INSERM U388, Institut Louis Bugnard, CHU Rangueil, 31403 Toulouse, France.
Screening of a foetal brain genomic DNA library allowed to isolate a 10-kb fragment of the gene encoding the human alpha2B-adrenergic receptor, that contained 5.5 kb of the 5'-flanking region, the open reading frame and 2.9 kb of the 3'-flanking region. The 1-kb fragment upstream from the start codon was rich in GC, lacked consensus TATA or CAAT box, but contained several Sp1-binding sites. Other potential cis-regulatory elements found in the 5'-flanking region included AP2, USF, Stat-6, NFkappaB and Olf-1. A single canonical polyadenylation signal (AATAAA) was found at position +3252/+3257 and the polyadenylation site was 3274 nucleotides downstream from ATG. Transfection experiments with chimeric luciferase constructs containing various truncated fragments of the 5'-region showed that the fragment -3160/+3 exhibited promoter activity in all tested cell lines and permitted the definition of a minimal 200-bp promoter (-603/-411) containing three putative Sp1-binding sites and two initiator elements. Transcriptional activity of this region was inhibited by the addition of mithramycin, a specific inhibitor of Sp1 binding to GC-rich sequences. The search for sequence variants within a fragment covering 1.7 kb of 5'-flanking region and the coding region allowed us to identify five novel single nucleotide polymorphisms. Interestingly, the G/C substitution at position -98 relative to the start codon was common and in complete linkage with a previously identified insertion/deletion polymorphism in the coding region which was showed to affect alpha2B-adrenergic receptor function. Based on transfection data and computer-assisted sequence analysis, the -98 G/C single nucleotide polymorphism was located within a portion of the 5'-UTR (-127/+3) affecting luciferase activity and it created additional putative binding site for Sp1. However, G/C substitution had no significant incidence on promoter activity in BHK-21 or HeLa cells.