© 1995 American Heart Association, Inc.
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Expression, Genomic Organization, and Transcription of the Mouse Angiotensin II Type 2 Receptor Gene
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 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
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