Human D2 Receptor Research Articles

Antagonist Binding Characteristics of the Ser311→Cys Variant of Human Dopamine D2Receptorin Vivoandin Vitro

We reportin vivoandin vitroantagonist binding characteristics of the naturally occurring Ser311→Cys variant of the human D2dopamine receptor. Striatal receptor binding characteristicsin vivowere measured with positron emission tomography and the D2antagonist [11C]raclopride. Thein vitroaffinity of raclopride for the Ser311→Cys variant and the wild type receptor was studied in membrane binding assays from stably transfected cell lines. One healthy male carrying the heterozygous Ser311→Cys (TCC→TGC) substitution was identified with denaturing gradient gel electrophoresis and DNA sequencing. The striatal D2receptor binding characteristicsin vivoin this subject were normal. This was supported by thein vitrodata as the Ki values of raclopride for the Ser311→Cys variant and the wild type receptor were identical. Our data suggest that the Ser311→Cys variant of the human D2receptor does not influence antagonist-receptor recognitionin vivoorin vitro.

Mapping the Binding-Site Crevice of the D2 Receptor

The binding sites of dopamine receptors are formed among their seven, mostly hydrophobic, membrane spanning segments and are accessible to charged, water-soluble agonists, like dopamine. Thus, each of these binding sites is contained within a water accessible crevice, the binding-site crevice, extending from the extracellular surface of the receptor into the plane of the membrane. The surface of this crevice is formed by residues that contact specific agonists and/or antagonists and other residues that may affect binding indirectly. To identify the residues that form the surface of the binding-site crevice in the human D2 receptor, the substituted-cysteine accessibility method (SCAM) is used. Consecutive residues in the membrane-spanning segments are mutated to cysteine, one at a time, and the mutant receptors are expressed in heterologous cells. If ligand binding to a cysteine-substitution mutant is near-normal, it is assumed that the structure of the mutant receptor, especially around the binding site, is similar to that of wild type and that the substituted cysteine lies in an orientation similar to that of the wild type residue. An additional advantage of this approach is that it can be determined whether a residue lines the binding-site crevice, even if mutation of the residue produces no functional change in the properties of the receptor.

P-234 - Effects of perospirone(SM-9018), a novel 5-HT2 and D2 antagonist, on human D2 and D4 receptors in CHO cells

P-234 - Effects of perospirone(SM-9018), a novel 5-HT2 and D2 antagonist, on human D2 and D4 receptors in CHO cells

NGD 94-I: A Specific Dopamine-4-Receptor Antagonist

NGD 94-1 (2-phenyl-4(5)-[4-(2-pyrimidinyl)-piperazin-1-yl)-methyl]-imidazole dimaleate) is a highly selective agent at the D4 receptor—a D4 antagonists. NGD 94-1 possesses a preclinical profile that is at once similar to clozapine and also quite different from clozapine. Its affinity for D4 receptors is 3 nM, while its affinity at D1, D2, D3, and D5 receptors is greater than 2 pM, NGD 94-1 has no significant affinity for a wide variety of monoamine or other neurotransmitter receptor or modulatory sites. The only other significant receptor activity is found at 5HT1A receptors with an affinity of 180 nM. The nonspecific agonist, quinpirole, is a specific D4 agonist and inhibits forskolin-stimulated adenylate cyclase activity in a cell line stably expressing only D4 receptors. NGD 94-1 inhibits this agonist effect of quinpirole without possessing significant agonist activity of its own. Thus, NGD 94-1 appears to be a D4 receptor antagonist in this system. In direct binding experiments, [ 3 H]NGD 94-1, prepared by the reductive tritiation of a halogenated precursor, binds to a single set of receptors in transformed cells expressing the human D4 receptor. The pharmacological binding profile of [ 3 H]NGD 94-1 is consistent with expected D4 pharmacology.

2-Phenyl-4(5)-[[4-(pyrimidin-2-yl)piperazin-1-yl]methyl]imidazole. A highly selective antagonist at cloned human D4 receptors.

2-Phenyl-4(5)-[[4-(pyrimidin-2-yl)piperazin-1-yl]methyl]imidazole. A highly selective antagonist at cloned human D4 receptors.

Two intracellular signaling pathways for the dopamine D3 receptor: opposite and synergistic interactions with cyclic AMP.

As cerebral neurons express the dopamine D1 receptor positively coupled with adenylyl cyclase, together with the D3 receptor, we have investigated in a heterologous cell expression system the relationships of cyclic AMP with D3 receptor signaling pathways. In NG108-15 cells transfected with the human D3 receptor cDNA, dopamine, quinpirole, and other dopamine receptor agonists inhibited cyclic AMP accumulation induced by forskolin. Quinpirole also increased mitogenesis, assessed by measuring [3H]thymidine incorporation. This effect was blocked partially by genistein, a tyrosine kinase inhibitor. Forskolin enhanced by 50-75% the quinpirole-induced [3H]thymidine incorporation. This effect was maximal with 100 nM forskolin, occurred after 6-16 h, was reproduced by cyclic AMP-permeable analogues, and was blocked by a protein kinase A inhibitor. Forskolin increased D3 receptor expression up to 135%, but only after 16 h and at concentrations of > 1 microM. Thus, in this cell line, the D3 receptor uses two distinct signaling pathways: it efficiently inhibits adenylyl cyclase and induces mitogenesis, an effect possibly involving tyrosine phosphorylation. Activation of the cyclic AMP cascade potentiates the D3 receptor-mediated mitogenic response, through phosphorylation by a cyclic AMP-dependent kinase of a yet unidentified component. Hence, transduction of the D3 receptor can involve both opposite and synergistic interactions with cyclic AMP.

YM-50001

We investigated some in vitro pharmacological properties of a novel human dopamine D2-like receptor antagonist, YM-50001 [(R)-5-chloro-4-cyclopropylacarbonylamino-2-methoxy-N-[1-(3-methox ybenzyl)- 3-pyrrolidinyl]benzamide monooxalate]. Receptor binding studies revealed that YM-50001 had a potent affinity for human D4 receptors (Ki = 5.62 nM). YM-50001 displayed weak or negligible affinity for other neurotransmitter receptors including human D2 and D3 receptors. YM-50001 shifted the dopamine response curve on each human D2-like receptor subtype-mediated low-Km GTPase activity to the right. YM-50001 also exhibited good D4 selectivity with respect to D2-like receptor antagonism in the functional assay. These results indicate that YM-50001 is a novel, potent and selective D4 receptor antagonist.

Endogenous dopamine limits the binding of antipsychotic drugs to D3 receptors in the rat brain: a quantitative autoradiographic study.

[3H]7-hydroxy-N,N-di-n-propyl-2-aminotetralin was used as a radioligand for the autoradiographic measurements of dopamine D3 receptors in rat and human brain. Preincubation of the brain sections was necessary to obtain binding of the radioligand in the islands of Calleja and in the nucleus accumbens, but not in cerebellar lobules 9/10 of the rat. D3 receptors were also totally occluded in unwashed sections of the human striatum. The radioligand binding to D3 receptors was maximal after preincubating the sections for at least 10 min. Pretreatment of the animals with reserpine or tetrabenazine, which results in a severe depletion of endogeneous monoamines, strongly reduces the occlusion of D3 receptors in unwashed brain sections. The occlusion of dopamine D3 receptors in brain sections suggests that the in vivo access to D3 receptors may be locally inhibited by endogenous dopamine. The in vitro binding affinities of 12 antipsychotic drugs for D2 and D3 receptors were evaluated in competition binding experiments, using both rat and cloned human receptors. Most of the compounds showed only a slightly lower affinity for D3 than for D2 receptors in vitro. Affinities of the antipsychotic drugs for cloned human D21 and D3 receptors were very close to their affinities for the rat receptors. In vivo occupancy of these receptors in the rat brain was measured ex vivo by quantitative autoradiography, 2 hours after subcutaneous drug administration. For most compounds, occupancy of D3 receptors, as compared to D2 receptor occupancy, was lower than expected from the corresponding in vivo affinity ratios. For the new antipsychotic risperidone, in vivo occupancy of D3 receptors was measured both in the islands of Calleja and in the cerebellar lobules 9/10. This compound was three times less potent for the occupancy of D3 receptors in the islands of Calleja than in the cerebellum, an area lacking endogenous dopamine (ED50 = 28 and 10 mg kg-1, respectively). Based on the observations in the rat brain, it may reasonably be supposed that therapeutic dosages of antipsychotic drugs will induce in patients only a minor occupancy of D3 receptors in brain areas containing high dopamine concentrations. The role of dopamine D3 receptors as a target of antipsychotic drugs may therefore be less important than previously thought.

Functional Analysis of the Human D2 Dopamine Receptor Missense Variants

The human dopamine D2 receptor gene (DRD2) has three polymorphic variants that predict the amino acid substitutions Val96 --> Ala, Pro310 --> Ser, and Ser311 --> Cys in the receptor protein. We have investigated the ligand binding and signal transduction properties of these human D2 receptor variants by stably expressing them in cultured mammalian cells. The Cys311 and Ser310 variants of the human D2 receptor, which involve substitutions located in the third cytoplasmic loop, were markedly less effective in inhibiting cAMP synthesis than the most prevalent form (Pro310, Ser311). Despite this difference, the Cys311 and Ser310 variants couple to G proteins in CHO-K1 (Chinese hamster ovary) cells. The impairment of the Cys311 and Ser310 variants to inhibit cAMP levels thus appears to result from a reduced ability of those variant receptors to activate the appropriate Gi-like protein. The demonstration of substantial functional differences between DRD2 gene variants found in the human population might have important pharmacological implications given the widespread use of D2 receptor blocking drugs in the treatment of schizophrenia and other psychiatric disorders.

Identification of a vitamin D3-response element that overlaps a unique inverted TATA box in the rat bone sialoprotein gene.

Bone sialoprotein (BSP), an early marker of osteoblast differentiation, has been implicated in the nucleation of hydroxyapatite during bone formation de novo. Our studies, using the osteoblastic cell line ROS 17/2.8, have revealed that rat BSP gene expression is suppressed by 1,25-dihydroxyvitamin D3[1,25(OH)2D3], which is a powerful regulator of bone formation and resorption. To determine the molecular basis of the transcriptional suppression of BSP gene transcription by 1,25(OH)2D3, we have conducted transient transfection analyses with chimaeric constructs of the rat BSP gene promoter linked to a luciferase reporter gene. 1,25(OH)2D3 suppressed expression in all constructs, including a short construct (pLUC 3; nt -116 to +60) that contained a putative vitamin D3-response element (VDRE; AGGGTTTATAGGTCA; nt -28 to -14) that overlaps a unique inverted TATA (TTTATA) box. Mobility shift assays demonstrated strong binding of recombinant human vitamin D3 receptor protein (hVDR) to the VDRE. Point mutations introduced into each half-site and analysed for 1,25(OH)2D3-mediated suppression of transcription and for hVDR binding either decreased or increased both transcriptional suppression and binding. In comparison with activating VDREs, the rat BSP VDRE bound VDR-VDR homodimers more avidly than VDR-RXR alpha heterodimers (where RXR is retinoid X receptor). These studies have therefore identified a novel 1,25(OH)2D3 suppressor element that overlaps the inverted TATA box in the rat BSP gene and indicate that transcriptional suppression of the rat BSP gene by 1,25(OH)2D3 might involve competition between the VDR and the TATA binding protein (TBP).

Inverse agonism at dopamine D2 receptors haloperidol-induced prolactin release from GH4C1 cells transfected with the human D2 receptor is antagonized by R(-)-n-propylnorapomorphine, raclopride, and phenoxybenzamine

Our earlier observation that the antipsychotic drug haloperidol in the absence of dopamine increases cAMP formation and prolactin release in two prolactin-producing cell lines expressing rat dopamine D2 receptors (GH3, GH4ZR7), but not in similar cells devoid of D2 receptors (GH4C1), prompted us to suggest that haloperidol may act as an inverse (or negative) agonist, rather than as a neutral antagonist, at the D2 receptor (Nilsson and Eriksson 1993). In the present study it is shown that haloperidol elicits a dose-dependent increase in prolactin release also in prolactin-producing GH4C1 cells transfected with the human dopamine D2 receptor (short isoform) (GH4C1-hD2s); in addition, it is shown that another antipsychotic drug, flupenthixol, also causes prolactin release per se in this cell line. The effect of haloperidol on prolactin release in GH4C1-hD2s is calcium dependent and counteracted by pretreatment either with the D2 receptor agonist R(-)-n-propylnorapomorphine or with a D2 receptor antagonist that does not affect prolactin release per se (raclopride). In addition, pretreatment with the alkylating compound phenoxybenzamine at a concentration causing a marked reduction of D2 receptor density in GH4C1-hD2s cells significantly counteracted haloperidol-induced prolactin release.

Regulation of dopamine D2 receptor affinity by cholecystokinin octapeptide in fibroblast cells cotransfected with human CCKB and D2L receptor cDNAs.

Alteration in dopamine (DA) and/or cholecystokinin (CCK) transmission in the CNS may be of relevance for schizophrenia. Previous findings in striatal membranes give indications of a modulation of DA D2 receptor affinity by CCKB receptor activation. In the present study receptor binding studies were performed in a mouse fibroblast cell line (L-hD2l/CCK), expressing both human D2 receptors (long form, D2L) and human CCKB receptors, and binding sites for [3H]CCK-8S (sulfated CCK octapeptide), the D2 agonist [3H]NPA and the D2 antagonist [3H]raclopride were found and characterized in saturation and competition experiments. 1 nM of CCK-8 caused a significant 38% increase in the KD value of the D2 agonist [3H]NPA binding sites in the L-hD2l/CCK cell membranes. This change was blocked by the CCKB receptor antagonist PD 134308 (50 nM). Furthermore, 1 nM of CCK-8 increased the KD value of the D2 antagonist [3H]raclopride binding sites by 34% (P < 0.05) in the L-hD2l/CCK cell membranes. Control cells (L-hD2l cells) expressing D2L receptors showed no specific [3H]CCK-8S binding sites and no modulation by CCK-8 of the D2L receptors. These findings indicate a modulation of the D2L receptor affinity by activation of the CCKB receptor also when they are coexpressed in a fibroblast cell line. One possible explanation of these data may include a receptor-receptor interaction between the CCKB and D2L receptors.

Dopamine D 2, D 3, D 4 receptors in human postmortem brain sections: Comparison between normals and schizophrenics

Within the dopamine receptor family, the D3 dopamine receptor's function remains inadequately described. The D3 receptor has been shown to couple to inhibition of adenylyl cyclase, stimulation of mitogenesis, and regulation of K+ and Ca2+ currents, all in a pertussis toxin (PTX)-sensitive manner. Here we report D3 receptor activation of the phospholipase D (PLD) enzyme in HEK 293 cells heterologously expressing the human D3 receptor. Activation by agonist is dose dependent and displays the pharmacology expected of the D3 receptor. The D3 receptor specific antagonists AJ-76 and U99194A ablated the increase in activity by the preferring D3 agonist (+) 7-OH DPAT. In addition, the D3 receptor-mediated activation of PLD is not mediated by G-proteins of the Gi/Go family, as pretreatment with PTX had no effect. PLD activation is a novel finding for the D3 receptor, and is the first example of an effector system where D3 signals without Gi/Go protein intermediates.

Synthesis and Purification of Soluble Ligand Binding Domain of the Human Vitamin D3Receptor

We expressed and purified milligram quantities of the ligand binding domain of the human 1,25-dihydroxyvitamin D3receptor using a glutathione-S-transferase (GST) fusion protein expression system. Amino acids 105–427 were expressed inE. colias a GST fusion protein at a reduced (20°C) temperature and purified on glutathione sepharose. The fusion protein adsorbed to glutathione sepharose was cleaved with thrombin to yield soluble 105–427 human 1,25-dihydroxyvitamin D3receptor. The 105–427 human 1,25-dihydroxyvitamin D3receptor was further purified by Mono Q ion exchange chromatography and was characterized as a single band on SDS–polyacrylamide gel electrophoresis. The 105–427 human 1,25-dihydroxyvitamin D3receptor bound 1,25-dihydroxyvitamin D3with high affinity (Kdapproximately 10−9M) and with a binding capacity of 47 pmoles/nmole protein. Large scale expression of 105–427 human 1,25-dihydroxyvitamin D3receptor will provide human 1,25-dihydroxyvitamin D3receptor ligand binding domain suitable for structural studies.

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.

1-Phenyl-3-(aminomethyl)pyrroles as potential antipsychotic agents. Synthesis and dopamine receptor binding.

A series of 1-phenyl-3-(aminomethyl)pyrroles were prepared in two steps from aniline and their affinities for D2, D3, and D4 dopamine receptor subtypes determined. A 15-fold selectivity for cloned human D4 receptors over cloned African Green monkey D2 receptors was observed with 1-(2-pyridyl)-4-[[3-(1-phenylpyrrolyl)]methyl]piperazine.

Hydrophobic Residues of the D2 Dopamine Receptor Are Important for Binding and Signal Transduction

Dopamine receptors belong to the seven transmembrane helix-containing, G protein-coupled receptor superfamily. Mutagenesis studies suggest that dopamine and its analogues interact with aspartate-114 in helix 3 and two helix 5 serines (194 and 197) of the D2 receptor. In addition to these amino acids, hydrophobic residues within the receptor core may be important not only for binding but also for receptor activation. Described is a site-directed mutagenesis investigation into the roles of these hydrophobic residues in the long isoform of the human D2 receptor. Replacement of helix 6 phenylalanines (389 or 390) with alanines resulted in disrupted binding to several agonists and antagonists and impaired inhibition of adenylyl cyclase activity. Replacement of the helix 5 phenylalanine-198 with an alanine selectively disrupted [3H]N-0437 binding, whereas the affinities for other agonists and antagonists remained unchanged. This mutant remained functionally intact when stimulated with dopamine or bromocriptine. Replacement of the helix 7 phenylalanine-411 or the helix 6 leucine-387 with alanines produced receptors that bound agonists well but were unable to inhibit adenylyl cyclase. Based on these data, two conserved helix 6 phenylalanines (389 and 390) appear to be crucial for ligand binding, and phenylalanine-411 in helix 7 and leucine-387 in helix 6 may be important for propagating conformational changes from the agonist binding site(s) to G protein coupling domain(s) of the D2 receptor.

Characterization of binding of [ 3H]PD 128907, A selective Dopamine D 3 receptor agonist ligand, to CHO-K1 cells

PD 128907 [4a R, 10 b R-(+)- trans- 3, 4, 4a, 10 b - tetrahydro - 4- n-propy12 H,5 H-[1] benzopyrano[4,3-b]1,4-oxazin-9-ol.], a selective dopamine (DA) D3 receptor agonist ligand exhibits about a 1000-fold selectivity for human D3 receptors (K i, 1 nM) versus human D 2 receptors (K i, 1183 nM) and a 10000-fold selectivity versus human D 4 receptors (K i, 7000 nM) using [ 3H]spiperone as the radioligand in CHO-K1-cells. Studies with [ 3H]PD 128907, showed saturable, high affinity binding to human D 3 receptors expressed in CHO-K1 cells (CHO-K1-D 3) with an equilibrium dissociation constant (K d) of 0.99 nM and a binding density (B max) of 475 fmol/mg protein. Under the same conditions, there was no significant specific binding in CHO-K1-cells expressing human D 2 receptors (CHO-K1-D 2). The rank order of potency for inhibition of [ 3H]PD 128907 binding with reference DA agents was consistent with reported values for D 3 receptors. These results indicate that [ 3H]PD 128907 is a new, highly selective D 3 receptor ligand with high specific activity, high specific binding and low non-specific binding and therefore should be useful for further characterizing the DA D 3 receptors.

The human dopamine D5 receptor gene: cloning and characterization of the 5'-flanking and promoter region.

Genomic and overlapping cDNA clones encompassing the entire 5'-untranslated region of the human D5 receptor gene were cloned and sequenced. Comparison of these human D5 receptor genomic and cDNA clones revealed the presence of two exons separated by a small and variably sized intron (of either 179 or 155 bp). We have determined that the major site of transcription initiation of the D5 gene is 2125 bp upstream from the translational initiation start site. The region 5' to the transcription initiation site lacked conventional TATA and CAAT sequences, but contained several putative binding sites for transcription factors, such as Sp1 and Ap1. Luciferase reporter gene constructs containing D5 gene sequence information up to 500 bp 5' of the transcription initiation site were able to stimulate transcription only in SK-N-SH cells but not in COS-7, CHO, P19EC, NB41A3, and SK-N-MC cell lines. Promoter deletion analysis indicated that the D5 gene promoter contained a positive modulator at 119-182 and a negative modulator 251-500 bases upstream from the site of transcription initiation. In addition, in order to detect the expression of functional D5 receptor mRNAs and not those of its expressed pseudogenes, in situ hybridization analysis of monkey and human brain using a 5' D5-specific riboprobe revealed that D5 receptor mRNA was most abundant in discrete cortical areas (layers II, IV, and VI), the dentate gyrus, and hippocampal subfields with very little message detected in the striatum. Unexpectedly, D5 mRNA antisense riboprobes labeled discrete cell bodies in the pars compacta of the substantia nigra. The characterization of the genomic organization of the D5 receptor gene and of those factors involved in its transcriptional regulation may aid in our understanding of the role this gene product plays in the generation and maintenance of dopamine D1-like receptor-mediated events.

The relationships between language production and thought disorder in schizophrenia

Administration of the N-methyl-d-aspartate (NMDA) antagonist S-ketamine in normals produces a psychosis-like syndrome including several positive and negative symptoms of schizophrenic disorders (Abi-Saab WM, D'Souza DC, Moghaddam B, Krystal JH. The NMDA antagonist model for schizophrenia: promise and pitfalls. Pharmacopsychiatry 1998;31:104–109). Given the clinical efficacy of dopamine (DA) D2 receptor antagonists in the treatment of positive symptoms, it is conceivable that S-ketamine-induced psychotic symptoms are partially due to a secondary activation of dopaminergic systems. To date, animal and human studies of the effects of NMDA antagonists on striatal DA levels have been inconsistent. The present study used positron emission tomography (PET) to determine whether a psychotomimetic dose of S-ketamine decreases the in vivo binding of [11C]raclopride to striatal DA D2 receptors in humans (n=8). S-ketamine elicited a psychosis-like syndrome, including alterations in mood, cognitive disturbances, hallucinations and ego-disorders. S-ketamine decreased [11C]raclopride binding potential (BP) significantly in the ventral striatum (−17.5%) followed by the caudate nucleus (−14.3%) and putamen (−13.6%), indicating an increase in striatal DA concentration. The change in raclopride BP in the ventral striatum correlated with heightened mood ranging from euphoria to grandiosity. These results provide evidence that the glutamatergic NMDA receptor may contribute to psychotic symptom formation via modulation of the DA system.