Molecular structure and transcriptional function of the rat vascular AT1a angiotensin receptor gene

Rat vascular angiotensin receptors (AT1a receptors) are encoded by two mRNA transcripts sharing an identical receptor coding sequence but differing in their 5' and 3' untranslated sequences. We screened male Sprague-Dawley rat genomic libraries to clone the vascular AT1a receptor gene. Two sets of overlapping clones were isolated that encode over 90 kb of genomic sequence around the AT1a receptor gene. Four overlapping clones were identified from the 5' flanking portion of the gene. These contain the promoter region and two exons, 141 bp and 89 bp in size, respectively, encoding the alternatively spliced 5' untranslated mRNA sequence. Six additional clones overlap each other but do not overlap the set of clones from the 5' flanking region of the gene. These contain a single 1977-bp exon that encodes 900 bp of the 5' and 3' untranslated sequences in addition to a 1077-bp open reading frame identical to that found in vascular smooth muscle cell AT1a receptor cDNAs. Primer extension and RNase protection studies indicate that the transcription start site for this gene begins 9 bp upstream from the most 5' sequence found within the AT1a receptor cDNAs. Our mapping studies of the cloned gene, which so far includes an uncloned gap within the second intron, indicate that the transcription start site is no less than 67 kb upstream from the receptor coding exon. Promoter-reporter assays were performed by transfection of vascular smooth muscle cells with deletions of a 3.2-kb promoter region fused to a luciferase cDNA reporter plasmid. Relatively strong basal transcriptional activity is observed from the 5'-most 2 kb of the promoter and diminishes markedly with deletions within 1 kb of the early promoter region, suggesting strong promoter elements in the more upstream regions of the gene. Deletion of a 53-bp early promoter region containing the transcription start site and a putative TATA box completely abolishes the ability of upstream elements to drive transcription of the luciferase cDNA. These results indicate that we have isolated the AT1a receptor gene and its functional promoter.