Molecular cloning and characterization of the 5'-flanking and promoter region of the human dopamine D5 receptor gene

Genomic and overlapping cDNA clones encompassing the entire 5$\sp\prime$-untranslated 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 intron (of either 179 or 155 bp). In this thesis it was demonstrated that the major site of transcription initiation of the D5 gene is 2125 bp upstream from the translational initiation start site. The region 5$\sp\prime$ to the transcription-initiation site lacked conventional TATA and CAAT sequences, but contained several putative binding sites for transcription factors, such as Sp1 and AP1. Luciferase reporter gene constructs containing D5 gene sequence information up to 500 bp 5$\sp\prime$ of the transcription initiation site were able to stimulate transcription in SK-N-SH and GH3 cells but not in COS-7, CHO, P19EC, NB41A3, and SK-N-MC cell lines. Promoter deletion analysis indicated that the D5 gene promoter contained positive modulators between nucleotides $-$52 to $-$119 and $-$120 to $-$182 upstream from the site of transcription initiation. Furthermore, a negative modulator $-$251 to $-$500 nucleotides upstream from the site of transcription initiation was also indicated. Within the positive modulatory region there existed a small dinucleotide repeat, termed (TC)$\sb{13}$. Genomic DNAs from 18 unrelated schizophrenic (n = 7), Parkinson's diseased (n = 2), Huntington's diseased (n = 6) and normal healthy individuals (n = 3) were analysed using a single-strand conformation polymorphism (SSCP) assay. SSCP analysis revealed the existence of two additional alleles, termed (TC)$\sb{12}$ and (TC)$\sb{14}$. Neither form significantly altered D5 promoter-mediated luciferase activity when compared to that of the wild-type control, suggesting that differences in the number of dinucleotide repeats are not likely of any functional consequence for D5 transactivation. Proteins from GH3 and SK-N-SH cell nuclear extracts were able to bind oligonucleotide probes directed to the positive and negative modulator regions of the D5 promoter in electrophoretic mobility shift experiments. Neither of the Sp1 consensus sequences appeared to bind to nuclear proteins and the involvement of Pit-1 and AP1 are currently under investigation. In addition, in order to detect the expression of functional D5 receptor mRNAs and not those of its expressed pseudogenes, in situ hybridisation analysis of monkey and human brain using a 5$\sp\prime$ DS-specific riboprobe revealed that D5 receptor mRNA was most abundant in discrete cortical areas (layers II, IV and VI), the dentate gyrus, and hippocampal subfields with very little message detected in the striatum. Unexpectedly, D5 mRNA antisense riboprobes labelled discrete cell bodies in the pars compacta of the substantia nigra. The characterization of the genomic organization of the D5 receptor gene and of those factors involved in its transcriptional regulation may aid in our understanding of the role this gene product plays in the generation and maintenance of dopamine D1-like receptor mediated events.