(Received for publication, April 10, 1995; and in revised form, June 20, 1995)
From the
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.
Arginine vasopressin (AVP) ()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.
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-B, 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.
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. V1a 2208 indicates the CAT construct lacking the sequence
between -348 and -222 from V1a 2208. The CAT activity
levels, normalized with
-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.
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.
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-B (-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.
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
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.
The CAT
activities normalized with -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
V1a 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.
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.
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.
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) .
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.
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 (-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
, D
, and D
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,
D
, and D
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.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank[GenBank].