CHARACTERIZATION OF THE RAT A2A ADENOSINE RECEPTOR GENE

To understand the molecular basis for the regulation of rat A2a adenos ine receptor (A2a-R) expression, we have characterized the rat A2a-R g ene and defined its promoter regions, Through a combination of restric tion mapping and sequence analysis, we have demonstrated that the rat A2a-R gene is composed of two exons interrupted by a 7,2-kb intron, Pr imer extension and RNase protection on RNA isolated from PC12 cells su ggested that the A2a-R gene encoded two clusters of alternative transc ripts, The most upstream transcription start site was designated as fl , The sequence of the proximal 1.5 kb of 5'-flanking region demonstrat ed no potential TATA box, CCAAT box, or initiator element in the appro priate location, Varying lengths of 5'-flanking regions were inserted into a transient expression vector (pGL(2)-basic), which contained bac terial luciferase as the reporter gene, to determine its promoter regi on(s) in PC12 cells, CHOP cells, and C6 cells, Consistent with two clu sters of transcription start sites, two independent functional promote r regions (designated P1, -67/-1; and P2, +272/+304) for the rat A2a-R gene were identified, Although both promoters are in use in PC12 cell s, only P2 is active in CHOP cells, indicating possible cell line-spec ific usage of these two promoters.