D2L Receptors Research Articles

Receptor density influences the recruitment bias of aripiprazole and brexpiprazole at the dopamine D2L receptor.

Aripiprazole, brexpiprazole, and cariprazine are dopamine D2 receptor ligands considered as effective and tolerable antipsychotics. Brain imaging studies showed that schizophrenia is characterized by elevated dopamine receptor density, which is exacerbated by antipsychotic treatments. Despite the complexity of translating in vitro studies to human neurobiology, overexpression experiments in transfected cells provide a proof-of-concept model of the influence of receptor density on antipsychotic treatments. Since receptor density was demonstrated to influence the signaling profile of dopaminergic ligands, we hypothesized that high dopamine D2 receptor expression levels could influence the recruitment of Gi1 and β-arrestin2 in response to partial agonists used as antipsychotics. A nanoluciferase complementation assay was used to monitor β-arrestin2 and Gi1 recruitment at the dopamine D2L receptor in response to aripiprazole, brexpiprazole, and cariprazine. This was performed in transfected cells carrying a doxycycline-inducible system allowing to manipulate the expression of the dopamine D2L receptors. Increasing D2L receptor density reoriented aripiprazole's preferential recruitment from Gi1 to β-arrestin2. With respect to brexpiprazole, which showed inverse agonism for β-arrestin2 recruitment at the lower receptor density tested, inverse agonism for Gi1 recruitment was observed when tested at a high receptor expression level. At variance, cariprazine evoked a potent partial agonism for β-arrestin2 recruitment only, in all the tested conditions. D2L receptor density appears to shape the recruitment bias of aripiprazole and brexpiprazole, but not cariprazine. This suggests that changes in receptor expression level could qualitatively influence the functional response of partial agonists used in psychiatry.

Allosteric modulation of dopamine D2L receptor in complex with Gi1 and Gi2 proteins: the effect of subtle structural and stereochemical ligand modifications

BackgroundAllosteric modulation of G protein-coupled receptors (GPCRs) is nowadays one of the hot topics in drug discovery. In particular, allosteric modulators of D2 receptor have been proposed as potential modern therapeutics to treat schizophrenia and Parkinson’s disease.MethodsTo address some subtle structural and stereochemical aspects of allosteric modulation of D2 receptor, we performed extensive in silico studies of both enantiomers of two compounds (compound 1 and compound 2), and one of them (compound 2) was synthesized as a racemate in-house and studied in vitro.ResultsOur molecular dynamics simulations confirmed literature reports that the R enantiomer of compound 1 is a positive allosteric modulator of the D2L receptor, while its S enantiomer is a negative allosteric modulator. Moreover, based on the principal component analysis (PCA), we hypothesized that both enantiomers of compound 2 behave as silent allosteric modulators, in line with our in vitro studies. PCA calculations suggest that the most pronounced modulator-induced receptor rearrangements occur at the transmembrane helix 7 (TM7). In particular, TM7 bending at the conserved P7.50 and G7.42 was observed. The latter resides next to the Y7.43, which is a significant part of the orthosteric binding site. Moreover, the W7.40 conformation seems to be affected by the presence of the positive allosteric modulator.ConclusionsOur work reveals that allosteric modulation of the D2L receptor can be affected by subtle ligand modifications. A change in configuration of a chiral carbon and/or minor structural modulator modifications are solely responsible for the functional outcome of the allosteric modulator.Graphical abstract

Dopamine D2L receptor density influences the recruitment of β-arrestin2 and Gi1 induced by antiparkinsonian drugs

IntroductionBrain imaging studies have highlighted that the density of dopamine D2 receptors markedly fluctuates across the stages of Parkinson's disease and in response to pharmacological treatment. Moreover, receptor density constitutes a molecular determinant for the signaling profile of D2 receptor ligands. We therefore hypothesized that variations in receptor expression could influence D2 receptor response to antiparkinsonian drugs, most notably with respect to the recruitment bias between Gi1 and β-arrestin2. MethodsThe recruitment bias of dopamine, pramipexole, ropinirole, and rotigotine was examined using a nanoluciferase-based biosensor for probing the interactions of the D2L receptor with either Gi1 or β-arrestin2. The characterization of the functional selectivity of these D2 receptor agonists was performed at two distinct D2L receptor densities by taking advantage of a cell model carrying an inducible system that enables the overexpression of the D2L receptor when exposed to doxycycline. ResultsA high receptor density oriented the balanced signaling profile of dopamine towards a preferential recruitment of Gi1. It also moderated the marked Gi1 and β-arrestin2 biases of pramipexole and rotigotine, respectively. At variance, the Gi1 bias of ropinirole appeared as not being influenced by D2L receptor density. ConclusionsTaken together, these observations highlight receptor density as a key driver of the signaling transducer recruitment triggered by antiparkinsonian agents. Moreover, given the putative beneficial properties of β-arrestin2 in promoting locomotion, this study provides molecular insights that position the arrestin-biased ligand rotigotine as a putatively more beneficial D2 receptor agonist for the treatment of early and late Parkinson's disease.

Receptor density influences ligand-induced dopamine D2L receptor homodimerization

Chronic treatments with dopamine D2 receptor ligands induce fluctuations in D2 receptor density. Since D2 receptors tend to assemble as homodimers, we hypothesized that receptor density might influence constitutive and ligand-induced homodimerization. Using a nanoluciferase-based complementation assay to monitor dopamine D2L receptor homodimerization in a cellular model enabling the tetracycline-controlled expression of dopamine D2L receptors, we observed that increasing receptor density promoted constitutive dopamine D2L receptor homodimerization. Receptor full agonists promoted homodimerization, while antagonists and partial agonists disrupted dopamine D2L receptor homodimers. High receptor densities enhanced this inhibitory effect only for receptor antagonists. Taken together, our findings indicate that both receptor density and receptor ligands influence dopamine D2L receptor homodimerization, albeit excluding any strict correlation with ligands’ intrinsic activity and highlighting further complexity to dopaminergic pharmacology.

Preclinical Evaluation of the Effects of Trazpiroben (TAK-906), a Novel, Potent Dopamine D2/D3 Receptor Antagonist for the Management of Gastroparesis.

Current therapies for gastroparesis metoclopramide and domperidone carry risks of extrapyramidal symptoms and life-threatening cardiac arrhythmias. Trazpiroben, a novel, potent dopamine D2/D3 receptor antagonist, has low brain permeation and very low affinity for human ether-à-go-go-related gene (hERG) channel inhibition, potentially improving on safety profiles of existing therapies. Trazpiroben demonstrated the following receptor affinities: high for D2 and D3, moderate for D4, and minimal for D1 and D5 It demonstrated moderate affinity for adrenergic α 1B (α 1B) and 5-hydroxytryptamine (5HT) 2A receptors and low potential for off-target adverse events (AEs). Trazpiroben potently inhibited dopamine-activated D2L receptor activation of cognate G-proteins in human embryonic kidney 293 cell membranes and was a neutral D2L receptor antagonist. In vivo, trazpiroben dose-dependently increased prolactin release in orally dosed rat (0.1-1 mg/kg). Additionally, multiple oral doses in the rat (100 mg/kg) and dog (50 mg/kg) for 3 days produced robust plasma exposures and prolactin increases in both species. Trazpiroben inhibited retching/vomiting in the dog with apomorphine-induced emesis with a potency (0.1-1 mg/kg) like that of trazpiroben-mediated prolactin increases in rat. Oral trazpiroben (1, 10, and 30 mg/kg) did not affect rat rotarod performance, suggesting low brain penetration. Trazpiroben concentrations were low in cerebrospinal fluid versus plasma after multiple oral doses for 4 days in rat and dog. Trazpiroben weakly inhibited the hERG channel current (concentration causing half-maximal inhibition of control-specific binding of 15.6 µM), indicating little potential for disrupting cardiac rhythm. Overall, trazpiroben is a potent D2/D3 receptor antagonist designed to avoid the serious potential AEs associated with current gastroparesis therapies. SIGNIFICANCE STATEMENT: Trazpiroben is a novel, potent dopamine D2/D3 selective receptor antagonist designed to avoid adverse effects associated with the current pharmacological therapies metoclopramide and domperidone. Preclinical studies have demonstrated low brain penetration and weak affinity for the hERG channel, indicating that trazpiroben is not expected to be associated with central nervous system or cardiovascular safety issues. With these pharmacological properties, trazpiroben may represent a viable new treatment option for gastroparesis because of a potentially improved safety profile relative to existing therapies.

Cannabinoid 2 receptors regulate dopamine 2 receptor expression by a beta-arrestin 2 and GRK5-dependent mechanism in neuronal cells

We have previously reported that the repeated exposure to cannabinoids upregulates and enhances the activity of serotonin 2A (5-HT2A) and dopamine 2 (D2) receptors and facilitates the formation of D2-5-HT2A receptor heterodimers in the rat prefrontal cortex and two neuronal cell lines. Because the repeated exposure to cannabinoids has been associated with adverse neuropsychiatric disorders, this study investigated the mechanisms that underly the cannabinoid-mediated regulation of D2 receptor expression in a neuronal cell model, CLU213 cells.We initially tested the effects of repeated exposure (72 h) to a non-selective cannabinoid agonist (1 nM CP55940), a selective CB1 receptor agonist (15 nM ACEA), or a selective CB2 receptor drug (1 nM GP1a) on the expression of postsynaptic D2 (D2L) receptors in CLU213 cells. Repeated CP55940, GP1a, or ACEA treatments significantly increased D2L receptor protein levels (99 % ± 7%, 30 % ± 7%, and 39 % ± 5% increases compared with control levels, respectively). Repeated exposure to both GP1a and ACEA increased D2L receptor protein levels by 73 % ± 8%. Interestingly, CP55940 and GP1a, but not ACEA, upregulated D2 mRNA. Using cells that were stably transfected with short-hairpin RNA (shRNA) lentiviral particles targeting CB2 receptors, G protein-coupled receptor kinase 5 (GRK5), and β-arrestin 2, we found that CB2 receptors regulated D2 expression through a mechanism that is dependent on GRK5, β-arrestin 2, and extracellular signal-related kinase (ERK)1/2. We also found that repeated exposure to either ACEA or GP1a selectively stimulated the protein and mRNA expression of GRK proteins. ACEA significantly upregulated GRK2 proteins, whereas GP1a upregulated GRK5 protein expression.Our results identified mechanisms associated with the upregulation of D2 receptors in neuronal cells after the repeated exposure to cannabinoids. These data can shed light on the mechanisms that can be targeted to prevent potential adverse effects, while simultaneously determining the therapeutic benefits of cannabinoids.

Dopamine D2 Long Receptors Are Critical for Caveolae-Mediated α-Synuclein Uptake in Cultured Dopaminergic Neurons

α-synuclein accumulation into dopaminergic neurons is a pathological hallmark of Parkinson’s disease. We previously demonstrated that fatty acid-binding protein 3 (FABP3) is critical for α-synuclein uptake and propagation to accumulate in dopaminergic neurons. FABP3 is abundant in dopaminergic neurons and interacts with dopamine D2 receptors, specifically the long type (D2L). Here, we investigated the importance of dopamine D2L receptors in the uptake of α-synuclein monomers and their fibrils. We employed mesencephalic neurons derived from dopamine D2L−/−, dopamine D2 receptor null (D2 null), FABP3−/−, and wild type C57BL6 mice, and analyzed the uptake ability of fluorescence-conjugated α-synuclein monomers and fibrils. We found that D2L receptors are co-localized with FABP3. Immunocytochemistry revealed that TH+ D2L−/− or D2 null neurons do not take up α-synuclein monomers. The deletion of α-synuclein C-terminus completely abolished the uptake to dopamine neurons. Likewise, dynasore, a dynamin inhibitor, and caveolin-1 knockdown also abolished the uptake. D2L and FABP3 were also critical for α-synuclein fibrils uptake. D2L and accumulated α-synuclein fibrils were well co-localized. These data indicate that dopamine D2L with a caveola structure coupled with FABP3 is critical for α-synuclein uptake by dopaminergic neurons, suggesting a novel pathogenic mechanism of synucleinopathies, including Parkinson’s disease.

Tannic acid acts as an agonist of the dopamine D2L receptor, regulates immune responses, and ameliorates experimentally induced colitis in mice

Tannic acid (TA) is an herbal polyphenol containing a galloyl group that has been prescribed to treat gastroenteritis, diarrhea, and irritable bowel syndrome. TA has anti-inflammatory, anti-cancer, and anti-viral properties; however, the molecular mechanisms of these potential therapeutic effects are still largely unknown. Here, we examined the ability of TA to induce anti-inflammatory responses. TA was found to be an agonist of the dopamine D2L receptor. TA reduced interferon (IFN)-γ and interleukin (IL)-1β secretion but upregulated tumor necrosis factor α and IL-10 secretion from lipopolysaccharide (LPS)-stimulated mouse splenocytes. TA also reduced IFN-γ secretion but enhanced IL-10 secretion from anti-cluster of differentiation (CD) 3/CD28 antibody-stimulated splenocytes. An immune subset study confirmed that TA regulated cytokine secretion by various types of immune cells in the context of stimulation with LPS or anti-CD3/CD28 antibodies. Administration of TA to mice with experimentally induced colitis strikingly suppressed weight loss, colon shrinkage, and IL-17 secretion from mesenteric lymph node lymphocytes in response to CD3/CD28 stimulation. These data suggest that TA suppresses inflammatory responses in colitis by regulating cytokine secretion by immune cells in the colon.

Preferential Coupling of Dopamine D2S and D2L Receptor Isoforms with Gi1 and Gi2 Proteins-In Silico Study.

The dopamine D2 receptor belongs to rhodopsin-like G protein-coupled receptors (GPCRs) and it is an important molecular target for the treatment of many disorders, including schizophrenia and Parkinson’s disease. Here, computational methods were used to construct the full models of the dopamine D2 receptor short (D2S) and long (D2L) isoforms (differing with 29 amino acids insertion in the third intracellular loop, ICL3) and to study their coupling with Gi1 and Gi2 proteins. It was found that the D2L isoform preferentially couples with the Gi2 protein and D2S isoform with the Gi1 protein, which is in accordance with experimental data. Our findings give mechanistic insight into the interplay between isoforms of dopamine D2 receptors and Gi proteins subtypes, which is important to understand signaling by these receptors and their mediation by pharmaceuticals, in particular psychotic and antipsychotic agents.

Preclinical pharmacodynamic and pharmacokinetic characterization of the major metabolites of cariprazine.

Introduction: Cariprazine, a dopamine D3-preferring D3/D2 receptor partial agonist and serotonin 5-HT1A receptor partial agonist, has two major human metabolites, desmethyl-cariprazine (DCAR) and didesmethyl-cariprazine (DDCAR). The metabolite pharmacology was profiled to understand the contribution to cariprazine efficacy.MethodsIn vitro receptor binding and functional assays, electrophysiology, animal models, microdialysis, and kinetic-metabolism approaches were used to characterize the pharmacology of DCAR and DDCAR. ResultsSimilar to cariprazine, both metabolites showed high affinity for human D3, D2L, 5-HT1A, 5-HT2A, and 5-HT2B receptors, albeit with higher selectivity than cariprazine for D3 versus D2 receptors. In [35S]GTPγS binding assays, cariprazine and DDCAR were antagonists in membranes from rat striatum and from cells expressing human D2 and D3 receptors, and were partial agonists in membranes from rat hippocampus. In cAMP signaling assays, cariprazine, DCAR, and DDCAR acted as partial agonists at D2 and D3 receptors; cariprazine and DDCAR were full agonists, whereas DCAR was a partial agonist at 5-HT1A receptors. Cariprazine, DCAR, and DDCAR were pure antagonists at human 5-HT2B receptors. Cariprazine and DDCAR increased rat striatal dopamine and reduced cortical serotonin turnover. Cariprazine and DDCAR showed similar in vivo D3 receptor occupancy in rat brain; however, cariprazine was more potent for D2 receptor occupancy. Both cariprazine and DDCAR dose-dependently but partially suppressed the spontaneous activity of midbrain dopaminergic neurons in rats, with the parent compound being more potent but shorter acting than its metabolite. Consistent with the D2 receptor occupancy profile, DDCAR was 3- to 10-fold less potent than cariprazine in rodent models of antipsychotic-like activity. Following acute cariprazine administration, DDCAR was detected in the rodent brain but at much lower levels than cariprazine.ConclusionOverall, in vitro and in vivo pharmacological profiles of DCAR and DDCAR demonstrated high similarity with cariprazine, suggesting that the major metabolites of cariprazine contribute significantly to its clinical efficacy.

Intravenous immunoglobulin for the treatment of autoimmune encephalopathy in children with autism

The identification of brain-targeted autoantibodies in children with autism spectrum disorder (ASD) raises the possibility of autoimmune encephalopathy (AIE). Intravenous immunoglobulin (IVIG) is effective for AIE and for some children with ASD. Here, we present the largest case series of children with ASD treated with IVIG. Through an ASD clinic, we screened 82 children for AIE, 80 of them with ASD. IVIG was recommended for 49 (60%) with 31 (38%) receiving the treatment under our care team. The majority of parents (90%) reported some improvement with 71% reporting improvements in two or more symptoms. In a subset of patients, Aberrant Behavior Checklist (ABC) and/or Social Responsiveness Scale (SRS) were completed before and during IVIG treatment. Statistically significant improvement occurred in the SRS and ABC. The antidopamine D2L receptor antibody, the anti-tubulin antibody and the ratio of the antidopamine D2L to D1 receptor antibodies were related to changes in the ABC. The Cunningham Panel predicted SRS, ABC, parent-based treatment responses with good accuracy. Adverse effects were common (62%) but mostly limited to the infusion period. Only two (6%) patients discontinued IVIG because of adverse effects. Overall, our open-label case series provides support for the possibility that some children with ASD may benefit from IVIG. Given that adverse effects are not uncommon, IVIG treatment needs to be considered cautiously. We identified immune biomarkers in select IVIG responders but larger cohorts are needed to study immune biomarkers in more detail. Our small open-label exploratory trial provides evidence supporting a neuroimmune subgroup in patients with ASD.

Homeostatic regulation of basal ganglia circuit for flexible behavior

Behavioral inflexibility has been reported in various psychiatric disorders including drug addiction and schizophrenia. Considerable evidence has demonstrated a critical role for the basal ganglia in the flexibility of behavioral strategies. These processes are guided by the activity of two discrete neuron types, dopamine D1- or D2-receptor expressing medium spiny neurons (D1-/D2-MSNs) in the basal ganglia circuit. We used a reversible neurotransmission blocking technique to examine the role of D1- and D2-MSNs in the acquisition and reversal learning of a place discrimination task in the IntelliCage. We demonstrated that D1- and D2-MSNs do not mediate the acquisition of the task, but that suppression of activity in D2-MSNs impairs reversal learning and increased perseverative errors. Additionally, global knockout of the dopamine D2L receptor isoform produced a similar behavioral phenotype to D2-MSN-blocked mice. We also showed that D2L receptors are necessary for visual discrimination and reversal learning. These results suggest that D2L receptors and D2-MSNs have a critical role in the homeostatic regulation of basal ganglia circuit for flexible behaviors.

Effects of Ovariectomy and 17<i>β</i>-Estradiol Replacement on Dopamine D2 Receptors in Female Rats: Consequences on Sucrose, Alcohol, Water Intakes and Body Weight

Background: Mechanisms underlying overeating-induced obesity in post-menopausal woman include functional lack of 17β-estradiol dysregulating dopamine D2 receptors, thereby inducing food addiction, glucose craving or alcohol dependence through reward circuitry. This study aimed at further understanding 17β-estradiol and dopamine D2 receptors interferences in the etiology of woman obesity. Method: Seventy-two Wistar female rats weighing 200 - 205 g, individually-housed, were divided into non-ovariectomized control (C = 6 groups) and ovariectomized rats (OVX = 6 groups) which were concurrently subjected to the following treatments: Non-drug-treated (DMSO vehicle), 17β-estradiol (E2, 5 μg/kg, s.c.), sulpiride (SUL, 20 mg/kg, i.p.), bromocriptine (BR, 0.1 mg/kg, i.p.), E2 + SUL or E2 + BR, designating the 6 constitutive groups of either control or ovariectomy. Within each experimental group, consumption of different solutions (10% alcohol, 10% sucrose and water) as well as food intake and body weight were daily measured, for 10 consecutive days. Results: This study indicated that D2S was a specific inducer of alcohol and food intakes, but reduced sugar consumption. In addition, 17β- estradiol regulated the body weight set point, modulating D2S functions towards increased food intake at lower weights and decreased food intake at higher weights. D2S met the slow genomic actions induced by 17β-estradiol. Conversely, D2L inhibited alcohol and food intakes, but induced specifically sugar consumption, thereby regulating blood glucose levels and promoting energy expenditure in reducing body weight. Indeed, 17β-estradiol exerted a tonic inhibition on D2L which was released by OVX, exacerbating sugar intake and increasing body weight. D2L mediated the rapid metabolic effects of 17β-estradiol. Conclusion: Our results supported physiological data reporting that activation of the mostly expressed presynaptically D2S-class autoreceptors decreased dopamine release stimulating food intake, whereas activation of the predominantly postsynaptic isoform D2L receptors increased dopamine activity inhibiting food intake. Our studies indicated that 17β-estradiol acted on the two types of D2 receptors showing opposite functions to equilibrate energy intake vs. expenditure for weight set point regulation. Our data also supported biochemical findings reporting that 17β-estradiol induced D2 genes transcriptional regulation, thereby involving both types of D2 receptors in the etiology of obesity. The combined dysregulated effects of D2L and D2S receptors, as 17β-estradiol was lacking, would be causal factors underlying the etiology of obesity.

Distinctive binding properties of the negative allosteric modulator, [3H]SB269,652, at recombinant dopamine D3 receptors

Recently, employing radioligand displacement and functional coupling studies, we demonstrated that SB269,652 (N-[(1r,4r)−4-[2-(7-cyano-1,2,3,4-tetrahydroisoquinolin-2-yl)ethyl]cyclohexyl]−1H-indole-2-carboxamide) interacts in an atypical manner with dopamine D3 receptor displaying a unique profile reminiscent of a negative allosteric ligand. Here, we characterized the binding of radiolabelled [3H]SB269,652 to human dopamine D3 receptor stably expressed in Chinese Hamster Ovary cells. Under saturating conditions, SB269,652 showed a KD value of ≈ 1nM. Consistent with high selectivity for human dopamine D3 receptor, [3H]SB269,652 binding was undetectable in cells expressing human dopamine D1, D2L or D4 receptors and absent in synaptosomes from dopamine D3 receptor knockout vs. wild-type mice. In contrast to saturation binding experiments, the dissociation kinetics of [3H]SB269,652 from human dopamine D3 receptors initiated with an excess of unlabelled ligand were best fitted by a bi-exponential binding model. Supporting the kinetic data, competition experiments with haloperidol, S33084 (a dopamine D3 receptor antagonist) or dopamine, were best described by a two-site model. In co-transfection experiments binding of SB269,652 to dopamine D3 receptor was able to influence the functional coupling of dopamine D2 receptor, supporting the notion that SB269,652 is a negative allosteric modulator across receptor dimers. However, because SB269,652 decreases the rate of [3H]nemonapride dissociation, the present data suggest that SB269,652 behaves as a bitopic antagonist at unoccupied dopamine D3 receptor, binding simultaneously to both orthosteric and allosteric sites, and as a pure negative allosteric modulator when receptors are occupied and it can solely bind to the allosteric site.

Visualization of ligand-induced dopamine D2S and D2L receptor internalization by TIRF microscopy

G protein-coupled receptors (GPCRs), including the dopamine receptors, represent a group of important pharmacological targets. Upon agonist binding, GPCRs frequently undergo internalization, a process that is known to attenuate functional responses upon prolonged exposure to agonists. In this study, internalization was visualized by means of total internal reflection fluorescence (TIRF) microscopy at a level of discrete single events near the plasma membrane with high spatial resolution. A novel method has been developed to determine the relative extent of internalized fluorescent receptor-ligand complexes by comparative fluorescence quantification in living CHO cells. The procedure entails treatment with the reducing agent sodium borohydride, which converts cyanine-based fluorescent ligands on the membrane surface to a long-lived reduced form. Because the highly polar reducing agent is not able to pass the cell membrane, the fluorescent receptor-ligand complexes located in internalized compartments remain fluorescent under TIRF illumination. We applied the method to investigate differences of the short (D2S) and the long (D2L) isoforms of dopamine D2 receptors in their ability to undergo agonist-induced internalization.

Tuning the allosteric regulation of artificial muscarinic and dopaminergic ligand-gated potassium channels by protein engineering of G protein-coupled receptors

Ligand-gated ion channels enable intercellular transmission of action potential through synapses by transducing biochemical messengers into electrical signal. We designed artificial ligand-gated ion channels by coupling G protein-coupled receptors to the Kir6.2 potassium channel. These artificial channels called ion channel-coupled receptors offer complementary properties to natural channels by extending the repertoire of ligands to those recognized by the fused receptors, by generating more sustained signals and by conferring potassium selectivity. The first artificial channels based on the muscarinic M2 and the dopaminergic D2L receptors were opened and closed by acetylcholine and dopamine, respectively. We find here that this opposite regulation of the gating is linked to the length of the receptor C-termini, and that C-terminus engineering can precisely control the extent and direction of ligand gating. These findings establish the design rules to produce customized ligand-gated channels for synthetic biology applications.

Nucleus accumbens dopamine D2-receptor expressing neurons control behavioral flexibility in a place discrimination task in the IntelliCage.

Considerable evidence has demonstrated a critical role for the nucleus accumbens (NAc) in the acquisition and flexibility of behavioral strategies. These processes are guided by the activity of two discrete neuron types, dopamine D1- or D2-receptor expressing medium spiny neurons (D1-/D2-MSNs). Here we used the IntelliCage, an automated group-housing experimental cage apparatus, in combination with a reversible neurotransmission blocking technique to examine the role of NAc D1- and D2-MSNs in the acquisition and reversal learning of a place discrimination task. We demonstrated that NAc D1- and D2-MSNs do not mediate the acquisition of the task, but that suppression of activity in D2-MSNs impairs reversal learning and increased perseverative errors. Additionally, global knockout of the dopamine D2L receptor isoform produced a similar behavioral phenotype to D2-MSN-blocked mice. These results suggest that D2L receptors and NAc D2-MSNs act to suppress the influence of previously correct behavioral strategies allowing transfer of behavioral control to new strategies.

99mTc-Cyclopentadienyl Tricarbonyl Chelate-Labeled Compounds as Selective Sigma-2 Receptor Ligands for Tumor Imaging

We have designed and synthesized a series of cyclopentadienyl tricarbonyl rhenium complexes containing a 5,6-dimethoxyisoindoline or a 6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline pharmacophore as σ2 receptor ligands. Rhenium compound 20a possessed low nanomolar σ2 receptor affinity (K(i) = 2.97 nM) and moderate subtype selectivity (10-fold). Moreover, it showed high selectivity toward vesicular acetylcholine transporter (2374-fold), dopamine D2L receptor, NMDA receptor, opiate receptor, dopamine transporter, norepinephrine transporter, and serotonin transporter. Its corresponding radiotracer [(99m)Tc]20b showed high uptake in a time- and dose-dependent manner in DU145 prostate cells and C6 glioma cells. In addition, this tracer exhibited high tumor uptake (5.92% ID/g at 240 min) and high tumor/blood and tumor/muscle ratios (21 and 16 at 240 min, respectively) as well as specific binding to σ receptors in nude mice bearing C6 glioma xenografts. Small animal SPECT/CT imaging of [(99m)Tc]20b in the C6 glioma xenograft model demonstrated a clear visualization of the tumor at 180 min after injection.

Dysbindin-1 modifies signaling and cellular localization of recombinant, human D₃ and D₂ receptors.

Dystrobrevin binding protein-1 (dysbindin-1), a candidate gene for schizophrenia, modulates cognition, synaptic plasticity and frontocortical circuitry and interacts with glutamatergic and dopaminergic transmission. Loss of dysbindin-1 modifies cellular trafficking of dopamine (DA) D2 receptors to increase cell surface expression, but its influence upon signaling has never been characterized. Further, the effects of dysbindin-1 upon closely related D3 receptors remain unexplored. Hence, we examined the impact of dysbindin-1 (isoform A) co-expression on the localization and coupling of human D2L and D3 receptors stably expressed in Chinese hamster ovary or SH-SY5Y cells lacking endogenous dysbindin-1. Dysbindin-1 co-transfection decreased cell surface expression of both D3 and D2L receptors. Further, while their affinity for DA was unchanged, dysbindin-1 reduced the magnitude and potency of DA-induced adenylate cylase recruitment/cAMP production. Dysbindin-1 also blunted the amplitude of DA-induced phosphorylation of ERK1/2 and Akt at both D2L and D3 receptors without, in contrast to cAMP, affecting the potency of DA. Interference with calveolin/clathrin-mediated processes of internalization prevented the modification by dysbindin-1 of ERK1/2 and adenylyl cyclase stimulation at D2L and D3 receptors. Finally, underpinning the specificity of the influence of dysbindin-1 on D2L and D3 receptors, dysbindin-1 did not modify recruitment of adenylyl cyclase by D1 receptors. These observations demonstrate that dysbindin-1 influences cell surface expression of D3 in addition to D2L receptors, and that it modulates activation of their signaling pathways. Accordingly, both a deficiency and an excess of dysbindin-1 may be disruptive for dopaminergic transmission, supporting its link to schizophrenia and other CNS disorders. Dysbindin-1, a candidate gene for schizophrenia, alters D2 receptors cell surface expression. We demonstrate that dysbindin-1 expression also influences cell surface levels of D3 receptors. Further, Dysbindin-1 reduces DA-induced adenylate cylase recruitment/cAMP production and modifies major signaling pathways (Akt and extracellular signal-regulated kinases1/2 (ERK1/2)) of both D2 and D3 receptors. Dysbindin-1 modulates thus D2 and D3 receptor signaling, supporting a link to schizophrenia.

Autoimmunity against dopamine receptors in neuropsychiatric and movement disorders: a review of Sydenham chorea and beyond.

Antineuronal autoantibodies are associated with the involuntary movement disorder Sydenham chorea (SC) and paediatric autoimmune neuropsychiatric disorders associated with streptococcal infections (PANDAS) which are characterized by the acute onset of tics and/or obsessive compulsive disorder (OCD). In SC and PANDAS, autoantibodies signal human neuronal cells and activate calcium calmodulin-dependent protein kinase II (CaMKII). Animal models immunized with group A streptococcal antigens demonstrate autoantibodies against dopamine receptors and concomitantly altered behaviours. Human monoclonal antibodies (mAbs) derived from SC target and signal the dopamine D2L (long) receptor (D2R). Antibodies against D2R were elevated over normal levels in SC and acute-onset PANDAS with small choreiform movements, but were not elevated over normal levels in PANDAS-like chronic tics and OCD. The expression of human SC-derived anti-D2R autoantibody V gene in B cells and serum of transgenic mice demonstrated that the human autoantibody targets dopaminergic neurones in the basal ganglia and other types of neurones in the cortex. Here, we review current evidence supporting the hypothesis that antineuronal antibodies, specifically against dopamine receptors, follow streptococcal exposures and may target dopamine receptors and alter central dopamine pathways leading to movement and neuropsychiatric disorders.