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(Circulation Research. 1995;76:693-700.)
© 1995 American Heart Association, Inc.

Articles

Expression, Genomic Organization, and Transcription of the Mouse Angiotensin II Type 2 Receptor Gene

Toshihiro Ichiki, Tadashi Inagami

From the Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, Tenn.

Correspondence to Tadashi Inagami, PhD, Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, TN 37232.

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
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.

Key Words: angiotensin II type 2 receptor • gene expression • reverse transcriptase–polymerase chain reaction • genomic DNA

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
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 CGP42112A^{4 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

Top Abstract Introduction Materials and Methods Results Discussion References

 
Reagents
Ang I, Ang II, Ang III, and Sarile ([Sar¹, Ile⁸] Ang II) were purchased from Peninsula Laboratories Inc. ³²P--dCTP and ³²P--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.

View larger version (32K): [in this window] [in a new window]   Figure 7. Schematic representation of the genomic organization of the mouse angiotensin II type 2 receptor (AT2) gene. A, Three boxes indicate the exons of the mouse AT2 gene. The shaded region indicates the coding region. Arrows shown below the first exon indicate two transcription initiation sites. The restriction map was deduced from the restriction sites of the genomic DNA clone of the AT2 gene and Southern blot analysis. B indicates BamHI; E, EcoRI; K, Kpn I; and S, Sac I restriction endonucleases. B, Detailed restriction map of the promoter region of the mouse AT2 gene. P indicates Pvu II; A, Acc I. Some consensus cis DNA elements are shown below. One of the transcription initiation sites located upstream is designated +1. C, Design of deletion mutant constructs of the promoter region of the mouse AT2 gene. DNA fragments -1497 through +56 bp, -874 through +56 bp, -427 through +56 bp, -284 through +56 bp, and -47 through +56 bp were fused to the luciferase gene and designated D1, D2, D3, D4, and D5, respectively. D, Relative luciferase activity in R3T3 cells of the deletion mutants shown in C. Ten micrograms of the AT2 promoter–luciferase constructs and 5 µg of pSVß-galactosidase were transfected to R3T3 cells by the calcium precipitation method. After 48 hours of transfection, cells were lysed and luciferase and ß-galactosidase activity were measured. The luciferase activity was normalized in reference to the ß-galactosidase activity. The relative luciferase activity in the D1 construct was set to 100%. A mock transfection was performed using the same amount of a promoterless luciferase gene with SV40 enhancer. pSV-Luc is driven by SV40 promoter and enhancer, and this construct was used as a positive control. Data are representative of three independent experiments and are shown as mean±SEM.

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 10x SSC buffer (1x 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, 5x SSC, 80 mmol/L sodium phosphate (pH 7.5), 2x 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 2x SSC for 5 minutes at room temperature followed by two washes with 2x 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.

View larger version (33K): [in this window] [in a new window]   Figure 1. Expression of mouse angiotensin II type 2 receptor (AT2) mRNA in fetal and adult mice examined by Northern blot analysis. Poly (A)+ RNA (1 µg) was electrophoresed in 1% agarose gel, transferred to a nylon membrane, and hybridized to a 32P-labeled mouse AT2 probe. A, RNA from eviscerated fetus carcasses and brains of the indicated gestation days (E) and 2-day-old newborn mice was examined. B, RNA of various organs from 10-week-old mice was examined. RNA size markers are shown at left. Similar results were obtained in the other two independent experiments.

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--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).

View larger version (95K): [in this window] [in a new window]   Figure 4. Nucleotide sequence of the mouse angiotensin II type 2 receptor (AT2) gene. A 4.5-kb EcoRI fragment of the mouse AT2 gene was cloned into the pBluescript vector, and nucleotide sequence was determined. Regions in boxes are exons of the AT2 gene. Solid triangles indicate the transcription initiation sites determined by primer extension experiments (Fig 5). A TATA box–like sequence (42 through 46 bp), the primer used in the primer extension experiments (111 through 130 bp) and reverse transcriptase–polymerase chain reaction (309 through 328 bp, 2646 through 2665 bp), and the exon-intron boundary consensus sequences (139 through 140 bp, 293 through 294 bp, 355 through 356 bp, 1548 through 1549 bp) are underlined. The initiation codon (ATG) and termination codon (TAA) in the third exon are in bold type. Genebank accession number U 00768.

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, 4x10⁵ 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 pSVß-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 ß-galactosidase activity in the same sample was measured spectrophotometrically according to Sambrook et al¹⁹ and used to normalize the luciferase activity.

Animals
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.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
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.

View larger version (81K): [in this window] [in a new window]   Figure 2. Expression of mouse angiotensin II type 2 receptor (AT2) mRNA in adult organs and in R3T3 cells examined by reverse transcriptase–polymerase chain reaction (RT-PCR). Poly (A)+ RNA (1 µg) from the organs indicated below and total RNA (20 µg) from R3T3 cells were reverse transcribed using an oligo-dT primer, and cDNAs were amplified by PCR. The resultant products were electrophoresed in 1% agarose gel and stained with ethidium bromide. Lane 1, brain; lane 2, heart; lane 3, lung; lane 4, liver; lane 5, kidney; and lane 6, R3T3 cells. M indicates molecular weight marker (lambda DNA-BstEII-digest). Arrow indicates predicted size (1.2 kb) of PCR products of cDNA.

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.

View larger version (49K): [in this window] [in a new window]   Figure 3. Southern blot analysis of the mouse angiotensin II type 2 receptor (AT2) gene. High-molecular-weight DNA was prepared from the liver of BALB/c (lanes 1 through 4) and C57B/6 (lanes 5 through 8) mice, digested with restriction endonucleases indicated in the figure, electrophoresed, blotted, and hybridized to a 32P-labeled mouse AT2 probe. DNA size markers (lambda DNA-HindIII digest) are indicated at left.

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).

View larger version (47K): [in this window] [in a new window]   Figure 5. Photograph showing transcription initiation sites of the angiotensin II type 2 receptor (AT2) gene. Poly (A)+ RNA from eviscerated carcasses of 18-day-gestation mouse fetus (lane 1) and tRNA (lane 2) were reverse transcribed with a 32P-labeled primer specific for the first exon of the AT2 gene (Fig 4, nucleotide 111 through 130 bp). The products were electrophoresed in 6% acrylamide/8 mol/L urea gel. The nucleotide sequence was obtained with the same primer used for the determination of transcription initiation site. Two arrows indicate the 5' end (transcription initiation site) of the mRNA of the AT2 gene. Similar results were obtained in the other two independent experiments.

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.

View larger version (22K): [in this window] [in a new window]   Figure 6. Blot showing expression of angiotensin II type 2 receptor (AT2) mRNA in R3T3 cells. R3T3 cells were cultured in the medium with 10% fetal calf serum until they were confluent (lane 1), then serum was depleted for 1 day (lane 2) and 2 days (lane 3). Total RNA was prepared by the acid guanidinium-phenol-chloroform extraction method. Total RNA (15 µg) was applied in each lane and examined by Northern blot analysis as described in Fig 1. The positions of 28S and 18S ribosomal RNA are shown at left. Similar results were obtained in the other two independent experiments.

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 ß-galactosidase activity expressed by cotransfected pSVß-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.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
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 GTPS. 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.

	Acknowledgments

 
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.

	References

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