Abstract Although the rat angiotensin II type 2 receptor (AT2) was cloned and shown to be a member of the seven transmembrane domain–type receptor family, its signaling mechanism and biological roles have not been established. To acquire additional information on the structure and functions of AT2 genomic DNA, we cloned the mouse AT2 gene and examined its expression, transcription, and genomic organization. The amino acid sequence of the mouse AT2 cDNA showed a 98.5% sequence identity with the rat AT2. In mouse fetus, mRNA of the AT2 was highly expressed in the eviscerated carcass and brain. This expression decreased rapidly after birth. In 10-week-old mice, mRNA of the AT2 could be detected in the brain by Northern blot analysis. However, reverse transcription–polymerase chain reaction showed that mRNA of the AT2 was expressed in all organs examined, indicating that the AT2 is expressed at a low level in other organs. Southern blot analysis of the genomic DNA of the mouse liver digested with BamHI, EcoRI, and HindIII resulted in single bands, indicating that the AT2 gene probably exists at a single locus in the mouse genome. The nucleotide sequence of the AT2 gene (4.5 kb of the EcoRI fragment) revealed the presence of three exons. An entire coding sequence was included in the third exon. Primer extension experiments showed the presence of two transcription initiation sites in the mouse AT2 gene. A DNA segment of about 1.5 kb of the promoter region (−1497 to +56 bp) of the mouse AT2 gene was fused to a luciferase reporter gene. This promoter-luciferase construct was functional as a promoter when transfected into R3T3 cells. The promoter region contains several transcription cis elements, such as AP1 and C/EBP, in the −1497- to −874-bp segment of the promoter region. Deletion analysis showed that this segment of the DNA accounted for 70% of the promoter activity. The shortest deletion segment (−47 to +56 bp), which contains the TATA box, contributed about 15% of the promoter activity. These results clarified several functional features of the mouse AT2 gene at a molecular level.
- angiotensin II type 2 receptor
- gene expression
- reverse transcriptase–polymerase chain reaction
- genomic DNA
Angiotensin (Ang) II plays an important role in blood pressure regulation, fluid homeostasis, and drinking behavior. In addition, a growing body of evidence indicates that Ang II is a growth factor.1 2 Physiological effects of Ang II on target cells such as adrenal gland and vascular smooth muscle cells are mediated by Ang II receptors. The presence of two isoforms of Ang II receptor was originally proposed on the basis of the differential sensitivity of receptor-ligand binding to dithiothreitol (DTT).3 The recent development of isoform-specific antagonists for Ang II receptors provided a firm support for the presence of at least two isoforms of the Ang II receptor.4 5 The type 1 receptor (AT1), which is sensitive to DTT and losartan, was cloned in 1991 and shown to have the structural features of a putative seven transmembrane domain receptor.6 7 AT1 was shown to mediate all the biological effects traditionally ascribed to Ang II, such as vasoconstriction, aldosterone release, facilitation of adrenergic outflow, water drinking, and cellular proliferation and growth.1 2
The type 2 receptor (AT2) is insensitive to DTT and has a high affinity for PD123319 and CGP42112A4 5 and a low affinity for losartan. Binding studies using radiolabeled Ang II and isoform-specific antagonists showed that AT2 is expressed in fetal tissues, most conspicuously mesenchymal tissues8 and specific brain nuclei of the rat.9 Its expression decreases rapidly after birth. In adult rats, AT2 is expressed in adrenal medulla,5 heart,10 brain nuclei (eg, inferior olive, thalamus, and locus ceruleus),11 and myometrium.4 These studies suggest developmental, neurological, and reproductive roles of Ang II via AT2. However, signaling mechanisms and biological function remain to be established.12 13 14 15 Recently we16 and Mukoyama et al17 cloned the cDNA of the rat AT2 and showed that it has the putative seven transmembrane domain structure but with a minimum of homology with AT1.
The pattern of the expression of the AT2 gene is quite different from that of AT1. A complete characterization of the AT2 gene is essential to begin to understand its biological roles and unique tissue-specific expression. We previously cloned the cDNA and genomic DNA of the mouse AT2.18 In the present study, we provide an extended report of the expression, genomic organization, and transcription of the mouse AT2 gene.
Materials and Methods
Ang I, Ang II, Ang III, and Sarile ([Sar1, Ile8] Ang II) were purchased from Peninsula Laboratories Inc. 32P-α-dCTP and 32P-γ-ATP were from DuPont-NEN. Losartan was a gift from DuPont-Merck, and PD123319 was a gift from Warner Lambert-Parke Davis Co. BALB/c and C57B/6 mice were purchased from Harlan Laboratories.
Cloning and Nucleotide Sequencing of the Genomic DNA of Mouse AT2
A genomic DNA library of the 129SV mouse strain was purchased from Stratagene. Five hundred thousand of the recombinant phages were screened by a conventional plaque hybridization method19 using a 32P-labeled full-length cDNA probe of mouse AT2, resulting in eight positive clones. A 4.5-kb EcoRI fragment and a 1.5-kb BamHI–Sac I fragment (see Fig 7A⇓) were subcloned into the pBluescript vector (Stratagene). Deletion mutants of this 4.5-kb EcoRI fragment were prepared by an Erase-A-Base kit (Promega), and their nucleotide sequences were determined by a dideoxy chain termination method using a Sequenase kit (USB) in both the sense and antisense directions.
Northern Blot Analysis
Poly (A)+ RNA was prepared by a Fast Track kit (In Vitrogen). Preparations were made from three BALB/c female mice and three fetuses at each gestational stage as shown in Fig 1⇓. Total RNA was prepared from R3T3 cells by the acid guanidinium-phenol-chloroform extraction method.20 One microgram of poly (A)+ RNA or 15 μg of total RNA was electrophoresed in 1% agarose/1% formaldehyde gel and transferred to Hybond N+ membrane (Amersham) by capillary transfer in 10× SSC buffer (1× SSC=300 mmol/L NaCl, 30 mmol/L sodium citrate) followed by baking for 2 hours at 80°C. Prehybridization and hybridization were performed in a buffer containing 50% formamide, 5× SSC, 80 mmol/L sodium phosphate (pH 7.5), 2× Denhardt solution, 1% SDS, and 100 ng/mL of heat-denatured herring sperm DNA for 2 hours and 16 hours, respectively, at 42°C. The 32P-labeled full-length cDNA of mouse AT2 was used as a probe. The hybridized filter was washed twice with 2× SSC for 5 minutes at room temperature followed by two washes with 2× SSC/1% SDS for 30 minutes at 55°C. The filter was then exposed to Kodak X-OMAT film at −70°C. The hybridized filter was stripped by boiling in 0.5% SDS solution and hybridized to a 32P-labeled GAPDH cDNA to obtain a reference for the amount of applied RNA.
Reverse Transcriptase–Polymerase Chain Reaction
For reverse transcriptase–polymerase chain reaction (RT-PCR), 1 μg of poly (A)+ RNA from various organs and 20 μg of total RNA from R3T3 cells were reverse transcribed with an oligo-dT primer. The resultant cDNAs were amplified by PCR using an oligonucleotide with the sequence 5′-GCTGAGTAAGCTGATTTATG-3′ as a forward primer and another oligonucleotide (5′-TTAAGACACAAAGGTGTCCA-3′) as a reverse primer. The reaction was run for 35 cycles of 1 minute of denaturation at 94°C, 1 minute of annealing at 58°C, and 2 minutes of polymerization at 72°C. One fifth of the reaction mixture (10 μL) was subjected to electrophoresis in 1% agarose gel and stained with ethidium bromide. Because the forward primer is specific for the second exon and the reverse primer is specific for the third exon, the predicted size of PCR product from AT2 cDNA is about 1.2 kb, and that from genomic DNA is about 2.3 kb.
Southern Blot Analysis
High-molecular-weight DNA was prepared from mouse liver. The liver was homogenized in a buffer containing 20 mmol/L Tris-HCl, 0.1 mol/L NaCl, and 1.5 mmol/L MgCl2. After centrifugation at 1500 rpm for 5 minutes, the pellet was resuspended in a buffer containing 10 mmol/L Tris-HCl, 0.1 mol/L EDTA, 0.5% SDS, and 100 μg/mL proteinase K and incubated for 16 hours at 50°C. The solution was phenol-extracted, ethanol-precipitated, and suspended in a 10 mmol/L Tris-HCl/1 mmol/L EDTA (TE) buffer. High-molecular-weight DNA (20 μg) was digested with several different restriction endonucleases overnight at 37°C and electrophoresed in 1% agarose gel. Transfer to Hybond N+ membrane, hybridization, and washing of the filter were done as described for Northern blot analysis.
Primer Extension Method
A 20-mer primer specific to the first exon of the AT2 gene (5′-GCAGGCTGAAGTAAGCTTTC-3′, nucleotide 111 through 130 bp in Fig 4⇓) was end-labeled with 32P-γ-ATP and T4 polynucleotide kinase, then purified by ammonium acetate/ethanol precipitation. mRNA (1 μg) from mouse fetus carcass or tRNA was reverse transcribed by use of this 32P-labeled primer and Moloney’s murine leukemia virus reverse transcriptase (NEB). The resultant product was phenol-extracted, ethanol-precipitated, and resuspended in 4 μL of a loading buffer (95% formamide, 20 mmol/L EDTA) and electrophoresed in 6% acrylamide/8 mol/L urea gel after heat denaturation. Sequencing ladders were obtained by the same primer by use of a Sequenase kit (USB).
Preparation of AT2 Promoter–Luciferase Gene Construct
Five deletion fragments of the promoter region of the AT2 gene were prepared by digestion with restriction endonuclease (as shown in Fig 7B⇓) except for D4, which was prepared by an Erase-A-Base deletion mutant kit (Promega). These fragments were cloned into the pGL2E (Promega) luciferase reporter vector, which has an SV40 enhancer sequence 3′ to the luciferase gene. Plasmid DNAs were prepared with Qiagen plasmid kit (Qiagen Inc) and purified once by centrifugation over cesium chloride cushion followed by dialysis against TE (10 mmol/L Tris-HCl pH 7.5/1 mmol/L EDTA) buffer and ethanol precipitation.
Cell Culture, Transfection, and Luciferase Assay
R3T3 cells were a generous gift from Dr Dudley (Warner Lambert-Parke Davis Co) and were maintained in DMEM supplemented with 10% fetal calf serum (Gibco BRL), 1000 U/mL penicillin (Gibco BRL), and 1000 μg/mL streptomycin (Gibco BRL). This medium will be referred to as complete medium. The day before transfection, 4×105 cells were prepared in a 6-cm tissue culture dish. On the day of transfection, the medium was changed to fresh complete medium and incubated for 1 hour. Then the cells were transfected with 10 μg AT2 promoter–luciferase constructs and 5 μg pSVβ-galactosidase (Promega) by the calcium phosphate precipitation method with a Profection kit according to the manufacturer’s instructions (Promega). After 6 hours of transfection, the cells were washed once with HBSS, supplemented with 4 mL of fresh complete medium. The next day, medium was changed to DMEM supplemented with 0.2% bovine serum albumin. After 48 hours of transfection, the cells were washed twice with HBSS and lysed in 200 μL of lysis buffer (25 mmol/L Tris, pH 7.8, 2 mmol/L EDTA, 2 mmol/L DTT, 10% glycerol, and 1% Triton X-100). One hundred microliters of lysate was used for luciferase activity assay in an Opticomp I luminometer (MGM Instruments Inc). The assay was started by adding 100 μL of 470 mmol/L luciferin to cell lysate, and integrated peak luminescence was determined over a 45-second window after a 5-second delay. The β-galactosidase activity in the same sample was measured spectrophotometrically according to Sambrook et al19 and used to normalize the luciferase activity.
Mice were anesthetized by an inhalation of methoxyflurane (Pitman Moore) and killed by cervical dislocation. Then the fetus and organs were removed. This method was approved by the Vanderbilt University animal care committee.
Expression of Mouse AT2 mRNA
cDNA of mouse AT2 consisted of 2871 nucleotides,18 which contained an open reading frame with an initiation codon and an in-frame termination codon encoding 363 amino acid residues. Of the total 363 amino acid residues, 4 amino acid residues are different from rat AT2, which also contains 363 amino acid residues. Thus, AT2 has a 98.8% sequence identity between the two rodent species. The mouse AT2 shows 38% amino acid identity with mouse AT1.21 22 Binding characteristics of the cloned cDNA expressed in COS7 cells confirmed that this cDNA encoded an AT2 receptor. This full-length cDNA was used as a probe for the following Northern and Southern blot analyses.
Expression of the AT2 gene was examined by Northern blot analysis. 32P-labeled mouse AT2 cDNA was used as a probe. Eviscerated mouse fetal carcass and brain expressed abundant mRNA of mouse AT2 (Fig 1A⇑), but the expression of AT2 was very weak in the carcass and brain of 2-day-old newborn mice. These data are consistent with results of earlier binding studies on rat fetus using radiolabeled Ang II.8
In 10-week-old adult mice, mRNA of AT2 was present in a detectable quantity in the brain (Fig 1B⇑). To confirm the expression of AT2 mRNA in other organs, an RT-PCR was performed. Samples of mRNA used in the Northern blot analysis were reverse transcribed with an oligo-dT primer. The first-strand cDNA was amplified by 35 cycles of PCR. To distinguish the PCR product of cDNA from that of genomic DNA, the 5′ primer specific for the second exon and the 3′ primer specific for the third exon were used. The size of the predicted PCR product of cDNA is about 1.2 kb, and that of genomic DNA is about 2.3 kb. Although a faint 2.3-kb band was observed in some samples, 1.2-kb bands were observed in all organs examined, as shown in Fig 2⇓ (lanes 1 through 5). The RT-PCR confirmed that the mouse AT2 mRNA is expressed at a low level in heart, liver, kidney, and lung.
Southern Blot Analysis of Mouse AT2 Gene
To determine the genomic organization of the mouse AT2 gene, Southern blot analysis was performed. High-molecular-weight DNA was prepared from livers of two mouse strains, BALB/c and C57B/6, and digested separately with four different restriction endonucleases. The 32P-labeled full-length cDNA of mouse AT2 was used as a probe. Fig 3⇓ shows the result of the Southern blot analysis. The two mouse strains showed identical bands when their DNAs were digested with the same restriction endonucleases. BamHI (lanes 1 and 5), EcoRI (lanes 2 and 6), and HindIII (lanes 3 and 7) digestion each gave single bands.
Molecular Cloning of Mouse AT2 Genomic DNA
To determine the exon-intron organization of the mouse AT2 gene, the genomic DNA of the mouse AT2 gene was cloned and its nucleotide sequence determined. A mouse genomic DNA library was screened with the 32P-labeled full-length cDNA of mouse AT2. Eight positive genomic clones were obtained. A 4.5-kb EcoRI fragment from the positive clone, which probably corresponds to the 4.5-kb band of the Southern blot analysis of the EcoRI digestion (Fig 3⇑, lanes 2 and 6), was subcloned into the pBluescript vector, and its nucleotide sequences were determined (Fig 4⇓). Comparison of the nucleotide sequence of this 4.5-kb fragment with the cDNA sequence revealed three exons of the mouse AT2 gene. They are indicated by boxes in Fig 4⇓. The entire coding sequence is contained in the third exon. An initiation codon (ATG) and a termination codon (TAA) are printed in bold type in the third exon. Exon-intron boundary consensus sequences (GT for exon-intron boundary and AG for intron-exon boundary) are conserved and underlined.
Transcription Initiation Site of Mouse AT2 Gene
A transcription initiation site was determined by the primer extension method. mRNA of the whole fetus at day 18 of gestation was reverse transcribed by use of a 32P-labeled primer specific to the first exon of the mouse AT2 gene (nucleotide 111 to 130 bp, indicated by an underline in Fig 4⇑). Two primer extension products were observed when mRNA of a fetus was used (Fig 5⇓, lane 1). The two initiation sites are 16 bp apart, and their locations are indicated by solid triangles in Fig 4⇑. No product was observed when tRNA was reverse transcribed (lane 2).
Expression of AT2 mRNA in R3T3 cells
Dudley and Summerfelt23 reported that R3T3 cells expressed the AT2 receptor and that the expression of the AT2 receptor increased after cells were confluent. To use this cell line for the study on the promoter function of AT2 gene by reporter gene assay, we first confirmed that R3T3 cells express AT2 mRNA by Northern blot analysis. R3T3 cells were cultured in medium with 10% fetal calf serum until they were confluent (Fig 6⇓, lane 1), then serum was depleted for 1 day (lane 2) and 2 days (lane 3). Total RNA was prepared from these R3T3 cells. The mRNA of the AT2 receptor was hardly seen when the cells were confluent (lane 1), but it was increased after serum depletion (lanes 2 and 3). The result of the Northern blot analysis is in good agreement with the receptor-binding assay by Dudley and Summerfelt.23 RT-PCR analysis of mRNA from R3T3 cells that were cultured in serum-free medium for 1 day was also performed (Fig 2⇑, lane 6) and confirmed the presence of AT2 mRNA in R3T3 cells.
Identification of the Promoter Region of Mouse AT2 Gene
The promoter activity of the upstream region of the transcription initiation sites of the mouse AT2 gene was examined. About 1.5 kb of the BamHI–Sac I fragment shown in Fig 7A⇓ was cloned, and its nucleotide sequences were determined (data not shown). There are several consensus transcription cis elements such as AP1, C/EBP, and PEA-3 in this region (Fig 7B⇓). Five deletion mutants of this fragment were prepared and ligated to a pGL2E luciferase reporter vector (Fig 7C⇓). These constructs were introduced into R3T3 cells. Luciferase activity driven by the mouse AT2 gene promoter was normalized by reference to β-galactosidase activity expressed by cotransfected pSVβ-galactosidase. Results are shown in Fig 7D⇓. The luciferase activity of construct D1 (−1497 to +56 bp) was set to 100%. This activity was about one fifth of the activity of the SV40 promoter (Fig 7D⇓, pSV-Luc). Deletion of a segment between −1497 and −874 bp reduced the luciferase activity by about 70%. Further deletion up to −47 bp did not significantly change the luciferase activity. The shortest deletion mutant (D5, −47 to +56 bp) still retained about 15% of the luciferase activity.
The biological role of the AT2 remains to be established, and the signaling mechanism is controversial.12 13 14 15 Its regulated expression in the developing rat fetus and newborn, however, suggests a developmental role of Ang II via AT2. Relative abundance of AT2 in the adult brain and adrenal medulla may suggest certain neuronal functions of AT2.
This study shows that the expression of mouse AT2 is also developmentally regulated in the mesenchymal tissue and brain. The expression of the mouse AT2 gene transcript as examined by the Northern blot analysis was in good agreement with previous binding studies of the rat fetus.8 A rapid decrease of AT2 mRNA immediately after birth was confirmed in mouse eviscerated carcass and brain. However, biological significance of this rapid shutoff of AT2 is still unclear. Dudley and Summerfelt23 reported that the expression of AT2 is regulated by growth factors in R3T3 cells, which are a subclone of the mouse embryonic fibroblast 3T3 cell line. Therefore, growth factors may be responsible for this rapid shutoff of this receptor.
Earlier ligand-binding studies showed that AT2 is expressed in the myometrium,4 adrenal medulla,5 heart,10 and several limited brain regions11 of adult rats. The mRNA of AT2 was detectable by conventional Northern blot analysis in the brain of 10-week-old mice. RT-PCR, however, confirmed that mRNA of AT2 is expressed in other mouse organs, such as heart and kidney, at a low level, although not all the organs that had been reported to express an Ang II receptor that is sensitive to PD123319 were examined. In the present study, it was not determined whether the cells responsible for the expression of the AT2 are parenchymal cells or connective tissue cells in each organ. An in situ hybridization study is required to address this question. However, such a study in the adult rat kidney by Kakuchi et al24 did not find specific cell types in which the AT2 mRNA was concentrated. Recently, Nakajima et al25 reported that they could not detect any mRNA of the AT2 in adult tissue even by the RT-PCR method. The reason for the discrepancy between our results and those of Nakajima et al is not clear.
The Southern blot analysis of the mouse AT2 gene showed a single band when high-molecular-weight DNA was digested with EcoRI, BamHI, or HindIII. This suggests that the mouse AT2 gene exists at a single locus in the mouse genome. Although we looked for related genes by Southern blot analysis under a low-stringency hybridization condition using 35% formamide for hybridization, we could not detect any additional bands (data not shown). Based on these experiments, it is unlikely that another closely homologous subtype of the AT2 is present.
Tsutsumi and Saavedra26 proposed the presence of two subtypes of AT2 in the rat brain on the basis of the sensitivity of Ang II binding to the stable GTP analogue GTPγS. The contradictory observation between the genomic DNA analysis in the present study and ligand-binding assay may be explained by a tissue-specific posttranslational modification of the AT2 or the presence of a tissue-specific associated protein, or it may be just a difference between species. However, in the rat, a Southern blot analysis of the genomic DNA also showed a single band when digested with Xba I or Bgl II endonucleases (personal communication from Dr Claude Szpirer, 1994). Therefore, the last possibility is unlikely. At present, however, the possibility of the presence of a second PD123319-sensitive Ang II receptor could not be excluded.
Cloning and nucleotide sequencing revealed some features of the genomic structure of the AT2 gene: (1) The mouse AT2 gene is composed of three exons. The first (91-bp) and second (60-bp) exons are relatively short. (2) The third exon contains the entire coding region of the mouse AT2 gene, with a short segment of the 5′ untranslated region and the entire 3′ untranslated region. (3) The exon-intron boundary consensus sequences are conserved.
Primer extension experiments using fetus mRNA indicated the presence of two transcription initiation sites. The transcription initiation site is located 2 nucleotides downstream of the TATA box. The TATA box is usually located 25 to 30 bp upstream of the transcription initiation site. Therefore, the short distance between the TATA box and transcription initiation site in the mouse AT2 gene is unusual. Such cases, however, have been reported in the EMBL Nucleotide Sequence Database.27 These features and a restriction map are presented schematically in Fig 7A⇑. The restriction sites were determined by nucleotide sequence, Southern blot analysis of the genomic DNA, and genomic DNA clone.
As is frequently but not always the case with seven transmembrane domain–type receptors, the coding sequence of the mouse AT2 receptor gene is not interrupted by an intron. This feature was used in our recent cloning of the human AT2 gene from a human genomic DNA library.28
It is intriguing that the AT1 and AT2 receptors share certain common features. Both have seven transmembrane domains and a single exon for their coding region, and they bind to Ang II, although the amino acid sequence homology is low.
Rapid shutoff of the AT2 receptor expression after birth in some brain nuclei and mesenchymal tissue suggests the importance of this receptor in fetal development. Therefore, studies on the transcriptional control of AT2 receptor gene are essential for understanding the biological roles of the rapid shutoff of this receptor. In this study, we showed that a 1.5-kb stretch of the upstream region of the two transcription initiation sites of the mouse AT2 gene is functional as a promoter in R3T3 cells. Deletion of a DNA segment between −1497 and −874 bp reduced the relative luciferase activity by 70%, and further deletion up to −47 bp did not greatly affect the residual luciferase activity. The segment between −47 and +56 bp maintained about 15% of the relative luciferase activity. These data suggest that (1) within the segment between −47 and +56 bp, where there is a TATA box (Fig 7B⇑), the region may be responsible for the basal promoter activity; and (2) the segment between −1497 and −874 bp with strong promoter activity contains several cis-acting elements. There are some consensus sequences of cis DNA elements such as C/EBP, NF-IL6, and AP1 in this region (Fig 7B⇑). These elements may be important for the expression of AT2 gene in R3T3 cells. Dudley and Summerfelt23 reported that AT2 receptor in R3T3 cells was downregulated by fibroblast growth factor and bombesin. Kambayashi et al29 showed that the expression of the AT2 receptor in vascular smooth muscle cells was suppressed by platelet-derived growth factor. Changes in the humoral environment such as increase in growth factors after birth may be responsible for the rapid shutoff of this receptor after birth. Studies in search of the cis DNA element responsible for the growth factor–mediated downregulation of this receptor are important and are in progress in our laboratory.
This work was supported in part by research grants HL-14192 and HL-35323 from the US Public Health Service, National Institutes of Health. We especially thank T. Fitzgerald for her excellent technical assistance in cell culture and Dr Erwin J. Landon for critically reading the manuscript.
- Received September 28, 1994.
- Accepted February 1, 1995.
- © 1995 American Heart Association, Inc.
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