Chromosomal localization of three human D5 dopamine receptor genes

It is currently thought that genetic predisposition to imbalances in dopaminergic transmission may underlie several neurological disorders, including schizophrenia, manic depression, Tourette syndrome, Parkinson disease, Huntington disease, and alcohol abuse. Originally two receptors, D1 and D2, were thought to account for all of the pharmacological actions of dopamine. However, through homology screening three additional genes, D3, D4, and D5, and two pseudogenes closely related to D5 have been characterized. To begin our genomic and evolutionary analyses of the human D5 dopamine receptor gene and its two pseudogenes, we have mapped each of them to their respective chromosomes. By combining in situ hybridization results with sequence analysis of PCR products from microdissected chromosomes, somatic cell hybrids, and radiation hybrids, we have assigned DRD5 (the locus containing the functional human D5 receptor gene) to chromosome 4p16.1, DRD5P1 (the locus containing D5 pseudogene 1) to chromosome 2p11.1-p11.2, and DRD5P2 (the locus of D5 pseudogene 2) to chromosome 1q21.1.     

Localization of the D5 dopamine receptor gene to human chromosome 4p15.1-p15.3, centromeric to the Huntington's disease locus.

Genes encoding G-protein-coupled receptors, including dopamine, serotonin, muscarinic cholinergic, and adrenergic receptors, play an important role in neurotransmission and may be involved in the pathophysiology of diseases such as Alzheimer's disease, Parkinson's disease, or Huntington's disease (HD). We mapped the gene encoding the D5 dopamine receptor (DRD5) to human chromosome 4p, an area implicated in HD and the Wolf-Hirschhorn syndrome, using gene-specific amplification with the polymerase chain reaction on a panel of somatic cell hybrids carrying different human chromosomes. Further localization of the DRD5 gene was carried out through the isolation and analysis of yeast artificial chromosomes, fluorescence in situ suppression hybridization to human metaphase chromosomes, and analysis of a panel of somatic cell hybrids subdividing human chromosome 4 into nine regions. The human DRD5 gene is located at 4p15.1-p15.33, centromeric to the location of the Huntington's disease locus although not in the obligate area containing the HD gene. The localization of the DRD5 gene to 4p15.1-p15.33 suggests the possibility that cis-position effects could be responsible for the altered D1-type dopamine receptor number observed in HD tissues or that the DRD5 gene could be a candidate for some of the abnormalities associated with the Wolf-Hirschhorn syndrome.     

Human dopamine D5 receptor pseudogenes.

Molecular cloning studies have now identified five structurally homologous genes encoding the biosynthesis of the human dopamine receptors, DRD1, DRD2, DRD3, DRD4, and DRD5. Two of these dopamine receptors (DRD1 and DRD5) are encoded by intronless genes. To ascertain whether there are other intronless genes that share identity with the gene (DRD5) encoding the DRD5 receptor, we used a cloning method based on the polymerase chain reaction (PCR). Human genomic DNA was amplified by PCR with oligodeoxyribonucleotides (oligos) based on the DRD5 nucleotide (nt) sequence. Amplification of nt sequences between these oligos allowed the isolation of two independent intronless genes that share identity with DRD5. The full-length clones have also been isolated by screening human genomic libraries. The deduced amino acid sequences for these genes, PG-1 and PG-2, share 91% and 92% identity to DRD5, respectively. However, each of the genes contains differences in the coding regions that would render these genes incapable of encoding functional receptors. Thus, the human genome contains at least two DRD5 pseudogenes, consistent with in situ human chromosomal hybridization analysis which reveals the presence of two pseudogenes.     

Structure and linkage of the D2 dopamine receptor and neural cell adhesion molecule genes on human chromosome 11q23.

The gene encoding the D2 dopamine receptor (DRD2) is located on human chromosome 11q23 and has been circumstantially associated with a number of human disorders including Parkinson's disease, schizophrenia, and susceptibility to alcoholism. To determine the physical structure of the DRD2 gene, we utilized cosmid cloning, isolation of yeast artificial chromosomes (YACs), and pulsed-field gel electrophoresis to construct a long-range physical map of human chromosome 11q23 linking the genes for the DRD2 and neural cell adhesion molecule (NCAM). The D2 dopamine receptor gene extends over 270 kb and includes an intron of approximately 250 kb separating the putative first exon from the exons encoding the receptor protein. The resulting physical map spans more than 1.5 mb of chromosome band 11q23 and links the DRD2 gene with the gene encoding the NCAM located 150 kb 3' of the DRD2 gene and transcribed from the same DNA strand. We additionally located the sites of at least four hypomethylated HTF islands within the physical map, which potentially indicate the sites of additional genes. High-resolution fluorescent in situ suppression hybridization using cosmid and YAC clones localized this gene cluster between the ApoAI and STMY loci at the interface of bands 11q22.3 and 11q23.1.     

The human dopamine D2 receptor gene is located on chromosome 11 at q22-q23 and identifies a TaqI RFLP.

Human dopaminergic neurons are involved in the control of hormone secretion, voluntary movement, and emotional behavior. Mediating these effects are the dopamine D1 and D2 receptors. These macromolecules belong to a large family of related sequences known as the G protein-coupled receptors. The D2 receptors have been of special interest because they bind, with high affinity and specificity, many of the commonly prescribed antipsychotic drugs. We previously isolated a full-length cDNA clone of the rat D2 receptor. When a chromosome mapping panel was probed with the rat D2 receptor cDNA a 15-kb EcoRI restriction fragment was identified and localized to human chromosome 11. The rat cDNA was also used to clone a human genomic fragment, lambda hD2G1, which contains the last coding exon of the D2 receptor gene (DRD2) and 16.5 kb of 3' flanking sequence. Hybridization of lambda hD2G1 to a chromosome 11 regional mapping panel localized DRD2 to 11q. In situ hybridization of lambda hD2G1 to metaphase chromosomes refined this assignment to the q22-q23 junction of chromosome 11. A search for RFLPs associated with D2DR identified a frequent two-allele TaqI RFLP.     

Genomic analysis and functional expression of canine dopamine D2 receptor

Dopamine D2 receptor (DRD2) is one of the five dopamine receptors with seven transmembrane domains that are coupled to the G protein. We have cloned and characterized the genomic and cDNA sequences of the canine DRD2 gene, which are 12.7 and 2.7 kb in size, respectively. The genomic DNA is composed of seven exons and six introns, encoding a 443 amino acid protein with 95% amino acid identity to other mammalian D2 receptors. A length polymorphism was detected in intron 3 of the receptor gene. We also characterized alternatively spliced forms of DRD2 cDNAs, DRD2L and DRD2S. They showed a higher level of expression in midbrain and thalamus. The ratio between the long and short form is similar in RT-PCR reaction. In human and rodent, the same two spliced forms are known to be coupled to G(i)-type heterotrimeric GTP binding protein, thereby opening an inwardly rectifying potassium channel, GIRK1. When the canine DRD2L and DRD2S were heterologously expressed in Xenopus oocytes, both forms activated GIRK1 potassium channels through coupling with G(i) protein. This activation was dose-dependent, demonstrating its ligand specificity.     

Parkin Expression Profile in Dopamine D3 Receptor Knock-Out Mice Brains

Patients affected by autosomic recessive juvenile parkinsonism (ARJP) exhibit parkin gene mutations with brain decrease in dopamine D2/D3 binding sites. To date, there are no data indicating whether the reduction in dopamine D3 receptors (DRD3) may be associated with the expression of specific parkin variants. In the present study we investigated parkin expression profile in DRD3 knock-out mice brains. RT-PCR analysis was performed to assess qualitative changes in parkin isoforms’ distribution pattern and in exons’ expression both in wild type controls and dopamine D3 receptor’s knock-out mice. Real-time PCR was performed to quantify single exons mRNA. Results demonstrated that exons 1, 2, 4, 6, 7, 8, were more expressed in wild type compared to dopamine D3 receptor KO mice brains while some other (3, 9, 10) were lower expressed. The expression levels of exons 5, 11 and 12 did not change in both animal groups. Our analysis was confirmed by western blot, which showed that parkin protein levels were influenced by the absence of DRD3.     

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.     

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.     

DRD2/CHRNA5 Interaction on Prefrontal Biology and Physiology during Working Memory

Background

Prefrontal behavior and activity in humans are heritable. Studies in animals demonstrate an interaction between dopamine D2 receptors and nicotinic acetylcholine receptors on prefrontal behavior but evidence in humans is weak. Therefore, we hypothesize that genetic variation regulating dopamine D2 and nicotinic acetylcholine receptor signaling impact prefrontal cortex activity and related cognition. To test this hypothesis in humans, we explored the interaction between functional genetic variants in the D2 receptor gene (DRD2, rs1076560) and in the nicotinic receptor α5 gene (CHRNA5, rs16969968) on both dorsolateral prefrontal cortex mediated behavior and physiology during working memory and on prefrontal gray matter volume.

Methods

A large sample of healthy subjects was compared for genotypic differences for DRD2 rs1076560 (G>T) and CHNRA5 rs16969968 (G>A) on prefrontal phenotypes, including cognitive performance at the N-Back task, prefrontal physiology with BOLD fMRI during performance of the 2-Back working memory task, and prefrontal morphometry with structural MRI.

Results

We found that DRD2 rs1076560 and CHNRA5 rs16969968 interact to modulate cognitive function, prefrontal physiology during working memory, and prefrontal gray matter volume. More specifically, CHRNA5-AA/DRD2-GT subjects had greater behavioral performance, more efficient prefrontal cortex activity at 2Back working memory task, and greater prefrontal gray matter volume than the other genotype groups.

Conclusions

The present data extend previous studies in animals and enhance our understanding of dopamine and acetylcholine signaling in the human prefrontal cortex, demonstrating interactions elicited by working memory that are modulated by genetic variants in DRD2 and CHRNA5.     

A Gene X Environment Interaction Between DRD2 and Religiosity in the Prediction of Adolescent Delinquent Involvement in a Sample of Males

Human behavioral phenotypes are the result of complex interactions between genotype and the environment. Still, much remains unknown about the gene X environmental basis to adolescent delinquent involvement. Using data from the National Longitudinal Study of Adolescent Health, we examine whether a polymorphism in the dopamine D2 receptor (DRD2) gene interacts with religiosity to predict variation in adolescent delinquent involvement. The results of the analyses revealed a gene X environment interaction between the A-1 allele of DRD2 and religiosity in the prediction of adolescent delinquency. Limitations are noted, and the implications of the findings are discussed.     

No evidence for strong recent positive selection favoring the 7 repeat allele of VNTR in the DRD4 gene

The human dopamine receptor D4 (DRD4) gene contains a 48-bp variable number of tandem repeat (VNTR) in exon 3, encoding the third intracellular loop of this dopamine receptor. The DRD4 7R allele, which seems to have a single origin, is commonly observed in various human populations and the nucleotide diversity of the DRD4 7R haplotype at the DRD4 locus is reduced compared to the most common DRD4 4R haplotype. Based on these observations, previous studies have hypothesized that positive selection has acted on the DRD4 7R allele. However, the degrees of linkage disequilibrium (LD) of the DRD4 7R allele with single nucleotide polymorphisms (SNPs) outside the DRD4 locus have not been evaluated. In this study, to re-examine the possibility of recent positive selection favoring the DRD4 7R allele, we genotyped HapMap subjects for DRD4 VNTR, and conducted several neutrality tests including long range haplotype test and iHS test based on the extended haplotype homozygosity. Our results indicated that LD of the DRD4 7R allele was not extended compared to SNP alleles with the similar frequency. Thus, we conclude that the DRD4 7R allele has not been subjected to strong recent positive selection.     

Characterization and mapping of bovine dopamine receptors 1 and 5

A cDNA encoding the bovine dopamine receptor 1 (DRD1) was isolated from a bovine cDNA library, cloned and completely sequenced. The coding region showed 93 and 91% sequence identity on DNA level and 96 and 94% on protein level with its respective porcine and human orthologs. The bovine DRD1 and dopamine receptor 5 (DRD5) were mapped, respectively, to BTA10 and 6 by radiation hybrid mapping. One SNP was found in DRD1 and four in DRD5. Using polymerase chain reaction-restriction fragment length polymorphism, 11 different European cattle breeds were screened for the presence of the DRD1 and DRD5 substitutions. Allele frequencies for DRD1 and DRD5 alleles were very similar across all the breeds examined. Allele frequency discrepancies were found between Belgian Blue beef breed and the other breeds.     

Folding efficiency is rate-limiting in dopamine D4 receptor biogenesis

Dopamine receptors are G protein-coupled receptors that are critically involved in locomotion, reward, and cognitive processes. The D2 class of dopamine receptors (DRD2, -3, and -4) is the target for antipsychotic medication. DRD4 has been implicated in cognition, and genetic studies have found an association between a highly polymorphic repeat sequence in the human DRD4 coding region and attention deficit hyperactivity disorder. Using DRD4 as a model, we show that antipsychotics can function as potent pharmacological chaperones up-regulating receptor expression and can also rescue a non-functional DRD4 folding mutant. This chaperone-mediated up-regulation involves reduced degradation by the 26 S proteasome; likely via the stabilization of newly synthesized receptor in the endoplasmic reticulum. Dopamine itself can function as a chaperone when shuttled into the cell by means of the dopamine transporter. Furthermore, different repeat variants of DRD4 display differential sensitivity to this chaperone effect. These data suggest that folding efficiency may be rate-limiting for dopamine receptor biogenesis and that this efficiency differs between receptor variants. Consequently, the clinical profile of dopaminergic ligands, including antipsychotics, may include their ability to serve as pharmacological chaperones.     

Overexpression of dopamine D2 receptors reduces alcohol self-administration

The mechanism(s) underlying predisposition to alcohol abuse are poorly understood but may involve brain dopamine system(s). Here we used an adenoviral vector to deliver the dopamine D2 receptor (DRD2) gene into the nucleus accumbens of rats, previously trained to self-administer alcohol, and to assess if DRD2 levels regulated alcohol preference and intake. We show that increases in DRD2 (52%) were associated with marked reductions in alcohol preference (43%), and alcohol intake (64%) of ethanol preferring rats, which recovered as the DRD2, returned to baseline levels. In addition, this DRD2 overexpression similarly produced significant reductions in ethanol non-preferring rats, in both alcohol preference (16%) and alcohol intake (75%). This is the first evidence that overexpression of DRD2 reduces alcohol intake and suggests that high levels of DRD2 may be protective against alcohol abuse.     

5' UTR polymorphism of dopamine receptor D1 (DRD1) associated with severity and temperament of alcoholism

Multiple dopamine receptors in the dopaminergic system may be prime candidates for genetic influence on alcohol abuse and dependence due to their involvement in reward and reinforcing mechanisms. Genetic polymorphisms in dopamine receptor genes are believed to influence the development and/or severity of alcoholism. To examine the genetic effects of the Dopamine Receptor D1 (DRD) gene family (DRD1-DRD5) in the Korean population, 11 polymorphisms in the DRD gene family were genotyped and analyzed in 535 alcohol-dependent subjects and 273 population controls. Although none of the polymorphisms of DRD1-5 genes were found to be associated with the risk of alcoholism, one 5' UTR polymorphism in the DRD1 (DRD1-48A>G) gene was significantly associated with severity of alcohol-related problem, as measured by the Alcohol Use Disorders Identification Test (AUDIT) in a gene dose-dependent manner, i.e., 24.37 (+/-8.19) among patients with -48A/A genotype, 22.37 (+/-9.49) among those with -48A/G genotype, and 17.38 (+/-8.28) among those with -48G/G genotype (P=0.002). The genetic effects of DRD1-48A>G were further analyzed with other phenotypes among alcohol-dependent subjects. Interestingly, the DRD1-48A>A genotype was also found to be associated with novelty seeking (NC), harm avoidance (HA), and persistence (P) (P =0.01, 0.02, and 0.003, respectively). The information derived from this study could be valuable for understanding the genetic factors involved in alcoholic phenotypes and genetic distribution of the DRD gene family, and could facilitate further investigation in other ethnic groups.     

Additive effects of the dopamine D2 receptor and dopamine transporter genes on the error-related negativity in young children

The error-related negativity (ERN) is a negative deflection in the event-related potential that occurs approximately 50 ms following the commission of an error at fronto-central electrode sites. Previous models suggest dopamine plays a role in the generation of the ERN. We recorded event-related potentials (ERPs) while 279 children aged 5-7 years completed a simple Go/No-Go task; the ERN was examined in relation to the dopamine D2 receptor (DRD2) and dopamine transporter (DAT1) genes. Results suggest an additive effect of the DRD2 and DAT1 genotype on ERN magnitude such that children with at least one DRD2 A1 allele and children with at least one DAT1 9 allele have an increased (i.e. more negative) ERN. These results provide further support for the involvement of dopamine in the generation of the ERN.