Basal level transcriptional regulation of the human angiotensin II type 1 receptor gene

The peptide hormone angiotensin II regulates a variety of physiological responses which are mediated by its interaction with high affinity G protein-coupled receptors localized on the surface of target cells. To gain insights into the transcriptional regulation of the human angiotensin II type 1 receptor (hAT(1)R) gene, we have isolated 1 kb of the 5'-flanking sequence of this gene. Expression constructs containing various 5'-deletions of the hAT(1)R promoter region, fused upstream to the luciferase reporter gene, were transiently transfected into H295-R, HEC-1B and A549 cells. It was demonstrated that a 145 bp sequence within the promoter region was required for basal level expression of the hAT(1)R gene in all of the three cell lines investigated. Computer analysis indicated the existence of numerous putative transcription factor binding sites in this region. Further detailed deletion data suggested essential transcription factor binding sites between -98 and -79 bp. Electrophoretic mobility shift assays revealed that four protein-DNA complexes were formed within the -98 to -79 bp region of the hAT(1)R gene when incubated with H295-R cell nuclear extract. Site-directed mutagenesis experiments showed that a putative Sp1 binding site was critical for the basal level expression of the hAT(1)R gene.