THE HUMAN DOPAMINE D5 RECEPTOR GENE - CLONING AND CHARACTERIZATION OFTHE 5'-FLANKING AND PROMOTER REGION

Genomic and overlapping cDNA clones encompassing the entire 5'-untrans lated region of the human D5 receptor gene were cloned and sequenced. Comparison of these human D5 receptor genomic and cDNA clones revealed the presence of two exons separated by a small and variably sized int ron (of either 179 or 155 bp). We have determined that the major site of transcription initiation of the D5 gene is 2125 bp upstream from th e translational initiation start site. The region 5' to the transcript ion initiation site lacked conventional TATA and CAAT sequences, but c ontained several putative binding sites for transcription factors, suc h as Spl and Apl. Luciferase reporter gene constructs containing D5 ge ne sequence information up to 500 bp 5' of the transcription initiatio n site were able to stimulate transcription only in SK-N-SH cells but not in COS-7, CHO, PI9EC, NB41A3, and SK-N-MC cell lines. Promoter del etion analysis indicated that the D5 gene promoter contained a positiv e modulator at 119-182 and a negative modulator 251-500 bases upstream from the site of transcription initiation. In addition, in order to d etect the expression of functional D5 receptor mRNAs and not those of its expressed pseudogenes, irt situ hybridization analysis of monkey a nd human brain using a 5' DS-specific riboprobe revealed that D5 recep tor mRNA was most abundant in discrete cortical areas (layers II, TV, and VI), the dentate gyrus, and hippocampal subfields with very little message detected in the striatum. Unexpectedly, D5 mRNA antisense rib oprobes labeled discrete cell bodies in the pars compacta of the subst antia nigra. The characterization of the genomic organization of the D 5 receptor gene and of those factors involved in its transcriptional r egulation may aid in our understanding of the role this gene product p lays in the generation and maintenance of dopamine D1-like receptor-me diated events.