The human dopamine D4 receptor repeat sequences modulate expression.

Genetic studies have implicated a polymorphic repeat sequence in exon 3 of the human dopamine D4 receptor in various behavioral and psychiatric disorders. Functionally various repeat variants are nearly identical, but whether these have different effects on gene expression has not been studied. To study the role of the repeat sequences on expression independently from its structural and functional effects at the protein level, we introduced these sequences immediately upstream of the promoter and in the 3' untranslated region of a luciferase reporter vector. In this report, we demonstrate that the repeat sequence can both modulate promoter activity and alter expression post-transcriptionally. The repeat sequence can serve as a substrate for a nuclear binding factor and all the three repeat variants can suppress promoter activity. Placement of the three repeat variants downstream from the luciferase gene in the expression vector shows, however, that the D4.7 repeat sequence has significantly suppressed expression of the reporter compared to the D4.2 and D4.4 repeats, likely via mechanisms involving RNA stability or translational efficiency. These data indicate that the various D4 repeat sequences have different effects on expression, which may explain its potential role in behavioral disorders.

Immunohistochemical localization of the vasopressin V1b receptor in the rat brain and pituitary gland: anatomical support for its involvement in the central effects of vasopressin.

Biological effects of vasopressin (VP) are mediated by four different receptors, two of which (the V1a and the oxytocin receptors) have been well characterized in the rodent brain, suggesting that these are the main receptors responsible for the central effects of VP. However, transcripts of the V1b VP receptor (V1bR) have been detected throughout the rat brain by RT-PCR and in situ hybridization, indicating that the V1bR adds to the population of central VP receptors. Because there are no specific ligands for the V1bR, the receptor protein itself has been difficult to visualize. In the present study, the distribution of the V1bR protein was investigated in the rat forebrain, midbrain, hindbrain, and cerebellum by immunohistochemistry using an antiserum raised against a synthetic fragment of the carboxylterminal of the rat V1bR protein. Immunohistochemistry revealed the presence of the V1bR in pituitary corticotrophs as expected. In naive, untreated rats, fiber networks containing V1bR-immunoreactivity were mainly concentrated in the hypothalamus, amygdala, cerebellum, and particularly in those areas with a leaky blood brain barrier or close to the circumventricular organs (medial habenula, subfornical organ, organum vasculosum laminae terminalis, median eminence, and nuclei lining to the third and fourth ventricles). A strikingly dense network was present in the external zone of the median eminence. Colchicine treatment was required to reveal the localization of V1bR-immunoreactive cell bodies. V1bR-containing cell bodies and associated protrusions were mainly located in the hippocampus, caudate putamen, cortex, thalamus, olfactory bulb, and cerebellum. These results demonstrate the widespread distribution of the V1bR protein in the rat brain over multiple, functionally distinct neuronal systems. These data suggest that the V1bR mediates different physiological functions of VP in the brain.

Co-expression of human Kir3 subunits can yield channels with different functional properties.

To date, no comprehensive study has been done on all combinations of the human homologues of the Kir3.0 channel family, and the human homologue of Kir3.3 has not yet been identified. To obtain support for the contention that most of the functional data on non-human Kir3.0 channels can be extrapolated to human channels, we have cloned the human homologues of the Kir3.0 family, including the yet unidentified human Kir3.3, and the human Kir4.1. The expression pattern of these channels in various human brain areas and peripheral tissues, analysed by Northern blot analysis, allows for the existence of various homomeric and heteromeric forms of human Kir3.0 channels. Expression studies of all possible combinations in Xenopus oocytes indicated that in homomeric Kir3.2c and heteromeric Kir3.1/3.2c channels mediate, in our studies, inward currents with largest amplitude of any other Kir3.0 channel combinations, followed by heteromeric Kir3.1/3.4 and homomeric Kir4.1 channels. Channel combinations which include Kir3.3 are detrimental to the formation of functional channels. The co-expression experiments with different Kir channel subunits indicate the selective formation of certain channel combinations, suggesting that channel specificity is not solely dependent on spatial and temporal regulation of Kir subunit expression.

SH3 binding domains in the dopamine D4 receptor.

The dopamine D4 receptor is a G protein-coupled receptor (GPCR) that belongs to the dopamine D2-like receptor family. Functionally, the D2-like receptors are characterized by their ability to inhibit adenylyl cyclase. The dopamine D4 receptor as well as many other catecholaminergic receptors contain several putative SH3 binding domains. Most of these sites in the D4 receptor are located in a polymorphic repeat sequence and flanking sequences in the third intracellular loop. Here we demonstrate that this region of the D4 receptor can interact with a large variety of SH3 domains of different origin. The strongest interactions were seen with the SH2-SH3 adapter proteins Grb2 and Nck. The repeat sequence itself is not essential in this interaction. The data presented indicate that the different SH3 domains in the adapter proteins interact in a cooperative fashion with two distinct sites immediately upstream and downstream from the repeat sequence. Removal of all the putative SH3 binding domains in the third intracellular loop of the dopamine D4 receptor resulted in a receptor that could still bind spiperone and dopamine. Dopamine could not modulate the coupling of these mutant receptors to adenylyl cyclase and MAPK, although dopamine modulated receptor-G protein interaction appeared normal. The receptor deletion mutants show strong constitutive internalization that may account for the deficiency in functional activation of second messengers. The data indicates that the D4 receptor contains SH3 binding sites and that these sites fall within a region involved in the control of receptor internalization.

Promoter-independent regulation of cell-specific dopamine receptor expression.

Here we describe the construction of recombinant adenoviruses expressing dopamine D2 and D4 receptors, and their ability to mediate high levels of heterologous expression in a variety of cell types in vitro and in vivo for at least 7 days post infection. These experiments demonstrated that maximum receptor expression is achieved generally within 24 h and remains constant thereafter. Maximum expression levels were highly variable between cell lines and dependent on infection efficiency and promoter strength. Correction for these two variables revealed differences in relative expression levels between cell lines varying by two orders of magnitude. Our results indicate that in addition to gene transcription, post-transcriptional mechanisms play a dominant role in determining dopamine receptor levels in this system.

Biochemistry of vasopressin fragments.

Vasopressin (VP) undergoes a step-wise aminopeptidase conversion process in the brain, leading to accumulation of several metabolites. Some of these metabolites, in particular [pGlu4,Cyt6]VP 4-9 and 4-8, show behavioral effects comparable to VP, but are more potent and selective than VP. Most data favor the existence of a separate receptor for the VP metabolites distinct of the classical VP and oxytocin receptors, although its identity has remained obscure thus far. The characterization of this receptor is a major challenge to understand how the brain VP system generates and regulates divers central functions.

Genomic organization and promoter analysis of the human G-protein-coupled K+ channel Kir3.1 (KCNJ3/HGIRK1).

The class of G-protein-coupled inwardly rectifying K+ channels is composed of at least four members, Kir3.1, Kir3.2, Kir3.3, and Kir3.4. Here we describe the genomic organization of human Kir3.1 (locus designated KCNJ3; cDNA previously named HGIRK1) and the characterization of its major promoter used in hippocampus. The Kir3.1 gene contains three exons separated by two introns, and its total length exceeds 45 kb. The two transmembrane domains, pore region, and part of the putative carboxyl terminus are encoded by exon 1, whereas the remainder of the tail is encoded by exons 2 and 3. The mRNA transcription initiation site was established, and the first 1520 bp upstream were sequenced; this region lacked a traditional TATA or CAAT box, but contained a GC-rich region as well as various putative transcription factor-binding elements. The 1520 bp upstream and 84 bp downstream of the transcription initiation site were tested for promoter activity in GH4-C1 cells. This sequence of 1604 bp contains a number of fragments that either stimulate or repress transcription, as tested by transient expression of various Kir3.1 promoter/luciferase fusion gene constructs in GH4-C1 cells. To our knowledge, this is the first promoter that has been isolated and characterized for an inwardly rectifying potassium channel. Additional data suggest the existence of another promoter that can drive transcription of Kir3.1 mRNA from a distinct initiation site.

Familial dementia of frontal lobe type.

OBJECTIVE: To describe an unusual case of familial frontal lobe dementia (FLD) with probable spongiform encephalopathy (SE). METHOD: The patient's clinical presentation, neuropsychological test results, family history, and results on magnetic resonance imaging (MRI) and positron emission tomography (PET) scans, among other routine investigations, are described. His leucocyte deoxyriboneucleic acid (DNA) was isolated, and his Prp (prion protein) gene was amplified with the polymerase chain reaction and sequenced using the Sanger method. RESULTS: Clinically, the patient had a presenile frontal lobe dementia. Four of the 8 members of the generation preceding the patient had presenile dementia. Autopsies performed on 2 of these cases revealed SE, and a pathological diagnosis of Creutzfeldt-Jakob disease (CJD) was made. One other member of that generation died of a limbic encephalitis, although the neuropathological findings were atypical. Sequencing of the patient's Prp gene did not reveal the abnormalities expected in a familial case of CJD. The results of other investigations were compatible with FLD but were not specific to a particular diagnosis. CONCLUSIONS: This case is a novel type of SE. The patient presented clinically with FLD, but based on the family history and neuropathology, a diagnosis of prion dementia seems likely. There were no mutations found in the Prp gene, so it is possible that there are SEs caused by genes or other pathological processes unrelated to the Prp gene.

Cloning of a G protein-activated inwardly rectifying potassium channel from human cerebellum.

Based on sequence homology with the rat atrial G protein-coupled muscarinic potassium channel (GIRK1 or KGA1/KGB1), a human cDNA encoding a G protein-activated inwardly rectifying K+ channel (HGIRK1) was isolated. The cDNA encodes a protein of 501 amino acids and shares 99% identity to rat GIRK1 in its total amino acid sequence. Southern blot analysis of genomic DNA indicates a high degree of conservation among various species. In the human population a useful NlaIII restriction fragment length polymorphism was found in the coding sequence of HGIRK1. Co-expression of HGIRK1 and the 5-HT1A receptor in Xenopus oocytes resulted in opening of the channel upon treatment with serotonin. HGIRK1 currents showed strong inward rectification and could be blocked by extracellular Ba2+. Northern blot analysis shows that HGIRK1 expression in human is most abundant in the brain, while lower levels are round in kidney and heart.

Cryptic initiation at the human D4 receptor reveals a functional role for the amino terminus.

It was found that deletion of the initiator methionine of the D4 receptor results in the use of a cryptic initiation site in the putative first transmembrane region. We made use of this observation to investigate the role of the amino terminus of the D4 receptor. In vitro transcription and translation of D4.4 and a D4.4 deleted for the initiation codon (D4.4 delta NH2) resulted in the formation of protein products with a molecular mass of about 44 and 40.5 kDa, respectively. The molecular mass of 40.5 kDa suggests initiation in the putative first transmembrane region. Transient expression of various deletion mutants indicated that this receptor form can be expressed at up to 70% of the D4.4 control levels and provided support for the existence for an alternative translation initiation site in the first transmembrane domain, most likely at nucleotide +112 (the initiator methionine codon is designated as +1). The D4.4 delta NH2 mutant was stably expressed in CHO cells. Pharmacological analysis demonstrated no major differences in antagonist binding with the regular D4.4 receptor, while dopamine and quinpirole binding affinities were about 5-fold decreased. The half-maximal level (EC50) for blocking forskolin-stimulated cAMP levels by dopamine was about 10-fold lower as compared to D4.4. Furthermore, the functional efficacy is decreased by about 40%. These data suggest that the amino-terminal domain is not essential for proper expression, but does interfere with the functional activity of the receptor, possibly through stabilization of the active state. To our knowledge this is the first demonstration that the amino terminus of a dopamine receptor is involved in signal transduction.

A PstI restriction fragment length polymorphism in the 5' untranslated region of DRD4 is not associated with schizophrenia.

We detected a PstI restriction fragment length polymorphism in the 5'-non-coding region of the dopamine D4 receptor gene (DRD4), making it the seventh known polymorphism for DRD4. DNA polymorphisms in the putative regulatory region of DRD4 are of interest because of the reported six-fold increase in D4 receptors in post-mortem schizophrenic brain tissue [Seeman P, Guan HC, Van Tol HHM (1993) Nature, 365, 441-445]. We found no difference in the PstI allele frequencies between DSM-III-R schizophrenia patients (0.76 and 0.24, n = 41), and matched control Caucasians (0.77 and 0.23, n = 46). The PstI DRD4 polymorphism has potential use in linkage and association studies with neuropsychiatric and cardiovascular disorders.

Long-term haloperidol elevates dopamine D4 receptors by 2-fold in rats.

Chronic treatment of rats with neuroleptics results in elevated numbers of dopamine D2-like receptors. The present study was done to determine whether neuroleptics altered the density of one type of dopamine D2-like receptors, namely the dopamine D4 receptor. We here describe the effect of a one-month treatment with haloperidol on dopamine D4 receptor mRNA and protein levels in rat striatum. Endogenous levels of dopamine D4 receptor mRNA in rat striatum are very low and, therefore, reverse transcription and subsequent amplification were used for quantification. Dopamine D4 receptor density was, because of the absence of a dopamine D4 receptor specific [3H]ligand, determined by the difference between the number of binding sites for [3H]nemonapride and [3H]raclopride. Scatchard analysis of [3H]nemonapride and [3H]raclopride binding show that treatment for one month with halperidol elevates the density of dopamine D4 receptors in rat striatum by approximately 2-fold, whereas dopamine D2 and D3 receptors together show a 19% higher receptor density. Dopamine D4 receptor mRNA was also approximately increased by 2-fold.

Dopamine D4 receptor repeat: analysis of different native and mutant forms of the human and rat genes.

Recent molecular characterization of the human D4 gene has revealed the existence of various polymorphic forms of this receptor. These variations are found in the putative third cytoplasmic loop region and encode a variable number of repeats of 16 amino acids in length. In the present study we have compared the pharmacological binding profiles of seven different polymorphic variants of the human D4 receptor, the rat D4 receptor, and two different human D4 receptor mutants that were deleted in the repeat sequence. For this purpose we cloned the rat D4 receptor gene and compared its gene structure and its pharmacological binding profile with those of the D4.4 and D4.7 genes. The rat and human D4 genes display a high degree of sequence similarity, especially in the coding regions. An Alu repeat sequence was identified in the first intron of the human D4 gene but is not present in the rat D4 gene. Furthermore, using the polymerase chain reaction we cloned 3-, 5-, 6-, and 9-fold repeat sequences. These cloned repeat sequences were used for the reconstruction of full length cDNAs encoding D4.3, D4.5, D4.6, and D4.9, respectively. These novel forms of the human D4 receptor, as well as the previously cloned D4.2, D4.4, and D4.7 forms, were transiently expressed in COS-7 cells. All of the different forms of the human and rat D4 receptors and repeat deletion mutants displayed similar binding profiles for all ligands tested, although small differences were observed. The affinity for dopamine could be decreased by guanosine-5'-(beta, gamma-imido)triphosphate with the different forms of the D4 receptor, including the two receptor mutants that were deleted in the repeat sequence. These data suggest that the polymorphic repeat sequence has little influence on D4 binding profiles and might not be essential for G protein interaction.