Biomedical subjects

B F O'Dowd

Publications and source records attributed to B F O'Dowd.

At least 19 recordsLinked to original sources

PubMed2001

Discovery and mapping of ten novel G protein-coupled receptor genes.

We report the identification, cloning and tissue distributions of ten novel human genes encoding G protein-coupled receptors (GPCRs) GPR78, GPR80, GPR81, GPR82, GPR93, GPR94, GPR95, GPR101, GPR102, GPR103 and a pseudogene, psi GPR79. Each novel orphan GPCR (oGPCR) gene was discovered using customized searches of the GenBank high-throughput genomic sequences database with previously known GPCR-encoding sequences. The expressed genes can now be used in assays to determine endogenous and pharmacological ligands. GPR78 shared highest identity with the oGPCR gene GPR26 (56% identity in the transmembrane (TM) regions). psi GPR79 shared highest sequence identity with the P2Y(2) gene and contained a frame-shift truncating the encoded receptor in TM5, demonstrating a pseudogene. GPR80 shared highest identity with the P2Y(1) gene (45% in the TM regions), while GPR81, GPR82 and GPR93 shared TM identities with the oGPCR genes HM74 (70%), GPR17 (30%) and P2Y(5) (40%), respectively. Two other novel GPCR genes, GPR94 and GPR95, encoded a subfamily with the genes encoding the UDP-glucose and P2Y(12) receptors (sharing >50% identities in the TM regions). GPR101 demonstrated only distant identities with other GPCR genes and GPR102 shared identities with GPR57, GPR58 and PNR (35-42% in the TM regions). GPR103 shared identities with the neuropeptide FF 2, neuropeptide Y2 and galanin GalR1 receptors (34-38% in the TM regions). Northern analyses revealed GPR78 mRNA expression in the pituitary and placenta and GPR81 expression in the pituitary. A search of the GenBank databases with the GPR82 sequence retrieved an identical sequence in an expressed sequence tag (EST) partially encoding GPR82 from human colonic tissue. The GPR93 sequence retrieved an identical, human EST sequence from human primary tonsil B-cells and an EST partially encoding mouse GPR93 from small intestinal tissue. GPR94 was expressed in the frontal cortex, caudate putamen and thalamus of brain while GPR95 was expressed in the human prostate and rat stomach and fetal tissues. GPR101 revealed mRNA transcripts in caudate putamen and hypothalamus. GPR103 mRNA signals were detected in the cortex, pituitary, thalamus, hypothalamus, basal forebrain, midbrain and pons.

Amino Acid Sequence↗

PubMed2001

FMRFamide-related neuropeptides are agonists of the orphan G-protein-coupled receptor GPR54.

We have isolated and determined the coding sequences of human and mouse orthologs of the rat orphan G-protein-coupled receptor GPR54. Mouse and rat GPR54 are nearly 95% identical to each other, and both are approximately 85% identical to human GPR54 at the amino acid level. Screening of agonists for GPR54 identified several invertebrate neuropeptides of the RFamide and RWamide family that were able to activate GPR54 at microM range through the G(alpha)q pathway. Substitution analysis showed that the C-terminal optimal sequence of GPR54-activating peptides is Gly-Leu-Arg-Trp-NH2. Northern analysis of human GPR54 detected expression in several peripheral tissues and many regions of the central nervous system.

Amino Acid Sequence↗

PubMed2001

Identification of a molecular target of psychosine and its role in globoid cell formation.

Globoid cell leukodystrophy (GLD) is characterized histopathologically by apoptosis of oligodendrocytes, progressive demyelination, and the existence of large, multinuclear (globoid) cells derived from perivascular microglia. The glycosphingolipid, psychosine (d-galactosyl-beta-1,1' sphingosine), accumulates to micromolar levels in GLD patients who lack the degradative enzyme galactosyl ceramidase. Here we document that an orphan G protein-coupled receptor, T cell death-associated gene 8, is a specific psychosine receptor. Treatment of cultured cells expressing this receptor with psychosine or structurally related glycosphingolipids results in the formation of globoid, multinuclear cells. Our discovery of a molecular target for psychosine suggests a mechanism for the globoid cell histology characteristic of GLD, provides a tool with which to explore the disjunction of mitosis and cytokinesis in cell cultures, and provides a platform for developing a medicinal chemistry for psychosine.

Calcium↗

PubMed2001

Prolonged fear responses in mice lacking dopamine D1 receptor.

Dopamine is an important neurotransmitter involved in learning and memory including emotional memory. The involvement of dopamine in conditioned fear has been widely documented. However, little is known about the molecular mechanisms that underlie contextual fear conditioning and memory consolidation. To address this issue, we used dopamine D1-deficient mice (D1-/-) and their wild-type (D1+/+) and heterozygote (D1+/-) siblings to assess aversive learning and memory. We quantified two different aspects of fear responses to an environment where the mice have previously received unsignaled footshocks. Using one-trial step-through passive avoidance and conditioned freezing paradigms, mice were conditioned to receive mild inescapable footshocks then tested for acquisition, retention and extinction of conditioned fear responses 5 min after and up to 45-90 days post-training. No differences were observed among any of the genotypes in the acquisition of passive avoidance response or fear-induced freezing behavior. However, with extended testing, D1-/- mice exhibited prolonged retention and delayed extinction of conditioned fear responses in both tasks, suggesting that D1-/- mice are capable of acquiring aversive learning normally. These findings demonstrate that the dopamine D1 receptor is not important for acquisition or consolidation of aversive learning and memory but has an important role in modulating the extinction of fear memory.

Animals↗

PubMed2001

Identification of four novel human G protein-coupled receptors expressed in the brain.

We report the discovery and tissue distributions of four novel human genes, GPR61, GPR62, GPR63 and GPR77, all of which encode G protein-coupled receptors (GPCRs). GPR61 was discovered in a search of the patent literature which retrieved a rabbit DNA sequence partially encoding a novel GPCR. This sequence was used to obtain a full-length human cDNA encoding GPR61, a receptor of 417 amino acid length. A search of the GenBank genomic sequence databases revealed three previously unrecognized intronless genes encoding the orphan GPCrs (oGPCRs) GPR62, GPR63 and GPR77, with respective amino acid lengths of 368, 419 and 337. Sequence analysis revealed that GPR61 and GPR62, and a published orphan receptor p47MNR, shared the highest level of identities to each other, ranging from 36 to 45% in the transmembrane (TM) domains. Together, these three oGPCRs appear to comprise a novel subfamily of GPCRs, most closely related to the serotonin 5-HT(6) receptor. Sequence analysis of GPR63 and GPR77 revealed highest sequence identities in the TM regions with the oGPCR PSP24 (58%) and the anaphylatoxin C5a receptor (49%) respectively. Tissue distribution analyses detected the expression of all four novel genes in the human brain. GPR61 mRNA expression was detected in the caudate, putamen and thalamus of human brain, with a more widespread expression pattern in rat brain, with mRNA signals in areas of the cortex, hippocampus, thalamus, hypothalamus and midbrain. GPR62 mRNA expression was detected in the basal forebrain, frontal cortex, caudate, putamen, thalamus and hippocampus. GPR63 mRNA expression was detected in the frontal cortex, with lower levels in the thalamus, caudate, hypothalamus and midbrain. Analysis of GPR77 mRNA expression revealed signals in the frontal cortex, hippocampus and hypothalamus with high transcript levels in the liver.

Animals↗

PubMed2001

Orphan G protein-coupled receptors in the CNS.

The majority of genes encoding G protein-coupled receptors were isolated by methods based on sequence similarities found throughout this family. Experimental techniques have exploited these similarities (including low-stringency hybridization, polymerase chain reaction and electronic database searching) to identify genes encoding many pharmacologically recognized receptors and their subtypes. Homology-based searches have revealed receptors for which the endogenous ligands were unknown and these were named orphan receptors. Many orphan receptors are expressed in the brain, suggesting the existence of unidentified neurotransmitters. Methods used to identify ligands for these orphan receptors resulted in the identification of novel ligands and succeeded in pairing previously identified ligands with their receptors. Similar successful strategies are required to characterize the physiological and pathological importance of the remaining orphan receptors to facilitate the discovery of novel drugs for these systems.

Animals↗

PubMed2001

Discovery of a novel member of the histamine receptor family.

We report the discovery, tissue distribution and pharmacological characterization of a novel receptor, which we have named H4. Like the three histamine receptors reported previously (H1, H2, and H3), the H4 receptor is a G protein-coupled receptor and is most closely related to the H3 receptor, sharing 58% identity in the transmembrane regions. The gene encoding the H4 receptor was discovered initially in a search of the GenBank databases as sequence fragments retrieved in a partially sequenced human genomic contig mapped to chromosome 18. These sequences were used to retrieve a partial cDNA clone and, in combination with genomic fragments, were used to determine the full-length open reading frame of 390 amino acids. Northern analysis revealed a 3.0-kb transcript in rat testis and intestine. Radioligand binding studies indicated that the H4 receptor has a unique pharmacology and binds [(3)H]histamine (K(d) = 44 nM) and [(3)H]pyrilamine (K(d) = 32 nM) and several psychoactive compounds (amitriptyline, chlorpromazine, cyproheptadine, mianserin) with moderate affinity (K(i) range of 33-750 nM). Additionally, histamine induced a rapid internalization of HA-tagged H4 receptors in transfected human embryonic kidney 293 cells.

Amino Acid Sequence↗

PubMed2000

Characterization of the human cysteinyl leukotriene 2 receptor.

The contractile and inflammatory actions of the cysteinyl leukotrienes (CysLTs), LTC(4), LTD(4), and LTE(4), are thought to be mediated through at least two distinct but related CysLT G protein-coupled receptors. The human CysLT(1) receptor has been recently cloned and characterized. We describe here the cloning and characterization of the second cysteinyl leukotriene receptor, CysLT(2), a 346-amino acid protein with 38% amino acid identity to the CysLT(1) receptor. The recombinant human CysLT(2) receptor was expressed in Xenopus oocytes and HEK293T cells and shown to couple to elevation of intracellular calcium when activated by LTC(4), LTD(4), or LTE(4). Analyses of radiolabeled LTD(4) binding to the recombinant CysLT(2) receptor demonstrated high affinity binding and a rank order of potency for competition of LTC(4) = LTD(4) LTE(4). In contrast to the dual CysLT(1)/CysLT(2) antagonist, BAY u9773, the CysLT(1) receptor-selective antagonists MK-571, montelukast (Singulair(TM)), zafirlukast (Accolate(TM)), and pranlukast (Onon(TM)) exhibited low potency in competition for LTD(4) binding and as antagonists of CysLT(2) receptor signaling. CysLT(2) receptor mRNA was detected in lung macrophages and airway smooth muscle, cardiac Purkinje cells, adrenal medulla cells, peripheral blood leukocytes, and brain, and the receptor gene was mapped to chromosome 13q14, a region linked to atopic asthma.

Adrenal Medulla↗

PubMed2000

Oligomerization of mu- and delta-opioid receptors. Generation of novel functional properties.

The existence of dimers and oligomers for many G protein-coupled receptors has been described by us and others. Since many G protein-coupled receptor subtypes are highly homologous to each other, we examined whether closely related receptors may interact with each other directly and thus have the potential to create novel signaling units. Using mu- and delta-opioid receptors, we show that each receptor expressed individually was pharmacologically distinct and could be visualized following electrophoresis as monomers, homodimers, homotetramers, and higher molecular mass oligomers. When mu- and delta-opioid receptors were coexpressed, the highly selective synthetic agonists for each had reduced potency and altered rank order, whereas endomorphin-1 and Leu-enkephalin had enhanced affinity, suggesting the formation of a novel binding pocket. No heterodimers were visualized in the membranes coexpressing mu- and delta-receptors by the methods available. However, hetero-oligomers were identified by the ability to co-immunoprecipitate mu-receptors with delta-receptors and vice versa using differentially epitope-tagged receptors. In contrast to the individually expressed mu- and delta-receptors, the coexpressed receptors showed insensitivity to pertussis toxin and continued signal transduction, likely due to interaction with a different subtype of G protein. In this study, we provide, for the first time, evidence for the direct interaction of mu- and delta-opioid receptors to form oligomers, with the generation of novel pharmacology and G protein coupling properties.

Animals↗

PubMed2000

The splice variant D3nf reduces ligand binding to the D3 dopamine receptor: evidence for heterooligomerization.

The D3 dopamine receptor belongs to the D2-like family of dopamine receptors. As with other members of this group, the D3 dopamine receptor gene contains introns which allow for alternative splicing of gene products. The best characterized of the human D3 dopamine receptor mRNA splice variants encodes a truncated protein called D3nf. The D3 dopamine receptor and D3nf were epitope-tagged and expressed in Sf9 insect cells by recombinant baculovirus infection. The D3 dopamine receptor showed saturable, high affinity binding of agonists and antagonists, consistent with reported D3 dopamine receptor pharmacology. When the D3 dopamine receptor and D3nf were co-expressed, the apparent density of D3 dopamine receptor expression, as determined by radioligand binding, was significantly lowered compared to D3 dopamine receptor expressed alone. This effect of D3nf was specific for the D3 dopamine receptor, since co-expression with the D2 dopamine receptor or beta2-adrenoceptor had no effect on binding. Confocal immunofluorescence studies were used to confirm that both D3 dopamine receptor and D3nf were well expressed on the cell surface and densitometric analysis of cell surface membrane protein confirmed that D3nf did not significantly alter the amount of D3 dopamine receptor expressed. Photoaffinity labelling with [125I]azidonemonapride showed that the amount of ligand bound by membranes co-expressing D3 dopamine receptor and D3nf was significantly less than that bound by membranes expressing D3 dopamine receptor alone. The greatest decrease in binding was observed in the D3 dopamine receptor oligomeric forms. Ligand binding to dimers and tetramers was reduced by 69 and 46%, respectively, indicating effects of a protein-protein interaction. Co-immunoprecipitation confirmed that the D3DR and D3nf interact with each other. These data indicate that D3nf heterodimerizes with the D3 dopamine receptor and decreases the capacity of D3 dopamine receptor to bind ligand.

Alternative Splicing↗

PubMed2000

Modification of dopamine D(1) receptor knockout phenotype in mice lacking both dopamine D(1) and D(3) receptors.

Experimental evidence suggests that dopamine D(1) and D(3) receptors may interact in an opposing or synergistic fashion. To investigate interactions between both receptors in behaviour, we have used dopamine D(1) and D(3) receptor knockout mice to generate mice lacking both receptors. D(1)(-/-)D(3)(-/-) mice were viable, fertile and showed no gross morphological abnormalities. In an open field, they exhibited lower activity than wild-type, D(1)(-/-) and D(3)(-/-) mice. D(1)(-/-)D(3)(-/-) mice performed equally poorly in the rotarod and Morris water maze tasks as their D(1)(-/-) littermates. Basal locomotor activity and anxiety-like behaviour were normal in D(1)(-/-)D(3)(-/-) mice. Combined deletion of both receptors abolished the exploratory hyperactivity and anxiolytic-like behaviour of dopamine D(3) receptor mutant phenotype and further attenuated the low exploratory phenotype of D(1)(-/-) mice. These results imply an interaction of both receptors in the expression of exploratory behaviour in a novel environment, and the need for the presence of intact dopamine D(1) receptor for the expression of certain behaviours manifested in dopamine D(3) receptor mutant phenotype. In addition, dopamine D(1) receptor, but not dopamine D(3) receptor, is involved in the ability to perform on the rotarod and spatial learning.

Analysis of Variance↗

PubMed2000

Characterization of a novel sphingosine 1-phosphate receptor, Edg-8.

Three G protein-coupled receptors (Edg-1, Edg-3, and Edg-5) for the lysolipid phosphoric acid mediator sphingosine 1-phosphate have been described by molecular cloning. Using a similar sequence that we found in the expressed sequence tag data base, we cloned and characterized of a fourth, high affinity, rat brain sphingosine 1-phosphate receptor, Edg-8. When HEK293T cells were co-transfected with Edg-8 and G protein DNAs, prepared membranes showed sphingosine 1- phosphate-dependent increases in [(35)S]guanosine 5'-(3-O-thio)triphosphate binding with an EC(50) of 90 nm. In a rat hepatoma Rh7777 cell line that exhibits modest endogenous responses to sphingosine 1-phosphate, this lipid mediator inhibited forskolin-driven rises in cAMP by greater than 90% when the cells were transfected with Edg-8 DNA (IC(50) 0.7 nm). This response is blocked fully by prior treatment of cultures with pertussis toxin, thus implicating signaling through G(i/o)alpha proteins. Furthermore, Xenopus oocytes exhibit a calcium response to sphingosine 1-phosphate after injection of Edg-8 mRNA, but only when oocytes are co-injected with chimeric G(q/i)alpha protein mRNA. Membranes from HEK293T and Rh7777 cell cultures expressing Edg-8 exhibited high affinity (K(D) = 2 nm) binding for radiolabeled sphingosine 1-phosphate. Rat Edg-8 RNA is expressed in spleen and throughout adult rat brain where in situ hybridization revealed it to be associated with white matter. Together our data demonstrate that Edg-8 is a high affinity sphingosine 1-phosphate receptor that couples to G(i/o)alpha proteins and is expressed predominantly by oligodendrocytes and/or fibrous astrocytes in the rat brain.

Amino Acid Sequence↗

PubMed2000

Role for the C-terminus in agonist-induced mu opioid receptor phosphorylation and desensitization.

Determining which domains and amino acid residues of the mu opioid receptor are phosphorylated is critical for understanding the mechanism of mu opioid receptor phosphorylation. The role of the C-terminus of the receptor was investigated by examining the C-terminally truncated or point-mutated mu opioid receptors in receptor phosphorylation and desensitization. Both wild-type and mutated receptors were stably expressed in Chinese hamster ovary (CHO) cells. The receptor expression was confirmed by receptor radioligand binding and immunoblottting. After exposure to 5 microM of DAMGO, phosphorylation of the C-terminally truncated receptor and the mutant receptor T394A was reduced to 40 and 10% of that of the wild-type receptor, respectively. Mutation effects on agonist-induced desensitization were studied using adenylyl cyclase inhibition assays. The C-terminally truncated receptor and mutant receptor T394A both showed complete loss of DAMGO-induced desensitization, while the mutant T/S-7A receptor only lost part of its ability to desensitize. Taken together, these results suggest that the C-terminus of the mu opioid receptor participates in receptor phosphorylation and desensitization with threonine 394, a crucial residue for both features. DAMGO-induced mu opioid receptor phosphorylation and desensitization are associated and appear to involve both the mu opioid receptor C-terminus and other domains of the receptor.

Adenylyl Cyclases↗

PubMed2000

Cloning and characterization of additional members of the G protein-coupled receptor family.

A search of the expressed sequence tag (EST) database retrieved a human cDNA sequence which partially encoded a novel G protein-coupled receptor (GPCR) GPR26. A human genomic DNA fragment encoding a partial open reading frame (ORF) and a rat cDNA encoding the full length ORF of GPR26 were obtained by library screening. The rat GPR26 cDNA encoded a protein of 317 amino acids, most similar (albeit distantly related) to the serotonin 5-HT(5A) and gastrin releasing hormone BB2 receptors. GPR26 mRNA expression analysis revealed signals in the striatum, pons, cerebellum and cortex. HEK293 and Rh7777 cells transfected with GPR26 cDNA displayed high basal cAMP levels, slow growth rate of clonal populations and derangements of normal cell shape. We also used a sequence reported only in the patent literature encoding GPR57 (a.k.a. HNHCI32) to PCR amplify a DNA fragment which was used to screen a human genomic library. This resulted in the cloning of a genomic fragment containing a pseudogene, psiGPR57, with a 99.6% nucleotide identity to GPR57. Based on shared sequence identities, the receptor encoded by GPR57 was predicted to belong to a novel subfamily of GPCRs together with GPR58 (a.k.a. phBL5, reported only in the patent literature), putative neurotransmitter receptor (PNR) and a 5-HT(4) pseudogene. Analysis of this subfamily revealed greatest identities (approximately 56%) between the receptors encoded by GPR57 and GPR58, each with shared identities of approximately 40% with PNR. Furthermore, psiGPR57, GPR58, PNR and the 5-HT(4) pseudogene were mapped in a cluster localized to chromosome 6q22-24. PNR and GPR58 were expressed in COS cells, however no specific binding was observed for various serotonin receptor-specific ligands.

1-Methyl-3-isobutylxanthine↗

PubMed2000

Serotonin transporter gene polymorphisms in alcohol dependence.

The serotonin transporter (5-HTT) gene is a candidate gene in alcohol dependence because serotonin reuptake inhibitors (SRIs) can alleviate alcohol withdrawal. Studies of the 5-HTT gene in alcohol dependence have not resulted in a consensus. Recent studies have examined the transcriptionally active promoter polymorphism, a 44-bp deletion resulting in short (S) or long (L) alleles. In this study, 131 alcohol-dependent patients of Northern and Western European descent were genotyped. Seventy of these patients were diagnosed with alcohol dependence without comorbid disorders. Sixty-one patients were diagnosed with alcohol dependence comorbid with Tourette syndrome (alcoholic-TS). We found an excess of the S allele in alcohol-dependent patients (47%) compared with 125 ethnically matched controls (39%). A similar trend was found in 150 ethnically matched TS patients without alcohol dependence comorbidity (51%). However, the statistical significance of this trend in the data was not present after Bonferroni correction. The data presented suggests a trend toward increased frequency of the S promoter allele in alcohol-dependent, alcoholic-TS and TS patients.

Adult↗

PubMed2000

Ontogenic profile of the expression of the mu opioid receptor gene in the rat telencephalon and diencephalon: an in situ hybridization study.

The developmental profile of mu (mu) opioid receptor gene expression has been characterized in the embryonic, postnatal and adult rat brain by in situ hybridization histochemistry. By ED12, mu opioid receptor mRNA was detectable in the deep neuroepithelium of the cortical plate. In the developing rat central nervous system (ED13-PD40), transcripts were seen over numerous telencephalic and diencephalic structures, such as the olfactory bulb, caudate-putamen, nucleus accumbens, amygdaloid complex, hippocampal formation, hypothalamus and thalamus. In the vast majority of brain regions examined, the developmental profile of the mu opioid receptor gene expression is similar to that of its translated protein as established using receptor autoradiography. Once a hybridization signal is detected in the prenatal period, it gradually increased to reach maximal levels during the second and third postnatal weeks. By the end of the third postnatal week, mu opioid receptor mRNA levels decreased to reach amounts seen in adulthood. Our study demonstrates that mu opioid receptor gene expression is seen very early on in the embryonic rat brain with transient increases observed during the critical period of neurogenesis, neuronal migration and synaptogenesis, suggesting a role of this opioid receptor subtype in brain developmental processes.

Animals↗

PubMed2000

Oligomerization of dopamine and serotonin receptors.

Until recently, it has largely been assumed that G protein-coupled receptors (GPCRs) function as monomeric entities. However, over the past few years, we and others have documented that GPCRs can form dimers and oligomers, leading to a re-evaluation of the mechanisms thought to mediate GPCR function. Despite the growing number of investigations into dimerization, little is known about the structural basis of receptor-receptor interactions and the functional consequences of dimer formation. Here, we present a brief review of some insights we have gained into the dimerization of dopamine and serotonin receptors. We have demonstrated that agonist-regulated trafficking is identical for receptor monomers and dimers, however, agonist treatment appears to stabilise the receptor oligomers. An investigation of the structural assembly between receptors involved in dimerization showed that there are several sites of interaction including hydrophobic transmembrane domain interactions and intermolecular disulphide bonds. We have also examined receptor hetero-oligomerization and demonstrated the potential for novel functions as a result of these associations. Finally, as a result of these observations, we have been able to present evidence that GPCRs function as oligomers in the cell.

Animals↗

PubMed2000

Human dopamine transporter gene: coding region conservation among normal, Tourette's disorder, alcohol dependence and attention-deficit hyperactivity disorder populations.

The dopamine transporter (DAT) provides major regulation of the synaptic levels of dopamine and is a principal target of psychostimulant drugs. Associations between DAT gene polymorphisms and human disorders with possible links to dopaminergic neurotransmission, including attention-deficit/hyperactivity disorder (ADHD) and consequences of cocaine and alcohol administration, have been reported. We now report approximately 60000 bp of genomic sequence containing the entire DAT gene. This sequence was used to amplify each of the 15 DAT gene exons and several introns and analyze these amplification products by single-stranded sequence conformation (SSCP) and/or direct sequencing. These results define silent allelic single nucleotide sequence variants in DAT gene exons 2, 6, 9 and 15. Rare conservative mutations are identified in amino acids encoded by DAT exons 2 and 8. Analyses of the common nucleotide variants and the previously reported VNTR in the non-coding region of exon 15 define the pattern of linkage disequilibrium across the DAT locus. These comprehensive analyses, however, fail to identify any common protein coding DAT sequence variant in more than 150 unrelated individuals free of neuropsychiatric disease, 109 individuals meeting City of Hope criteria for Tourette's syndrome, 64 individuals with DSM-IV diagnoses of ethanol dependence, or 15 individuals with ADHD. These data are consistent with substantial evolutionary conservation of the DAT protein sequence. They suggest that gene variants that alter levels of DAT expression provide the best current candidate mechanism for reported associations between DAT gene markers, ADHD and other more tentatively associated neuropsychiatric disorders.

Adolescent↗