Efficient transcription of the human angiotensin II type 2 receptor gene requires intronic sequence elements

To investigate mechanisms of human angiotensin II type 2 receptor (hAT2) ge ne regulation we functionally characterized the promoter and downstream reg ions of the gene. 5'-Terminal deletion mutants from -1417/+100 to -46/+100 elicited significant but low functional activity in luciferase reporter gen e assays with PC12W cells. Inclusion into the promoter constructs of intron 1 and the transcribed region of the hAT2 gene up to the translation start enhanced luciferase activity 6.7+/-1.6-fold and 11.6+/-1.7-fold (means+/-S. E.M.) respectively, whereas fusion of the promoter to the spliced 5' untran slated region of hAT2 cDNA did not, which indicated an enhancement caused b y intronic sequence elements. Reverse transcriptase-mediated PCR confirmed that the chimaeric hAT2-luciferase mRNA was regularly spliced in PC12W cell s. A Northern blot analysis of transfected cells showed levels of luciferas e mRNA expression consistent with the respective enzyme activities. Mapping of intron 1 revealed that a 12 bp sequence in the centre of the intron was required for the increase in promoter activity, whereas the 5' adjacent in tronic region mediated a decrease in luciferase activity. Mutation of the 1 2 bp region led to altered protein binding and markedly decreased luciferas e activity. Cloned into a promoterless luciferase vector, a 123 bp intron 1 fragment was able to direct reporter gene expression to the same activity as occurred in conjunction with the 5' flanking region. These results indic ate that sequence elements in intron 1 are necessary for efficient transcri ption of hAT2. In reporter gene assays, intron 1 might by itself function a s a promoter and initiate transcription from an alternative start point.