No direct effect of the -521 C/T polymorphism in the human dopamine D4 receptor gene promoter on transcriptional activity

Endogenous mRNA expression of the DRD4 gene was demonstrated in two neuroblastoma (SK-N-F1, IMR32) and one retinoblastoma cell line (Y79) by RT-PCR. In addition, very low DRD4 mRNA levels were also detected in HeLa cells. The transcriptional activity of a series of 5' promoter deletion mutants was determined by transient transfection of luciferase reporter constructs. The activity profile of these promoter fragments was similar in each of the cell lines tested. The highest luciferase reporter activity was obtained with a construct containing promoter sequences between nucleotides -668 to -389, while a putative silencer region was localised spanning from nucleotide -1571 to -800. Surprisingly, the -521 C/T polymorphism had no significant effect on transcriptional activity of the reporter construct with the highest activity (-668 to -389) in any of the three cell lines tested.Our results do not confirm previous data assigning different transcriptional activities to the -521 C/T alleles of the human DRD4 promoter. Furthermore, these findings highlight the need for further characterization of the 5' regulatory region of the DRD4 gene and identification of additional functional promoter polymorphic sites, especially in the context of haplotype.Dopamine, an important neurotransmitter in the brain, plays a major role in the control of motor functions and behavioral patterns via interacting with specific cell surface receptors. Dopamine receptors belong to the large family of G protein-coupled receptors and can be classified as D1-like (D1, D5) and D2-like (D2, D3, D4) subgroups based on their sequence homology and pharmacological characteristics [1]. The human dopamine D4 receptor (DRD4) gene was originally cloned by Van Tol et al. [2]. The 5' flanking region of the gene was characterized by Kamakura et al. [3] as a housekeeping gene-like promoter. The promoter lacks a TATA or CAAT box but several putative transcription factor binding sites and CpG islands have been identi