Association of seven polymorphisms of the D2 dopamine receptor gene with brain receptor-binding characteristics

Association of alleles at the Taql A, Taql B, intron 6, Taql D, exon 7, exon 8, and promoter-141C sites of the D2 dopamine receptor gene with D2 dopamine receptor binding characteristics in the caudate nucleus of Caucasian alcoholic and nonalcoholic subjects was determined. For the Taql D, exon 7, exon 8, and promoter-141C sites there were no significant allelic differences in Bmax (number of binding sites) or Kd (binding affinity) of the D2 dopamine receptors. However, subjects having the minor alleles at the Taql A, Taql B, and intron 6 sites had significantly lower Bmax than subjects not having them. None of these three polymorphisms had any significant effect on Kd. Highly significant linkage disequilibria were observed among the Taql A, Taql B, and intron 6 polymorphic sites, but linkage disequilibria between these three sites and each of the Taql D, exon 7, exon 8, and promoter-141C sites were of lesser or of no significance. Taken together, these findings suggest that the Taql A, Taql B, and intron 6 polymorphisms, but not the Taql D, exon 7, exon 8, and promoter-141C polymorphisms, are in linkage disequilibrium with a functional allelic variant that affects D2 dopamine receptor expression.     

Association of polymorphisms in the dopamine D4 receptor gene and the activity-impulsivity endophenotype in dogs

A variable number of tandem repeats (VNTR) polymorphism in exon 3 of the human dopamine D4 receptor gene ( DRD4 ) has been associated with attention deficit hyperactivity disorder (ADHD). Rodents possess no analogous repeat sequence, whereas a similar tandem repeat polymorphism of the DRD4 gene was identified in dogs, horses and chimpanzees. Here, we present a genetic association study of the DRD4 VNTR and the activity-impulsivity dimension of the recently validated dog-ADHD Rating Scale. To avoid false positives arising from population stratification, a single breed of dogs (German shepherd) was studied. Two DRD4 alleles (referred to as 2 and 3a ) were detected in this breed, and genotype frequencies were in Hardy–Weinberg equilibrium. For modelling distinct environmental conditions, 'pet' and 'police' German shepherds were characterized. Police German shepherds possessing at least one 3a allele showed significantly higher scores in the activity-impulsivity dimension of the dog-ADHD Rating Scale than dogs without this allele ( P  = 0.0180). This difference was not significant in pet German shepherds. To the best of our knowledge, this is the first report of an association between a candidate gene and a behaviour trait in dogs, and it reinforces the functional role of DRD4 exon 3 polymorphism.     

TaqIB allele polymorphism of the dopamine receptor D2 gene in patients with endogenous psychoses

The genotype at theTaqIB locus of the dopamine receptor D2 (DRD2) gene was established by PCR in 113 healthy donors and 340 patients with endogenous psychoses. The allele and genotype frequencies in patients with affective psychoses significantly differed from those in controls and in patients with schizophrenia. The proportion of B2/B2 homozygotes decreased with increasing contribution of the affective component to the clinical manifestation of schizo-affective and affective disorders. TheTaqIB polymorphism was assumed to play a role in mood aberrations in patients with mental disorders.     

Characterization of a novel D2-like dopamine receptor with a truncated splice variant and a D1-like dopamine receptor unique to invertebrates from Caenorhabditis elegans

We have cloned two novel Caenorhabditis elegans dopamine receptors, DOP-3 and DOP-4. DOP-3 shows high sequence homology with other D2-like dopamine receptors. As a result of alternative splicing, a truncated splice variant of DOP-3, DOP-3nf, was produced. Because of the in-frame insertion of a stop codon in the third intracellular loop, DOP-3nf lacks the sixth and seventh transmembrane domains that are found in the full-length DOP-3 receptor. Reporter gene assay showed that DOP-3 attenuates forskolin-stimulated cAMP formation in response to dopamine stimulation, whereas DOP-3nf does not. When DOP-3 was coexpressed with DOP-3nf, the ability to inhibit forskolin-stimulated cAMP formation was reduced. DOP-4 shows high sequence homology with D1-like dopamine receptors unique to invertebrates, which are distinct from mammalian D1-like dopamine receptors. Reporter gene assay showed that DOP-4 stimulates cAMP accumulation in response to dopamine stimulation. These two receptors provide new opportunities to understand dopaminergic signaling at the molecular level.     

Expression of D2 dopamine receptor in the mouse brain

The neurotransmitter, dopamine, binds to dopamine receptor (DR), and is involved in several functions of the brain, such as initiation and execution of movement, emotion, prolactin secretion, etc. Of all the five DRs, D2 dopamine receptor has maximal affinity for dopamine. D2 has a short isoform, D2S, and a long isoform D2L. D2L is longer than D2S by 29 amino acid residues. We studied the expression of the gene and protein of D2 receptor in the cerebral and cerebellar cortices of the brain of new born, developing, adult, and old male mice to find out: (i) at what stage of development, expression of the gene peaks and (ii) if it undergoes any changes as the animal ages, which may account for the neurodegenerative changes and symptoms of Parkinson's and other diseases seen in old age. RT-PCR and Western blot studies show that peak expression of D2 gene occurs in the cerebral and cerebellar cortices around 15-day after birth. We speculate that the majority of dopaminergic synapses are established and possibly become functional in the brain around 15-day after birth. The expression of D2 receptor is upregulated in the cerebral cortex in old mice. However, it is down-regulated in the cerebellar cortex.     

Polypyrimidine tract-binding protein 1 regulates the alternative splicing of dopamine receptor D(2)

Dopamine receptor D(2) (DRD2) has two splicing isoforms, a long form (D2L) and short form (D2S), which have distinct functions in the dopaminergic system. However, the regulatory mechanism of the alternative splicing of DRD2 is unknown. In this study, we examined which splicing factors regulate the expression of D2L and D2S by over-expressing several RNA-binding proteins in HEK293 cells. In a cellular splicing assay, the over-expression of polypyrimidine tract-binding protein 1 (PTBP1) reduced the expression of D2S, whereas the knockdown of PTBP1 increased the expression of D2S. We also identified the regions of DRD2 that are responsive to PTBP1 using heterologous minigenes and deletion mutants. Our results indicate that PTBP1 regulates the alternative splicing of DRD2. Considering that DRD2 inhibits cAMP-dependent protein kinase A, which modulates the intracellular localization of PTBP1, PTBP1 may contribute to the autoregulation of DRD2 by regulating the expression of its isoforms.     

The signaling pathway of dopamine D2 receptor (D2R) activation using normal mode analysis (NMA) and the construction of pharmacophore models for D2R ligands

G-protein-coupled receptors (GPCRs) are targets of more than 30% of marketed drugs. Investigation on the GPCRs may shed light on upcoming drug design studies. In the present study, we performed a combination of receptor- and ligand-based analysis targeting the dopamine D2 receptor (D2R). The signaling pathway of D2R activation and the construction of universal pharmacophore models for D2R ligands were also studied. The key amino acids, which contributed to the regular activation of the D2R, were in detail investigated by means of normal mode analysis (NMA). A derived cross-correlation matrix provided us an understanding of the degree of pair residue correlations. Although negative correlations were not observed in the case of the inactive D2R state, a high degree of correlation appeared between the residues in the active state. NMA results showed that the cytoplasmic side of the TM5 plays a significant role in promoting of residue–residue correlations in the active state of D2R. Tracing motions of the amino acids Arg219, Arg220, Val223, Asn224, Lys226, and Ser228 in the position of the TM5 are found to be critical in signal transduction. Complementing the receptor-based modeling, ligand-based modeling was also performed using known D2R ligands. The top-scored pharmacophore models were found as 5-sited (AADPR.671, AADRR.1398, AAPRR.3900, and ADHRR.2864) hypotheses from PHASE modeling from a pool consisting of more than 100 initial candidates. The constructed models using 38 D2R ligands (in the training set) were validated with 15 additional test set compounds. The resulting model correctly predicted the pIC₅₀ values of an additional test set compounds as true unknowns.     

Cloning and characterization of a Caenorhabditis elegans D2-like dopamine receptor

The neurotransmitter dopamine plays an important role in the regulation of behavior in both vertebrates and invertebrates. In mammals, dopamine binds and activates two classes of dopamine receptors, D1-like and D2-like receptors. However, D2-like dopamine receptors in Caenorhabditis elegans have not yet been characterized. We have cloned a cDNA encoding a putative C. elegans D2-like dopamine receptor. The deduced amino acid sequence of the cloned cDNA shows higher sequence similarities to vertebrate D2-like dopamine receptors than to D1-like receptors. Two splice variants that differ in the length of their predicted third intracellular loops were identified. The receptor heterologously expressed in cultured cells showed high affinity binding to [125I]iodo-lysergic acid diethylamide. Dopamine showed the highest affinity for this receptor among several amine neurotransmitters tested. Activation of the heterologously expressed receptor led to the inhibition of cyclic AMP production, confirming that this receptor has the functional property of a D2-like receptor. We have also analyzed the expression pattern of this receptor and found that the receptor is expressed in several neurons including all the dopaminergic neurons in C. elegans.     

Molecular dynamics simulation of biased agonists at the dopamine D2 receptor suggests the mechanism of receptor functional selectivity

The dopamine D2 receptor (D2R) is the primary target for antipsychotic drugs. Besides schizophrenia, this receptor is linked to dementia, Parkinson’s disease, and depression. Recent studies have shown that β-arrestin biased agonists at this receptor treat schizophrenia with less side effects. Although the high resolution structure of this receptor exists, the mechanism of biased agonism at the receptor is unknown. In this study, dopamine, the endogenous unbiased G-protein agonist, MLS1547, a G-protein biased agonist, and UNC9975, a G-protein antagonist and a β-arrestin biased agonist, were docked to a homology model of the whole D2R including all flexible loops, and molecular dynamics simulations were conducted to study the potential mechanisms of biased agonism. Our thorough analysis on the protein–ligand interaction, secondary structure, tertiary structure, structure dynamics, and molecular switches of all three systems indicates that ligand binding to transmembrane 3 might be essential for G-protein recruitment, while ligand binding to transmembrane 6 might be essential for β-arrestin recruitment. Our analysis also suggests changes in both the secondary and the tertiary structures of TM5 and TM7, molecular switches and ICL3 flexibility are important in biased signaling.

Communicated by Ramaswamy H. Sarma     

Genetic polymorphism in dopamine receptor D4 is associated with early body condition in a large population of greater flamingos, Phoenicopterus roseus

Body condition is an important determinant of fitness in many natural populations. However, as for many fitness traits, the underlying genes that regulate body condition remain elusive. The dopamine receptor D4 gene (DRD4) is a promising candidate as dopamine is known to play an important role in the regulation of food intake and the metabolism of both glucose and lipids in vertebrates. In this study, we take advantage of a large data set of greater flamingos, Phoenicopterus roseus, to test whether DRD4 polymorphism predicts early body condition (EBC) while controlling for whole-genome effects of inbreeding and outbreeding using microsatellite multilocus heterozygosity (MLH). We typed 670 of these individuals for exon 3 of the homologue of the human DRD4 gene and 10 microsatellite markers. When controlling for the effects of yearly environmental variations and differences between sexes, we found strong evidence of an association between exon 3 DRD4 polymorphisms and EBC, with 2.2-2.3% of the variation being explained by DRD4 polymorphism, whereas there was only weak evidence that MLH predicts EBC. Because EBC is most likely a polygenic trait, this is a considerable amount of variation explained by a single gene. This is to our knowledge, the first study to show an association between exon 3 DRD4 polymorphism and body condition in non-human animals. We anticipate that the DRD4 gene as well as other genes coding for neurotransmitters and their receptors may play an important role in explaining variation in traits that affect fitness.     

Potent activation of dopamine D3/D2 heterodimers by the antiparkinsonian agents, S32504, pramipexole and ropinirole

Recombinant, human dopamine D3 and D2 receptors form functional heterodimers upon co-expression in COS-7 cells. Herein, actions of the antiparkinsonian agents, S32504, ropinirole and pramipexole, at D3/D2L heterodimers were compared to their effects at the respective monomers and at split, chimeric D3trunk/D2tail and D2trunk/D3tail receptors: the trunk incorporated transmembrane domains (TDs) I-V and the tail TDs VI and VII. In binding assays with the antagonist [3H]nemonapride, all agonists were potent ligands of D3 receptors showing, respectively, 100-, 18- and 56-fold lower affinity at D2L receptors, mimicking the selective D3 receptor antagonist, S33084 (100-fold). At D3trunk/D2tail receptors, except for ropinirole, all drugs showed lower affinities than at D3 sites, whereas for D2trunk/D3tail receptors, affinities of all drugs were higher than at D2L sites. The proportion of high affinity binding sites recognized by S32504, pramipexole and ropinirole in membranes derived from cells co-expressing D3 and D2L sites was higher than in an equivalent mixture of membranes from cells expressing D3 or D2L sites, consistent with the promotion of heterodimer formation. In contrast, the percentage of high and low affinity sites (biphasic isotherms) recognized by S33084 was identical. Functional actions were determined by co-transfection of a chimeric adenylyl cyclase (AC)-V/VI insensitive to D3 receptors. Accordingly, D3 receptor-transfected cells were irresponsive whereas, in D2L receptor-transfected cells, agonists suppressed forskolin-stimulated cAMP production with modest potencies. In cells co-transfected with D3 and D2L receptors, S32504, ropinirole and pramipexole potently suppressed AC-V/VI with EC50s 33-, 19- and 11-fold lower than at D2L receptors, respectively. S32504 also suppressed AC-V/VI activity at split D3trunk/D2tail and D2trunk/D3tail chimeras transfected into COS-7 cells. In conclusion, antiparkinson agents behave as potent agonists at D3/D2'heterodimers', though any role in their actions in vivo remains to be demonstrated.     

双生子多巴胺D3受体基因多态性对儿童体成分的影响

均衡的体成分构成对维持机体的健康状态具有重要作用,体成分受遗传与环境因素的共同影响。多巴胺参与摄食、运动及认知等活动的调节,多巴胺D3受体（DRD3）对多巴胺神经通路起关键调节作用,进而对摄食功能发挥作用,从而可能对体成分产生影响。为了解遗传与环境因素对双生子儿童体成分的影响,并探讨DRD3基因单核苷酸多态性（SNP）与体成分的相关性,对160对4-12岁双生子肱三头肌皮褶厚度（d₁）、肩胛下皮褶厚度（d₂）、髂前上棘位皮褶厚度（d₃）和体质量（m）进行了测量,计算d₄(d₁+d₂)、d₅(d₂/d₁),体脂率（P_f）、瘦体质量（m_l）;从口腔拭子中提取全基因组DNA;通过Amp FISTR Sino filerPlus试剂盒分析确定卵型;采用SNaPshot技术对DRD3基因4个SNP位点进行检测;使用Mx软件估算各指标遗传度;运用广义估计方程模型分析各指标与DRD3基因SNP的相关性。校正年龄效应后,除个别指标（d₃, m_l）外,男女生指标遗传度（h）学龄前期总体偏低,且某些指标（d₂, d₄, P_f, m_l）的遗传度存在一定的性别差异。d₂分别与rs324029、rs226082存在相关（P<0.05）; d₃分别与rs2134655、rs226082存在相关（P<0.05）;d₅分别与rs2134655、rs167771存在相关（P<0.05）;P_f分别与rs226082、rs167771存在相关（P<0.05）;m_l分别与rs2134655、rs226082、rs167771存在相关（P<0.05）。本研究结果表明,遗传和环境因素对儿童体成分发育均有影响,但遗传效应可能存在一定的发育阶段和性别差异;DRD3基因SNPs与儿童的体成分可能存在一定的相关性。     

多巴胺D3受体(D3R)的神经科学新进展

多巴胺(DA)是脑内一种重要的神经递质,通过不同DA受体亚型调控运动功能、认知活动和药物成瘾等生理、病理过程。多巴胺D3受体(D3R)属于D2样受体,但其功能长期不明。近年来,人们对它在神经科学中的意义有了新的认识。首先,D3R的信号通路独特,它被激活后显示细胞增殖效应,但cAMP信号传导途径不明显。其次,D3R基因敲除小鼠研究提示,正常生理状态下D3R仅表现辅助功能：在特定病理条件下,D3R显示出重要的“平衡缓冲作用”,在精神分裂症、帕金森病(PD)治疗中运动障碍副作用LID的发生和毒品复吸等病理过程扮演了重要角色。因此,D3R是一个重要的药物靶标。D3R拮抗剂在精神分裂症治疗中显示了临床前景,D3R激动剂则对PD治疗和毒品复吸防治展示了应用价值。     

东方(鱼屯)生长激素基因内含子2的克隆与多态性分析

以暗纹东方鲀(Takifugu obscurus)、红鳍东方鲀(Takifugu rubripes)、星点东方鲀(Takifugu niphobles)共82个个体为对象,运用PCR产物电泳检测、单链构向多态性(SSCP)技术和克隆测序技术检测到生长激素(Growth Hormone, GH)基因内含子2的长度和序列多态性。结果表明,3个群体中GH基因内含子2存在9种长度类型,分别为A、B、C、D、E、F、G、H、I,变异频率达到24.22％。对这9种序列进行比对分析,(1)发现9种长度类型A、G、T、C的平均百分比为17.15％、20.77％、37.38％、24.70％,其中G+C(45.47％)含量与A+T(54.53%)的含量差异不显著;(2)9种序列分别长351 bp、327 bp、319 bp、303 bp、295 bp、291 bp、287 bp、283 bp和271 bp,引起长度变化的主要原因是短串联序列TCTG的重复 (重复次数从20到40不等); (3)发现4个突变位点,其中3个转换位点为83(C→T),101(A→G)296(G→A),一处颠换位点103(C→A)。用UPGMA法构建分子系统树,发现DD与II首先聚为一类,然后依次与GG、AA聚为一大类,BB与CC,EE与HH分别聚为一类,最后再与FF聚为一大类,由此可见GH基因内含子2在品种间的差异远大于品种内差异。     

Atomistic molecular dynamics simulations of typical and atypical antipsychotic drugs at the dopamine D2 receptor (D2R) elucidates their inhibition mechanism

Dopamine D2 receptor (D2R) plays a pivotal role in nervous systems. Its dysfunction leads to the schizophrenia, Parkinson’s diseases and drug addiction. Since the crystal structure of the D2R was not solved yet, discovering of potent and highly selective anti-psychotic drugs carry challenges for different neurodegenerative diseases. In the current study, we modeled the three-dimensional (3D) structure of the D2R based on a recently crystallized structure of the dopamine D3 receptor. These two receptors share a high amino acid sequence homology (>70%). The interaction of the modeled receptor with well-known atypical and typical anti-psychotic drugs and the inhibition mechanisms of drugs at the catalytic domain were studied via atomistic molecular dynamics simulations. Our results revealed that, class-I and class-II forms of atypical and typical D2R antagonists follow different pathways in the inhibition of the D2Rs.     

Alpha-synuclein aggregation and cell death triggered by energy deprivation and dopamine overload are counteracted by D2/D3 receptor activation

Progressive degeneration and intraneuronal Lewy bodies made of filamentous α-synuclein (α-syn) in dopaminergic cells of the nigrostriatal system are characteristics of Parkinson's disease (PD). Glucose uptake is reduced in some of the brain regions affected by PD neurodegenerative changes. Defects in mitochondrial activity in the substantia nigra have been observed in the brain of patients affected by PD and substantia nigra lesions can induce the onset of a secondary parkinsonism. Thus, energy starvation and consequently metabolic impairment to dopaminergic neurons may be related to the onset of PD. On this line, we evaluated the effect of nutrient starvation, reproduced ' in vitro ' by glucose deprivation (GD), in primary mesecephalic neuronal cultures and dopaminergic-differentiated SH-SY5Y cells, to evaluate if decreased glucose support to dopaminergic cells can lead to mitochondrial damage, neurodegeneration and α-syn misfolding. Furthermore, we investigated the effect of dopamine (DA) treatment in the presence of a DA-uptake inhibitor or of the D₂/D₃ receptor (D₂R/D₃R) agonist quinpirole on GD-treated cells, to evaluate the efficacy of these therapeutic compounds. We found that GD induced the formation of fibrillary aggregated α-syn inclusions containing the DA transporter in dopaminergic cells. These alterations were accompanied by dopaminergic cell death and were exacerbated by DA overload. Conversely, the block of DA uptake and D₂R/D₃R agonist treatment exerted neuroprotective effects. These data indicate that glucose starvation is likely involved in the induction of PD-related pathological changes in dopaminergic neurons. These changes may be counteracted by the block of DA uptake and by dopaminergic agonist treatment.