The transcription factors Sp1 and Sp3 are required for human angiotensin II type 1 receptor gene expression in H295-R cells

Abstract

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. Our previous studies have demonstrated that a 145 bp sequence within the promoter region was required for basal level expression of the human angiotensin II type 1 receptor (hAT 1R) gene. In the present study, deletional analysis of the hAT 1R promoter localized the major regulatory sequence to two overlapping GC boxes harbored within the −105 to −85 bp region relative to the transcription start site in H295-R cells. Electrophoretic mobility shift assays (EMSAs) using a double-stranded (ds) oligonucleotide corresponding to this region and H295-R cell nuclear extract resulted in five specific DNA–protein complexes. EMSAs performed with competitive ds-oligonucleotides which harbored the consensus binding site for Sp1 prevented the formation of the DNA–protein complexes. Supershift EMSAs also demonstrated that Sp1 and Sp3 could bind to the GC boxes present within the −105 to −85 bp region of the hAT 1R promoter. Transactivation experiments utilizing Drosophila SL2 cells, which lack endogenous Sp family transcription factors, demonstrated that Sp1 and Sp3 activated the hAT 1R promoter and that maximal activation was only achieved when both GC boxes were present. Taken together, these findings suggest that Sp1 and Sp3 are necessary for the expression of the hAT 1R gene in H295-R cells.