Polymorphism of the dopamine D2 receptor gene in populations from the Volga-Ural region

The PCR technique was used to analyze the TaqIA- and NcoI-polymorphisms at the dopamine D2 receptor gene (DRD2) in eight populations of the Volga-Ural region belonging to Turkic (Bashkirs, Tatars, and Chuvashes), Finno-Ugric (Maris, Komis, Mordovians, and Udmurts), and Eastern-Slavic (Russians) ethnic groups. Population-specific patterns of the main TaqIA- and NcoI-polymorphisms distribution were established. Specific trends in changes of genotype and allele frequency of the dopamine D2 receptor gene depending on the ethnicity of the population were revealed.     

Allelic variation of the dopamine receptor D4 gene polymorphic region in gibbons

We examined the tandem repeat sequence of the dopamine receptor D4 (DRD4) gene in 73 individuals derived from 8 species of gibbons (genusHylobates) in an attempt to assess the variability of this gene in gibbon species.H. syndactylus (subgenusSymphalangus) andH. concolor (subgenusNomascus), which were inferred to have diverged at an early time within the family Hylobatidae, shared only long repeat (7–8) alleles. On the other hand, DRD4 was highly polymorphic in gibbons of the subgenusHylobates, with 4-, 5-, 6-, 7-, and 8-repeat alleles being recognized. In this subgenus, 4- and 5-repeat alleles were found in the species distributed mainly in the southern islands such as Sumatra, Java, and Borneo but not in the species inhabiting the Asian continent. Sequence analysis indicated that the repeat structure of the gibbon DRD4 gene was quite complex but most of the 48-bp units could be classified into several groups across the species based on sequence similarities. However, the sequence of the 7-repeat allele ofH. muelleri was unique, since the repeat units had low similarities to other units of gibbons.     

Structure and expression of human and rat D2 dopamine receptor genes.

D2 dopamine receptor may be related with the pathogenesis of Parkinson's disease and schizophrenia. Furthermore, the antipsychotic drugs have high affinity for D2 dopamine receptor. We carried out the cloning of the genomic DNA for human D2 dopamine receptor and clarified the structure of this gene. Our isolated gene spans about 15 kbp and consists of seven exons interrupted by six introns. However, putative first exon was not yet identified. Spot blot hybridization analysis of cell sorter fractionated human chromosomal DNA with D2 receptor genomic DNA revealed the localization of this gene in the chromosome 11 fraction. We analyzed human genomic DNA by Southern blot hybridization with D2 dopamine receptor genomic DNA as a probe, but so far we could not find RFLP. Northern blot analyses of brain RNA of several animals and rat brain RNA after various treatments were carried out. Developmental changes of D2 dopamine receptor mRNA were observed in the rat brains.     

Purification of brain D2 dopamine receptor.

D2 dopamine receptors have been extracted from bovine brain using the detergent cholate and purified approximately 20,000-fold by affinity chromatography on haloperidol-sepharose and wheat germ agglutinin-agarose columns. The purified preparation contains D2 dopamine receptors as judged by the pharmacological specificity of [3H]spiperone binding to the purified material. The sp. act. of [3H]spiperone binding in the purified preparation is 2.5 nmol/mg protein. The purified preparation shows a major diffuse band at Mr 95,000 upon SDS-polyacrylamide gel electrophoresis and there is evidence for microheterogeneity either at the protein or glycosylation level. Photoaffinity labelling of D2 dopamine receptors also shows a species of Mr 95,000. The D2 dopamine receptor therefore is a glycoprotein of Mr 95,000.     

A new polymorphism in the gene for the dopamine D2 receptor

A new polymorphism has been identified in the gene for the dopamine D₂ receptor. The polymorphism was found by single stranded conformational polymorphism (SSCP) analysis. Sequencing revealed an insertion at a BsoF1 restriction site. This polymorphism in the 3- untranslated region was found at a frequency of 0.07 in Centre d'Etude du Polymorphisme Humain (CEPH) parents.     

Detection and characterization of additional DNA polymorphisms in the dopamine D2 receptor gene.

The gene encoding the dopamine D2 receptor (DRD2) has been suggested as a candidate gene for several mental disorders. We previously described the cloning and chromosomal mapping (to 11q22-q23) of a human DRD2 gene as well as its use for the detection of a two-allele TaqI RFLP with a minor allele frequency of 0.24, corresponding to a PIC of 0.30. Family linkage utilizing DRD2 would be facilitated if the PIC of the DRD2 locus were increased. To this end, we have used additional phage and cosmid clones in the vicinity of DRD2 to identify a new two-allele TaqI RFLP as well as a TG microsatellite polymorphism with a PIC of 0.62. We report localizations of the three polymorphisms on the restriction map of the DRD2 locus. The TaqI RFLPs are in apparent linkage equilibrium with the microsatellite, yielding a highly informative compound marker locus with a PIC of 0.76.     

Localization of dopamine D1 and D2 receptor mRNAs in the rat systemic and pulmonary vasculatures.

The present study was designed to evaluate the expression of dopamine D1 and D2 receptor mRNAs in systemic and pulmonary vasculatures. Using specific antisense riboprobes for dopamine D1 and D2 receptor cDNAs, in situ hybridization histochemistry was performed in the aorta, common carotid artery, vertebral artery, pulmonary artery, and superior vena cava of the adult male Sprague Dawley rat. In the case of the aorta, common carotid artery, and vertebral artery, dopamine D1 receptor mRNAs localized mainly in the smooth muscle cells of the tunica media. However, the signals of dopamine D2 receptor mRNAs were found in the endothelium and subendothelial layer of tunica intima, and interstitial cells of tunica adventitia. In the case of the pulmonary artery, signals of dopamine D1 receptor mRNAs were detected within the tunica intima, media, and adventitia. Expression of D2 receptor mRNAs was detected in the walls of small blood vessels within the tunica adventitia of the pulmonary artery. There were no detectable signals of dopamine D1 and D2 receptor mRNAs in the vein. The uneven distribution of dopamine D1 and D2 receptor mRNAs in the rat systemic vasculatures and pulmonary artery suggests that dopamine differentially regulates the vasodilation of the systemic and pulmonary arteries through the differential stimulation of dopamine D1 and D2 receptor.     

The distribution of a dopamine D2 receptor mRNA in rat brain

Based on the recently reported sequence of a dopamine D2 receptor cloned from rat brain, we prepared a series of cDNA probes to determine the distribution of mRNA encoding this receptor. Within the forebrain, D2 receptor mRNA is abundant in the caudate-putamen, accumbens nucleus and olfactory tubercle. Moderate to low levels of mRNA are observed in the medial habenular nucleus, diagonal band, lateral septal nucleus, claustrum, dorsal endopiriform nucleus, and entorhinal cortex. In the mesencephalon, D2 receptor mRNA is abundant within the substantia nigra, pars compacta, and the ventral tegmental area. Comparison of the distribution of the mRNA and ligand binding indicates that both presynaptic and postsynaptic D2 receptors of the nigrostriatal, mesolimbic and mesocortical pathways are derived from the same mRNA.     

Repeat sequences in the gene encoding the human D4 dopamine receptor.

The gene encoding the human D4 dopamine receptor has evolved by gaining at least five internal repeats which are located within exons 3 and 4, and in the intervening intron 3 sequence. The amino acid sequence in the cytoplasmic loop of the receptor, involved in G protein coupling, has been altered by these gene changes.     

An antibody to dopamine D2 receptor inhibits dopamine antagonist and agonist binding to dopamine D2 receptor cDNA transfected mouse fibroblast cells.

A polyclonal antibody to dopamine D2 receptor (D2-receptor) has been used to examine the immuno-inhibition in the binding of a D2 antagonist, [3H]YM09151-2 and an agonist, PPHT-fluorescein to dopamine receptor DNA transfected mouse fibroblast cells. The specific activity of the [3H]YM09151-2 binding to transfected (Ltk-RGB) cells is 4-5 fold higher than untransfected (Ltk-) cells. The antibody is able to inhibit the [3H]YM09151-2 binding to the cell membranes from Ltk-RGB cells (Bmax 110.56 +/- 5.26 and 76.20 +/- 5.18 fmoles/mg protein in the presence of preimmune and immune sera, respectively, with no change in the Kd). The flow cytometric analysis of the PPHT-fluorescein labeled Ltk- and Ltk-RGB cells indicated that ligand specific fluorescence is associated only with small Ltk-RGB cells (second peak) and autofluorescence with large cells (first peak). Preincubation of the Ltk-RGB cells with antibody, reduced the fluorescence intensity of the PPHT-fluorescein by 20-25% without changing the auto-fluorescence. These results suggest that peptide antibody recognize D2-receptor in both membranes and in intact cells and interact at or near the ligand binding site of the receptor.