Activator region analysis of the human D1A dopamine receptor gene.

Abstract

The human D1A dopamine receptor gene is transcribed from a TATA-less promoter, and its main activator is between nucleotides -1342 and -1102 relative to the first ATG (Minowa, M. T., Minowa, T., Monsma, F. J., Jr., Sibley, D. R., and Mouradian, M. M. (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 3045-3049). In the present study, additional serial deletion mutants fused with a reporter gene and used in transient transfections of the D1A-expressing cell line NS20Y further localized this activator to two regions: -1154 to -1137, which increases promoter activity 4-fold, and -1197 to -1154, which confers an additional 2-fold increase in transcriptional activity. Cell specificity of the D1A gene expression is contributed by both the -1197/-1154 region and the core promoter located downstream to -1102. DNase I footprinting and gel shift assays suggested that DNA-protein interactions occur primarily between -1197 and -1116. Competitive cotransfections using different portions of this segment, D1A-Act-1 (-1197 to -1152) and D1A-Act-2 (-1154 to -1116), confirmed the in vivo functional significance of these sequences in activating the D1A promoter. D1A-Act-1 has no consensus sequences for known transcription factors but binds to several proteins as a complex. Although D1A-Act-2 has AP2 consensus sequences and binds to recombinant AP2, the nuclear factors in NS20Y cells interacting with this region are distinct from AP2 itself. Despite the inability of Sp1 to directly bind to its consensus sequences in D1A-Act-2, Sp1 or another protein antigenically related to Sp1, as well as a novel nuclear factor, is included in the complex that binds to this activator.