes, such as CAUAAA, AAUAAC, AAUAAU, AAUACA, AAUUAA, AUUAAA, and that the secondary structure of RNA precursors rather than the AAUAAA sequence itself is more important for recognition by RNA processing enzymes. Thus, it is likely that the AACAAA sequence plays a role as a cleavage signal in the rat V1a receptor gene.

In conclusion, in this study we demonstrated the structural organization and characterized the 5`-flanking region of the rat V1a receptor gene and reported the complete cDNA sequence of its major transcript. We are the first to characterize the V1-type AVP receptor gene structure, which was unique among G protein-coupled receptor genes with respect to the location of the intron. Knowledge of the structural organization and promoter analyses of the V1 and V2 receptor genes will lead to a better understanding of the mechanisms of transcriptional regulation of the AVP-oxytocin receptor gene family and will provide clues in the search for genetic disorders involving this gene family.


FOOTNOTES

*
This study was supported in part by research grants from the Ministry of Education, Science and Culture of Japan and the Study Group of Molecular Cardiology in Japan. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank[GenBank].

§
To whom correspondence and reprint requests should be addressed. Fax: 81-6-998-6178.

(^1)
The abbreviations used are: AVP, arginine vasopressin; kb, kilobase(s); bp, base pair(s); nt, nucleotide(s); PCR, polymerase chain reaction; CAT, chloramphenicol acetyltransferase; PAGE, polyacrylamide gel electrophoresis; RACE, rapid amplification of cDNA ends.


ACKNOWLEDGEMENTS

We thank Dr. Yoshifumi Ogawa for excellent technical assistance.


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