Human D2 Research Articles

Differential regulation of human dopamine D2 and somatostatin receptor subtype expression by glucocorticoids in vitro

Dopamine agonists (DA) and somatostatin (SS) analogues have been proposed in the treatment of ACTH-producing neuro-endocrine tumours that cause Cushing's syndrome. Inversely, glucocorticoids (GCs) can differentially influence DA receptor D(2) or SS receptor subtype (sst) expression in rodent models. If this also occurs in human neuro-endocrine cells, then cortisol-lowering therapy could directly affect the expression of these target receptors. In this study, we investigated the effects of the GC dexamethasone (DEX) on D(2) and sst expression in three human neuro-endocrine cell lines: BON (carcinoid) and TT (medullary thyroid carcinoma) versus DMS (small cell lung cancer), which is severely GC resistant. In BON and TT, sst(2) mRNA was strongly down-regulated in a dose-dependent manner (IC(50) 0.84 nM and 0.16 nM), whereas sst(5) and especially D(2) were much more resistant to DEX treatment. Sst(2) down-regulation was abrogated by a GC receptor antagonist and reversible in time upon GC withdrawal. At the protein level, DEX also induced a decrease in the total number of SS (-52%) and sst(2)-specific (-42%) binding sites. Pretreatment with DEX abrogated calcitonin inhibition by sst(2)-preferring analogue octreotide in TT. In DMS, DEX did not cause significant changes in the expression of these receptor subtypes. In conclusion, we show that GCs selectively down-regulate sst(2), but not D(2) and only to a minor degree sst(5) in human neuro-endocrine BON and TT cells. This mechanism may also be responsible for the low expression of sst(2) in corticotroph adenomas and underwrite the current interest in sst(5) and D(2) as possible therapeutic targets for a medical treatment of Cushing's disease.

The d subunit plays a central role in human vacuolar H(+)-ATPases.

The multi-subunit vacuolar-type H+-ATPase consists of a V1 domain (A–H subunits) catalyzing ATP hydrolysis and a V0 domain (a, c, c′, c″, d, e) responsible for H+ translocation. The mammalian V0 d subunit is one of the least-well characterized, and its function and position within the pump are still unclear. It has two different forms encoded by separate genes, d1 being ubiquitous while d2 is predominantly expressed at the cell surface in kidney and osteoclast. To determine whether it forms part of the pump’s central stalk as suggested by bacterial A-ATPase studies, or is peripheral as hypothesized from a yeast model, we investigated both human d subunit isoforms. In silico structural modelling demonstrated that human d1 and d2 are structural orthologues of bacterial subunit C, despite poor sequence identity. Expression studies of d1 and d2 showed that each can pull down the central stalk’s D and F subunits from human kidney membrane, and in vitro studies using D and F further showed that the interactions between these proteins and the d subunit is direct. These data indicate that the d subunit in man is centrally located within the pump and is thus important in its rotary mechanism.

Targeting of PED/PEA-15 Molecular Interaction with Phospholipase D1 Enhances Insulin Sensitivity in Skeletal Muscle Cells

Phosphoprotein enriched in diabetes/phosphoprotein enriched in astrocytes (PED/PEA-15) is overexpressed in several tissues of individuals affected by type 2 diabetes. In intact cells and in transgenic animal models, PED/PEA-15 overexpression impairs insulin regulation of glucose transport, and this is mediated by its interaction with the C-terminal D4 domain of phospholipase D1 (PLD1) and the consequent increase of protein kinase C-alpha activity. Here we show that interfering with the interaction of PED/PEA-15 with PLD1 in L6 skeletal muscle cells overexpressing PED/PEA-15 (L6(PED/PEA-15)) restores insulin sensitivity. Surface plasmon resonance and ELISA-like assays show that PED/PEA-15 binds in vitro the D4 domain with high affinity (K(D) = 0.37 +/- 0.13 mum), and a PED/PEA-15 peptide, spanning residues 1-24, PED-(1-24), is able to compete with the PED/PEA-15-D4 recognition. When loaded into L6(PED/PEA-15) cells and in myocytes derived from PED/PEA-15-overexpressing transgenic mice, PED-(1-24) abrogates the PED/PEA-15-PLD1 interaction and reduces protein kinase C-alpha activity to levels similar to controls. Importantly, the peptide restores insulin-stimulated glucose uptake by approximately 70%. Similar results are obtained by expression of D4 in L6(PED/PEA-15). All these findings suggest that disruption of the PED/PEA-15-PLD1 molecular interaction enhances insulin sensitivity in skeletal muscle cells and indicate that PED/PEA-15 as an important target for type 2 diabetes.

Subnanomolar dopamine D3 receptor antagonism coupled to moderate D2 affinity results in favourable antipsychotic-like activity in rodent models: I. neurochemical characterisation of RG-15

RG-15 (trans-N-[4-[2-[4-(3-cyano-5-trifluoromethyl-phenyl)-piperazine-1-yl]-ethyl]-cyclohexyl]-3-pyridinesulfonic amide dihydrochloride) displayed subnanomolar affinity to human and rat dopamine D3 receptors (pKi 10.49 and 9.42, respectively) and nanomolar affinity to human and rat D2 receptors (pKi 8.23 and 7.62, respectively). No apparent interactions were found with the other 44 receptors and four channel sites tested in this study. RG-15 inhibited dopamine-stimulated [35S]GTPgammaS binding in membranes from rat striatum, in murine A9 cells expressing human D2L receptors and in CHO cells expressing human D3 receptors (IC50 values were 21.2, 36.7 and 7.2 nM, respectively). In these tests RG-15 showed the highest affinity toward D3 receptors when compared to amisulpride, haloperidol and SB-277011. RG-15, similar to haloperidol and amisulpride, dose-dependently inhibited in vivo [3H]raclopride binding in mouse striatum, enhanced dopamine turnover and synthesis rate in mouse and rat striatum and olfactory tubercle. SB-277011 did not change [3H]raclopride binding in mouse striatum nor biosynthesis or turnover rates in either region in mice or rats. RG-15 and haloperidol, but not SB-277011, antagonised dopamine synthesis inhibition induced by the D3/D2 full agonist 7-OH-DPAT in GBL-treated mice. RG-15, but not SB-277011, elevated plasma prolactin levels. In vitro receptor binding and functional experiments demonstrated that RG-15 had an antagonist profile on both D3 and D2 receptors. with high selectivity for dopamine D3 receptors over D2 receptors. However, in vivo, its neurochemical actions were similar to those of D2 receptor antagonists. Neurochemical comparison of RG-15 with antagonists having a different affinity and selectivity toward D3 and D2 receptors indicate that D3 receptors have little, if any, role in the control of presynaptic dopamine biosynthesis/release in dopaminergic terminal regions.

Investigations on the interactions of scorpion neurotoxins with the predicted structure of D1 dopamine receptor by protein–protein docking method. A bioinformatics approach

Dopamine receptors play a critical role in the cell signalling process responsible for information transfer in neurons functioning in the nervous system. Development of improved therapeutics for disorders like Parkinson's disease and schizophrenia would be significantly enhanced with the availability of the 3D structure for the dopamine receptors. Scorpion neurotoxins are unique source of structural templates from which new therapeutic agents might be developed. We report here the 3D structure of the human D1 dopamine receptor, predicted from primary sequence using computational techniques. The predicted structure of the human D1 dopamine receptor is used to understand the mechanism of interactions between scorpion neurotoxins through the protein–protein docking method. CHARMM force field was used for the energy minimization step before applying the docking method. To cite this article: C. Sudandiradoss et al., C. R. Biologies 331 (2008).

D1 Receptors Physically Interact with N-Type Calcium Channels to Regulate Channel Distribution and Dendritic Calcium Entry

Dopamine signaling through D1 receptors in the prefrontal cortex (PFC) plays a critical role in the maintenance of higher cognitive functions, such as working memory. At the cellular level, these functions are predicated to involve alterations in neuronal calcium levels. The dendrites of PFC neurons express D1 receptors and N-type calcium channels, yet little information exists regarding their coupling. Here, we show that D1 receptors potently inhibit N-type channels in dendrites of rat PFC neurons. Using coimmunoprecipitation, we demonstrate the existence of a D1 receptor-N-type channel signaling complex in this region, and we provide evidence for a direct receptor-channel interaction. Finally, we demonstrate the importance of this complex to receptor-channel colocalization in heterologous systems and in PFC neurons. Our data indicate that the N-type calcium channel is an important physiological target of D1 receptors and reveal a mechanism for D1 receptor-mediated regulation of cognitive function in the PFC.

Bioisosteric Heterocyclic Versions of 7-{[2-(4-Phenyl-piperazin-1-yl)ethyl]propylamino}-5,6,7,8-tetrahydronaphthalen-2-ol: Identification of Highly Potent and Selective Agonists for Dopamine D3 Receptor with Potent in Vivo Activity

In the current report, we extend the SAR study on our hybrid structure 7-{[2-(4-phenyl-piperazin-1-yl)ethyl]propylamino}-5,6,7,8-tetrahydronaphthalen-2-ol further to include heterocyclic bioisosteric analogues. Binding assays were carried out with HEK-293 cells expressing either D2 or D3 receptors with tritiated spiperone to evaluate inhibition constants (Ki). Functional activity of selected compounds in stimulating GTPgammaS binding was assessed with CHO cells expressing human D2 receptors and AtT-20 cells expressing human D3 receptors. The highest binding affinity and selectivity for D3 receptors were exhibited by (-)-34 (Ki=0.92 nM and D2/D3=253). In the functional GTPgammaS binding assay, (-)-34 exhibited full agonist activity with picomolar affinity for D3 receptor with high selectivity (EC50=0.08 nM and D2/D3=248). In the in vivo rotational study, (-)-34 exhibited potent rotational activity in 6-OH-DA unilaterally lesioned rats with long duration of action, which indicates its potential application in neuroprotective treatment of Parkinson's disease.

Recruitment of β-arrestin2 to the dopamine D2 receptor: Insights into anti-psychotic and anti-parkinsonian drug receptor signaling

Drugs acting at dopamine D2-like receptors play a pivotal role in the treatment of both schizophrenia and Parkinson's disease. Recent studies have demonstrated a role for G-protein independent D2 receptor signaling pathways acting through β-arrestin. In this study we describe the establishment of a Bioluminescence Resonance Energy Transfer (BRET) assay for measuring dopamine induced recruitment of human β-arrestin2 to the human dopamine D2 receptor. Dopamine, as well as the dopamine receptor agonists pramipexole and quinpirole, acted as full agonists in the assay as reflected by their ability to elicit marked concentration dependent increases in the BRET signal signifying β-arrestin2 recruitment to the D2 receptor. As expected from their effect on G-protein coupling and cAMP levels mediated through the D2 receptor RNPA, pergolide, apomorphine, ropinirole, bromocriptine, 3PPP, terguride, aripiprazole, SNPA all acted as partial agonists with decreasing efficacy in the BRET assay. In contrast, a wide selection of typical and atypical anti-psychotics was incapable of stimulating β-arrestin2 recruitment to the D2 receptor. Moreover, we observed that haloperidol, sertindole, olanzapine, clozapine and ziprasidone all fully inhibited the dopamine induced β-arrestin2 recruitment to D2 receptor (short variant) in a concentration dependent manner. We conclude that most anti-psychotics are incapable of stimulating β-arrestin2 recruitment to the dopamine D2 receptor, in accordance with their antagonistic properties at the level of G-protein coupling.

RGH-188, a d3/d2 dopamine receptor antagonist/partial agonist atypical antipsychotic candidate

Objectives:RGH-188 is an orally active, potent dopamine D3/D2 receptor antagonist/partial agonist atypical antipsychotic for the treatment of schizophrenia and bipolar mania.Results:RGH-188 displayed high affinity to human D3 receptors (Ki: 0.085 nM) and approximately six- and thirty-times less affinity to human D2, and 5-HT1A receptors. In various in vitro and in vivo assays RGH-188 behaved either as an antagonist or as a partial agonist on dopamine D3 and D2 receptors.RGH-188 displayed potent antipsychotic activity (0.1-0.8 mg/kg) in rodent models such as apomorphine-induced climbing, amphetamine- and phencyclidine-induced hypermotility, conditioned avoidance response. It significantly improved the learning performance of rats (0.02-0.2 mg/kg) impaired by scopolamine in a water-labyrinth learning paradigm. RGH-188 showed no EPS liability as it produced no catalepsy up to 100-fold therapeutic range.In a nonhuman primate positron emission tomography (PET) study using 11C-raclopride RGH-188 occupied striatal D2/D3 receptors in a dose dependent and saturable manner with an ED50 of 7 μg/kg iv. In healthy male subjects multiple administration of 1 mg RGH-188 resulted in over 70% D2/D3 receptor occupancy and the displacement showed correlation with RGH-188 and metabolites plasma levels.After single administration to healthy volunteers, Tmax for RGH-188 was 3-4 hours and the terminal disposition half-life was 5-6 days. Over the dose range of 0.5-2.5 mg AUC of the parent drug was approximately dose-proportional. Systemic exposure to the pharmacologically active metabolites, desmethyl- and didesmethyl-RGH-188 was 20-30% and 50-200% of that to the parent, respectively.

Opposite Regulation of Human D1‐like Dopaminergic Responsiveness by Protein Kinase C is Shaped by Two Critical Serine Residues of the Third Intracellular Loop of Human D5 Dopaminergic Receptor

Phorbol‐12‐myristate‐13‐acetate (PMA), an activator of serine/threonine protein kinase C, promotes in a strikingly opposite fashion sensitization and desensitization of the highly homologous human dopamine D1 and D5 receptors (D1R, D5R), respectively. We identified the third intracellular loop (IL3) as the main structural determinant controlling the opposite PMA‐mediated regulation of D1R and D5R responsiveness using a chimeric approach. Single‐point mutations were made to delineate the residues of D5R‐IL3 region implicated in PMA‐induced D5R desensitization. Dopamine responsiveness of cells transfected with wild‐type or mutant forms of D5R following PMA treatment was assessed using whole cell cAMP assays. Our results show that serine residues 261 and 271 of D5R‐IL3 (corresponding to alanine 230 and asparagine 240 in D1R‐IL3) impart PMA‐induced desensitization to D5R. In fact, D5R mutants harboring single serine‐to‐alanine substitution of residue 261 or 271 display a sensitized responsiveness following PKC activation that was reminiscent of D1R sensitization observed in PMA‐treated cells. Overall, our work suggests that the presence or absence of serine residues located at positions 261 and 271 of D5R‐IL3 or positions 230 and 240 of D1R‐IL3 control the fate of PMA‐mediated regulation of D1‐like responsiveness. Supported by CIHR grant (MOP‐81341 to MT) and FRSQ Graduate Scholarship (to BP).

D1 Dopamine Receptor Down‐regulates NADPH Oxidase Activity via a Protein Kinase C and Protein kinase A Cross‐talk in Human Kidney Cells

D1‐like dopamine receptors inhibit NADPH oxidase activity via stimulation of protein kinase A (PKA) and inhibition of phospholipase D. We tested the hypothesis that a cross‐talk between PKA and protein kinase C (PKC) is involved in the regulation of NADPH oxidase activity in HEK‐293 cells heterologously expressing human D1 dopamine receptor (hD1R) (n=6/group). The D1‐like receptor agonist, fenoldopam (Fen., 1 μM), decreased NADPH oxidase activity (63±5.0% vs. vehicle=101.6±6.2%) that was completely blocked by the D1‐like receptor antagonist, Sch23390 (5μM) (84±6.2%), which, by itself, had no effect. The PKA inhibitor, H89 (10 μM), by itself, had no effect on NADPH oxidase activity but prevented (85.6±5.0%) the inhibitory effect of fenoldopam (P<0.01, vs. others, ANOVA Newman‐Keuls test). The D1R‐mediated decrease in oxidase activity (65±3.0%) was also completely reversed by PKC inhibitors, e.g., bisinolylmaleimide I (1 μM)(94±6.0%) and staurosporine (10 nM) (93.4±7.8%), which, by themselves, had no effect (P<0.001). Moreover, the PKC activator, phobol‐12‐myristate‐13‐acetate (PMA,1 μM), inhibited oxidase activity (72.7±8.1%), that was accompanied by increased PKCθS647 phosphorylation, effects that were also abolished by H89 (103±7.4%), which, by itself, had no effect (P<0.05). We conclude that D1R inhibition of NADPH oxidase activity is dependent on both PKA and PKC (PKCθS647) signaling. Our studies demonstrate for the first time that a cross‐talk between PKA and PKC signaling pathways plays an important role in the D1R negative regulation of NADPH oxidase activity in human kidney cells.

Using pharmacokinetic–pharmacodynamic modelling as a tool for prediction of therapeutic effective plasma levels of antipsychotics

In the rat, selective suppression of conditioned avoidance response has been widely reported as a test with high predictive validity for antipsychotic efficacy. Recent studies have shown that the relationship between dopamine D2 receptor occupancy and the suppression of conditioned avoidance response behaviour correlates well with the relationship between human dopamine D2 receptor occupancy and clinical effect. The aim of the present study was to evaluate how pharmacokinetic/pharmacodynamic (PK/PD) predictions of therapeutic effective steady-state plasma levels by means of conditioned avoidance response behaviour in rodents, correlate with clinically relevant plasma exposure for the classical antipsychotic drug haloperidol and four second generation antipsychotics: sertindole, clozapine, risperidone and olanzapine, including selected metabolites. In order to confirm the validity of the present conditioned avoidance response procedure, in vivo striatal dopamine D2 receptor occupancy was determined in parallel using 3H-raclopride as the radioligand. The PK/PD relationship was established by modelling the time–response and time–plasma concentration data. We found the order of dopamine D2 receptor occupancy required to suppress conditioned avoidance response behaviour according to EC50 measurements to be sertindole (+dehydrosertindole)=dehydrosertindole=paliperidone (the metabolite of risperidone)=haloperidol=olanzapine>risperidone≫clozapine. Overall, a good agreement was observed between the rat dopamine D2 receptor occupancy levels providing 50% response in the conditioned avoidance response test and the dopamine D2 receptor occupancy levels reported from responding schizophrenic patients treated with antipsychotics. Predictions of therapeutically effective steady-state levels for sertindole (+dehydrosertindole) and olanzapine were 3–4-fold too high whereas for haloperidol, clozapine and risperidone the predicted steady-state EC50 in conditioned avoidance responding rats correlated well with the therapeutically effective plasma levels observed in patients. Accordingly, the proposed PK/PD model may act as a guide for determining effective plasma concentrations of potential antipsychotics in the clinical setting and thereby accelerating the overall drug development process.

Effects of recombinant human PLD2 on proliferation and apoptosis of HL‐60 cells

In the present study, we investigated the role of recombinant human phospholipase D2 (rhPLD2) on proliferation and apoptosis in human leukemia HL-60 cells which induced by camptothecin. Our research demonstrated that various concentrations of rhPLD2 inhibit the growth of HL-60 cells in a dose-dependent manner, and rhPLD2 plus camptothecin can produce a synergistic effect on growth inhibition of HL-60 cells in vitro. So, we conclude that rhPLD2 alone cannot induce apoptosis in HL-60 cells, but it can potentiate the apoptosis of HL-60 cells induced by camptothecin. Similarly, we show that both rhPLD2 and standard PLD were able to enhance camptothecin-induced apoptosis of HL-60 cells.

Pharmacological Analysis of Human D1 and D2 Dopamine Receptor Missense Variants

Drugs targeting dopamine receptors have been the focus of much research over the past 30 years, in large part because of their role in treating multiple pathological conditions including Parkinson's disease, schizophrenia, Tourette's syndrome, and hyperprolactinemia. Missense mutations in G protein-coupled receptors (GPCRs) can alter basal and/or ligand-induced signaling, which in turn can affect individuals' susceptibility to disease and/or response to therapeutics. To date, five coding variants in the human D1 receptor (hD1R; T37P, T37R, R50S, S199A, and A229T) and three in the human D2 receptor (hD2R; P310S, S311C, and T351A) have been reported in the NCBI single nucleotide polymorphism database. We utilized site-directed mutagenesis to generate cDNAs encoding these receptor isoforms. After expression in either HEK293 or neuronal GT1 cells, basal and ligand-induced signaling of each of these receptors was determined and compared to wild type. In addition, we investigated expression levels of each recombinant receptor and the effect of inverse agonist administration. Our data demonstrate that naturally occurring amino acid substitutions in the hD1R can lead to alterations in expression levels as well as in basal and ligand-induced signaling. The potency and efficacy of dopamine, synthetic agonists (i.e., fenoldopam, SKF-38393, SKF-82958, and SCH23390), and inverse agonists [i.e., flupenthixol and (+)butaclamol] were reduced at selected hD1R variants. Furthermore, inverse agonist induced effects on expression levels were sensitive to selected amino acid substitutions. In contrast to the hD1R variants, hD2R polymorphisms did not affect ligand function or receptor expression. The observation that the hD1R mutations induce significant alterations in pharmacologic properties may have implications both for disease susceptibility and/or therapeutic response to dopaminergic ligands.

D4 dopamine receptor enhances angiotensin II-stimulated aldosterone secretion through PKC-ε and calcium signaling

Aldosterone secretion is subjected to dopaminergic regulation. Our previous study showed that both human D2 and D4 dopamine receptors (D2R and D4R) modulate aldosterone secretion, but in opposing directions. The inhibitory effect of D2R is mediated by attenuating protein kinase C-micro (PKC-micro) and calcium-dependent signaling. The mechanism of D4R effect on angiotensin II (AII)-stimulated aldosterone secretion is explored in this study. Experiments were done with primary human adrenal cortical cells and human adrenocarcinoma (NCI-H295R) cells. Activation of different PKC isoforms was detected by specific phospho-PKC antibodies and PKC translocation. The role of calcium-dependent signaling was examined by measuring the cytoplasmic inositol 1,4,5-triphosphate (IP(3)) and calcium ([Ca(2+)](i)). The D4R agonist PD-168,077 enhanced AII-stimulated aldosterone synthesis and secretion as early as 30 min following exposure independently of the modulation of aldosterone synthase (CYP11B2) transcription. CYP11B2 mRNA level elevated by AII was augmented by D4R in the later period. These effects were reversed by the D4R antagonist L-745,870. AII activated PKC-alpha/betaII, -epsilon, and -micro but not PKC-delta, -theta, or -zeta/lambda of H295R cells. The D4R agonist selectively enhanced AII-stimulated PKC-epsilon phosphorylation and its translocation to the cell membrane. Furthermore, the D4R agonist enhanced the AII-stimulated elevation of intracellular IP(3) and [Ca(2+)](i). Inhibition of PKC-epsilon translocation by the PKC-epsilon-specific inhibitory peptide attenuated AII-stimulated aldosterone secretion, CYP11B2 mRNA expression, and elevation of intracellular IP(3) and [Ca(2+)](i). We conclude that D4R augmented aldosterone synthesis/secretion induced by AII. The mechanisms responsible for this augmentation are mediated through enhancing PKC-epsilon phosphorylation and [Ca(2+)](i) elevation.

Polymorphisms in human dopamine D2 receptor gene affect gene expression, splicing, and neuronal activity during working memory

Subcortical dopamine D2 receptor (DRD2) signaling is implicated in cognitive processes and brain disorders, but the effect of DRD2 variants remains ambiguous. We measured allelic mRNA expression in postmortem human striatum and prefrontal cortex and then performed single nucleotide polymorphism (SNP) scans of the DRD2 locus. A previously uncharacterized promoter SNP (rs12364283) located in a conserved suppressor region was associated with enhanced DRD2 expression, whereas previously studied DRD2 variants failed to affect expression. Moreover, two frequent intronic SNPs (rs2283265 and rs1076560) decreased expression of DRD2 short splice variant (expressed mainly presynaptically) relative to DRD2 long (postsynaptic), a finding reproduced in vitro by using minigene constructs. Being in strong linkage disequilibrium with each other, both intronic SNPs (but not rs12364283) were also associated with greater activity of striatum and prefrontal cortex measured with fMRI during working memory and with reduced performance in working memory and attentional control tasks in healthy humans. Our results identify regulatory DRD2 polymorphisms that modify mRNA expression and splicing and working memory pathways.

Further Structure–Activity Relationships Study of Hybrid 7-{[2-(4-Phenylpiperazin-1-yl)ethyl]propylamino}-5,6,7,8-tetrahydronaphthalen-2-ol Analogues: Identification of a High-Affinity D3-Preferring Agonist with Potent in Vivo Activity with Long Duration of Action

This paper describes an extended structure-activity relationships study of aminotetralin-piperazine-based hybrid molecules developed earlier for dopamine D2/D3 receptors. Various analogues as positional isomers have been developed where location of the phenolic hydroxyl group has been varied on the aromatic ring. Between two catechol derivatives, compound 6e with a two methylene linker length exhibited higher affinity and selectivity for D3 over D2 receptors over compound 6f with four methylene linkers (D2/D3 = 50.6 vs 7.51 for 6e and 6f, respectively). In general, the (-)-isomer was more potent than the (+)-isomeric counterpart. Binding results indicated highest selectivity for D3 receptors in compound (-)- 10 ( K i = 0.35 nM; D2/D3 = 71). In the 5-hydroxy series, highest selectivity for D3 receptors was exhibited by compound (-)- 25 ( K i = 0.82 nM; D2/D3 = 31.5). Most potent compounds exhibited binding and functional affinities at the sub-nanomolar level for the D3 receptor. Binding assays were carried out with HEK-293 cells expressing either D2 or D3 receptors by using tritiated spiperone as radioligand for competition studies to evaluate inhibition constants ( K i). A functional assay of selected compounds for stimulating GTPgammaS binding was carried out with CHO cells expressing human D2 receptors and AtT-20 cells expressing human D3 receptors. The functional assay results indicated partial to full agonist characteristics of test compounds. Compound (-)- 25 was selected further for in vivo study to evaluate its potency in producing contralateral rotations in rats with unilateral lesion in the nigrostriatal region induced by neurotoxin 6-OHDA, a Parkinsonian animal model. Compound (-)- 25 at 5 micromol/kg exhibited rotational activity that lasted beyond 12 h, whereas at a 1 micromol/kg dose the rotations lasted beyond 8 h.

Cyclin-Dependent Kinase Phosphorylation of RUNX1/AML1 on Three Sites Increases Trans-Activation Potency and Stimulates Cell Proliferation.

RUNX1/AML1 regulates lineage-specific genes during hematopoiesis and also stimulates G1 cell cycle progression. CBFβ-SMMHC or AML1-ETO dominantly inhibit RUNX1 and slow G1 progression in hematopoietic cell lines or in murine or human marrow progenitors, cdk4, cyclin D2, or c-Myc overcome inhibition of proliferation by these CBF oncoproteins, exogenous RUNX1 stimulates G1 progression, and stimulation of G1 via deletion of p16INK4a or expression of E7 cooperates with CBFβ-SMMHC or TEL-AML1 to induce acute leukemia in mice. Induction of cdk4 or cyclin D3 transcription may underlie stimulation of G1 progression by RUNX1. Remarkably, the C. elegans ortholog of RUNX1, RNT-1, also stimulates G1 progression and couples stem cell proliferation with differentiation. Not only does RUNX1 regulate cell cycle progression, but in addition RUNX1 levels increase as hematopoietic cells progress from G1 to S and from S to G2/M. Within RUNX1, S48, S303, and S424 fit the cdk phosphorylation consensus, (S/T)PX(R/K). Phosphorylation of RUNX1 by cyclin dependent kinases on serine 303 was shown to mediate destabilization of RUNX1 in G2/M. We now find that S48 and S424 are also phosphorylated by cdk1 or cdk6. S48, S303, or S424 phosphopeptide antiserum that we developed specifically recognized kinased GST-RUNX1 and interacted with RUNX1 expressed in 293T cells or in the Ba/F3 hematopoietic cell line. S48 phosphorylation of RUNX1 paralleled total RUNX1 levels during cell cycle progression, S303 was more effectively phosphorylated in G2/M, and S424 in G1. Single, double, and triple mutation to alanine or to the partially phosphomimetic aspartic acid progressively diminished or increased trans-activation, such that the tripleA mutant activated a RUNX1 reporter 5-fold less potently than the tripleD mutant. Aspartic acid does not perfectly mimic serine phosphorylation, as illustrated by the much greater affinity of our antisera for wild-type RUNX1 versus RUNX1(tripleD), suggesting that the biologic effect of RUNX1 cdk phosphorylation is even more significant. The p300 co-activator retained interaction with the tripleA variant. The tripleD RUNX1 mutant rescued Ba/F3 cells from inhibition of proliferation by CBFβ-SMMHC more effectively than the tripleA mutant. Cdk phosphorylation of RUNX1 on three sites increases its ability to active transcription and to stimulate proliferation, potentially coupling entry of stem/progenitors into cycle with induction of genes required for hematopoietic lineage progression, such as those encoding myeloperoxidase, neutrophil elastase, the M-CSF receptor, and PU.1.

Human and mouse cyclin D2 splice variants: transforming activity and subcellular localization

We have previously reported the identification of a novel 17 kDa truncated isoform of the cyclin D2 activated in 13% of the leukemias induced by the Graffi murine leukemia retrovirus. Retroviral integration in the Gris1 locus causes an alternative splicing of the mouse cyclin D2 gene and expression of a truncated protein of 159 amino acids that is detected at high levels in the Gris1 tumors and also in normal mouse tissues mainly the brain and ovaries. A truncated form of the cyclin D2 was also found in human. We show here that both mouse- and human-truncated cyclin D2 are able to transform primary mouse embryo fibroblasts (MEF) when co-expressed with an activated Ras protein. The truncated cyclin D2 localizes only to the cytoplasm of transfected cells. It has retained the ability to interact with cyclin-dependent kinases (CDKs), although it is a poor catalyst of pRb phosphorylation. Interestingly, the presence of a similar, alternatively spliced cyclin D2 mRNA was also detected in some human brain tumors.

Differences in agonist/antagonist properties at human dopamine D2 receptors between aripiprazole, bifeprunox and SDZ 208-912

Aripiprazole is the first dopamine D(2) receptor partial agonist approved for use in schizophrenia and bipolar disorder. Other partial agonists have failed in various stages of development, either for reasons of poor tolerability or lack of efficacy. We conducted an in vitro comparative analysis between aripiprazole, bifeprunox, SDZ 208-912, OPC-4392 and ACR16 in attempt to correlate specific pharmacological properties with clinical outcome. In vitro pharmacological assessment included inhibition of forskolin-stimulated cAMP accumulation and the reversal of this inhibition produced by dopamine in clonal CHO cell lines expressing high and low densities of human dopamine D(2L) and D(2S) receptors. In cells expressing high receptor densities, all drugs except ACR16 predominantly behaved as agonists. However, in cells expressing low receptor densities, all drugs showed significantly lower maximal effects than dopamine. Aripiprazole's intrinsic activity was lower than that observed with bifeprunox and OPC-4392, and higher than that of SDZ 208-912. Aripiprazole's antagonist activity was greater than that of bifeprunox and OPC-4392, and less than that of SDZ 208-912. In conclusion, our data suggests that aripiprazole's unique intrinsic activity profile may account for its demonstrated clinical efficacy in the treatment of both positive and negative symptoms of schizophrenia, as well as its demonstrated low liability for parkinsonism and hyperprolactinemia. A higher degree of intrinsic activity, and lower relative antagonist activity, such as that observed with bifeprunox and OPC-4392 may translate into a clinically suboptimal improvement of positive symptoms. SDZ 208-912's intrinsic activity may be lower than the optimal level needed to minimize extrapyramidal symptoms.