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Henry Wellcome Laboratories for Integrative Neuroscience and Endocrinology (LINE), Dorothy Hodgkin Building, Whitson Street, University of Bristol, Bristol BS1 3NY, UK

(Requests for offprints should be addressed to A-M O’Carroll; Email: A.M.OCarroll{at}bristol.ac.uk)

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), glucocorticoid receptor and CCAAT enhancer binding protein (C/EBP) 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.