Transcriptional regulation of the rat apelin receptor gene: promoter cloning and identification of an Sp1 site necessary for promoter activity

Abstract

The genomic structure and transcriptional regulation of the rat apelin receptor (APJR) were analysed by rapid amplification of 5′ cDNA ends (5′-RACE), transient expression assays and DNA–protein interaction. Analysis of the 5′-flanking region of a rat genomic clone shows no TATA box, but a putative CAAT box and several putative binding sites for transcription factors are present. Two transcriptional start sites were identified by 5′-RACE, RNase protection and primer extension analyses. Promoter activity was exhibited in the APJR- expressing SH-SY5Y cell line as well as in COS-7 and Chinese hamster ovary (CHO-K1) cells. Consecutive 5′-deletion analysis revealed the highest promoter activity in a region between bp −966 and −165. DNaseI footprint analysis revealed seven protected regions and electrophoretic mobility shift, super-shift and competition assays identified individual DNA–protein complexes capable of binding Sp1, estrogen receptor (ER)α, glucocorticoid receptor and CCAAT enhancer binding protein (C/EBP)γ transcription factors. Site-directed mutagenesis identified an individual Sp1 motif that plays a major role in activation of the APJR promoter and also demonstrated constitutive transcriptional regulation of the promoter by estrogen and glucocorticoid receptors. Promoter regulation by the cAMP-dependent signal cascade was also shown.