A NEGATIVE REGULATORY ELEMENT IN THE PROMOTER REGION OF THE RAT ALPHA(2)A-ADRENERGIC RECEPTOR GENE OVERLAPS AN SP1 CONSENSUS BINDING-SITE

Three subtypes of alpha(2)-adrenergic receptors (alpha(2)A, alpha(2)B and alpha(2)C) have been described that differ in their primary sequen ce and tissue-specific expression and are encoded by three distinct ge nes. Previous work has shown that the human alpha(2)A-adrenergic recep tor gene promoter consists of a TATA-box (TATAAA), palindromic sequenc e (CCCACGTGGG) and GC-box (GGGGCGG) motif. Sequence analysis of the pu tative promoter region of the rat alpha(2)A-adrenergic receptor gene s howed that these promoter regions are conserved in their sequence and relative location. We analysed the transcriptional activity of these r egions using RINm5F, a rat insulinoma cell line that expresses the end ogenous alpha(2)A-adrenergic receptor gene. These results showed that the region from -484 to -92 has a negative effect on transcription, as deletion of this region in alpha(2)A-adrenergic receptor gene-chloram phenicol acetyltransferase reporter constructs increased reporter gene activity. This region included the GC-box sequence which is a consens us binding site for the nuclear factor SP1, which is a positive activa tor of transcription. Gel-mobility-shift assays and supershift assays with an antibody that recognizes SP1 showed binding of the SP1 nuclear factor as well as other nuclear factors to this GC-box region. Additi onal nuclear factors bind to the downstream palindromic region. We sug gest that positive- and negative-acting nuclear factors contribute to the activity of the alpha(2)-adrenergic receptor promoter.