Characterization of the 5′ flanking region of the rat D3 dopamine receptor gene

The D-3 dopamine receptor has a restricted regional distribution in brain and is regulated by dopaminergic agents. Additionally, the D-3 gene is implicated in the pathogenesis of several neuropsychiatric disorders or in their response to pharmacological agents. Elucidating its transcription control mechanisms is therefore of interest in order to explain these biological features of the D-3 gene. In this study, the 5' flanking region of the rat D-3 gene was characterized by isolating the 5' end of its cDNA as well as 4.6 kb of genomic sequence. Analysis of this region revealed the presence of two new exons 196-bp and 120-bp long, separated by an 855-bp intron, located several kilobases upstream of the previously published coding exons. Thus, current evidence indicates that the rat D-3 gene is organized into eight exons. Transcription initiation site was determined by primer extension analysis and repeated rounds of 5' RACE and was found to localize at a pyrimidine-rich consensus 'initiator' sequence, similar to the rat D-2 gene. The D-3 promoter lacks TATA or CAAT boxes but unlike that of other dopamine receptor genes has only 52% GC content. Functional analysis of D-3 promoter deletion mutants fused to a reporter gene in TE671 cells, which endogenously express this gene, revealed strong transcriptional activity localized within 36 nucleotides upstream of transcription start site, and a potent silencer between bases -37 and -537. The D-3 promoter is inactive in C6 and COS7 cells. We conclude that the D-3 gene, similar to the closely related D-2 gene, is transcribed from a tissue specific promoter which is under intense negative control.