Characterization of the regulatory region ofAdra2c, the gene encoding the murine a2C adrenoceptor subtype

The 5′ flanking sequence (3,227 base pairs, bp) of the mouseAdra2c subtype gene was determined and characterized. The transcription start site was mapped to nucleotide ‘A’ of two initiator motifs in tandem array, i.e. 1,159 and 1,153 bp upstream from the initiation codon of the open reading frame (ORF) ofAdra2c, respectively. One structural feature salient to the 5′ regulatory region ofAdra2c is present in the sequence 1 kb immediately upstream from the receptor ORF, which is highly enriched in GC content (76%) and CpG island counts (i.e. CpG/GpC, 146:177), and thus rich in Sp1-binding motifs. At the 3′ flanking region, the polyadenylation signal was mapped to 481 bp downstream from the termination codon. The transcript defined by sequence data thereby is consistent with a size of 3 kb (brain form) determined by Northern blot analysis. The transgene,Adra2c-NN-lacZ, which links the promoter region ofAdra2c to thelacZ reporter gene, was constructed in order to evaluate the functional capacity of the promoter and the putative motifs residing within the defined regulatory region (1.9 kb upstream from the ORF) in directing the reporter gene expression in vitro in transiently transfected cells and in vivo in transgenic (Tg) mice. Permissive cell types toAdra2c-NN include those derived from neural and kidney lineages. SignificantAdra2c-NN-driven reporter expression in Tg mice established suggests that α2C adrenoceptor expression is permissive underAdra2c-NN in central (cerebral cortex, hippocampus, subthalamus, hypothalamus, superior colliculus, cerebellum, and brain stem) and peripheral (pancreatic β-islets) tissues.