Distinctive and synergistic signaling of human adenosine A2a and dopamine D2L receptors in CHO cells

Adenosine A(2a) receptor (A(2a)R) colocalizes with dopamine D(2) receptor (D(2)R) in the basal ganglia and modulates D(2)R-mediated dopaminergic activities. A(2a)R and D(2)R couple to stimulatory and inhibitory G proteins, respectively. Their opposing roles in regulating neuronal activities, such as locomotion and alcohol consumption, are mediated by their opposite actions on adenylate cyclase, which often serves as "co-incidence detector" of various activators. On the other hand, the neural actions of A(2a)R and D(2)R are also, at least partially, independent of each other, as indicated by studies using D(2)R and A(2a)R knock-out mice. Here we co-expressed human A(2a)R and human D(2L)R in CHO cells and examined their signaling characteristics. Human A(2a)R desensitized rapidly upon agonist stimulation. A(2a)R activity (80%) was diminished after 2 hr of pretreatment with its agonist CGS21680. In contrast, human D(2L)R activity was sustained even after 2 hr and 18 hr pretreatment with its agonist quinpirole. Long-term (18 hr) stimulation of human D(2L)R also increased basal cAMP levels in CHO cells, whereas long-term (18 hr) activation of human A(2a)R did not affect basal cAMP levels. Furthermore, long-term (18 hr) activation of D(2L)R dramatically sensitized A(2a)R-induced stimulation of adenylate cyclase in a pertussis toxin-sensitive way. Forskolin-induced cAMP accumulation was significantly increased after short-term (2 hr) human D(2L)R stimulation and further elevated after long-term (18 hr) D(2L)R activation. However, neither short-term (2 hr) nor long-term (18 hr) stimulation of A(2a)R affected the inhibitory effects of D(2L)R on adenylate cyclase. Co-stimulation of A(2a)R and D(2L)R could not induce desensitization or sensitization of D(2L)R either. In summary, signaling through A(2a)R and D(2L)R is distinctive and synergistic, supporting their unique and yet integrative roles in regulating neuronal functions when both receptors are present.     

Oligomerization of adenosine A2A and dopamine D2 receptors in living cells

We investigated whether oligomerization of adenosine A(2A) receptor (A(2A)R) and dopamine D(2) receptor (D(2)R) exists in living cells using modified bioluminescence resonance energy transfer (BRET(2)) technology. Fusion of these receptors to a donor, Renilla luciferase (Rluc), and to an acceptor, modified green fluorescent protein (GFP(2)), did not affect the ligand binding affinity, subcellular distribution, and coimmunoprecipitation of the receptors. BRET was detected not only between Myc-D(2)R-Rluc and A(2A)R-GFP(2) but also between HA-tagged A(2A)R-Rluc and A(2A)R-GFP(2). These results indicate A(2A)R, either homomeric or heteromeric with D(2)R, exists as an oligomer in living cells.     

Coaggregation, cointernalization, and codesensitization of adenosine A2A receptors and dopamine D2 receptors

Antagonistic and reciprocal interactions are known to exist between adenosine and dopamine receptors in the striatum. In the present study, double immunofluorescence experiments with confocal laser microscopy showed a high degree of colocalization of adenosine A(2A) receptors (A(2A)R) and dopamine D(2) receptors (D(2)R) in cell membranes of SH-SY5Y human neuroblastoma cells stably transfected with human D(2)R and in cultured striatal cells. A(2A)R/D(2)R heteromeric complexes were demonstrated in coimmunoprecipitation experiments in membrane preparations from D(2)R-transfected SH-SY5Y cells and from mouse fibroblast Ltk(-) cells stably transfected with human D(2)R (long form) and transiently cotransfected with the A(2A)R double-tagged with hemagglutinin. Long term exposure to A(2A)R and D(2)R agonists in D(2)R-cotransfected SH-SY5Y cells resulted in coaggregation, cointernalization and codesensitization of A(2A)R and D(2)R. These results give a molecular basis for adenosine-dopamine antagonism at the membrane level and have implications for treatment of Parkinson's disease and schizophrenia, in which D(2)R are involved.     

Behavioral control by striatal adenosine A2A‐dopamine D2 receptor heteromers

G protein‐coupled receptors (GPCR) exhibit the ability to form receptor complexes that include molecularly different GPCR (ie, GPCR heteromers), which endow them with singular functional and pharmacological characteristics. The relative expression of GPCR heteromers remains a matter of intense debate. Recent studies support that adenosine A_2A receptors (A_2AR) and dopamine D₂ receptors (D₂R) predominantly form A_2AR‐D₂R heteromers in the striatum. The aim of the present study was evaluating the behavioral effects of pharmacological manipulation and genetic blockade of A_2AR and D₂R within the frame of such a predominant striatal heteromeric population. First, in order to avoid possible strain‐related differences, a new D₂R‐deficient mouse with the same genetic background (CD‐1) than the A_2AR knock‐out mouse was generated. Locomotor activity, pre‐pulse inhibition (PPI) and drug‐induced catalepsy were then evaluated in wild‐type, A_2AR and D₂R knock‐out mice, with and without the concomitant administration of either the D₂R agonist sumanirole or the A_2AR antagonist SCH442416. SCH442416‐mediated locomotor effects were demonstrated to be dependent on D₂R signaling. Similarly, a significant dependence on A_2AR signaling was observed for PPI and for haloperidol‐induced catalepsy. The results could be explained by the existence of one main population of striatal postsynaptic A_2AR‐D₂R heteromers, which may constitute a relevant target for the treatment of Parkinson's disease and other neuropsychiatric disorders.     

Reduced D2-mediated signaling activity and trans-synaptic upregulation of D1 and D2 dopamine receptors in mice overexpressing the dopamine transporter

The dopamine transporter (DAT) regulates the temporal and spatial actions of dopamine by reuptaking this neurotransmitter into the presynaptic neurons. We recently generated transgenic mice overexpressing DAT (DAT-tg) that have a 3-fold increase in DAT protein levels which results in a 40% reduction of the extracellular DA concentration in the striatum. The aim of this study was to examine the effect of this reduction in dopaminergic tone on postsynaptic responses mediated by dopamine receptors. We report here that DAT-tg mice have increased levels of striatal D1 (30%) and D2 (approximately 60%) dopamine receptors with D1 receptor signaling components not significantly altered, as evidenced by unaffected basal or stimulated levels of phospho-GluR1 (Ser845) and phospho-ERK2. However, the novel D2 mediated Akt signaling is markedly altered in DAT-tg animals. In particular, there is a 300% increase in the basal levels of phospho-Akt in the striatum of DAT-tg, reflecting the reduced extracellular dopamine tone in these animals. This increase in basal pAkt levels can be pharmacologically recapitulated by partial dopamine depletion in WT mice treated with the selective tyrosine hydroxylase inhibitor alpha-methyl-para-tyrosine (alpha-MPT). Behaviorally, DAT-tg animals demonstrate an augmented synergistic interaction between up-regulated D1 and D2 receptors, which results in increased climbing behavior in transgenic mice after stimulation with either apomorphine or a co-administration of selective D1 and D2 receptor agonists. In sum, our study reveals that hypodopaminegia caused by up-regulation of DAT results in significant alterations at postsynaptic receptor function with most notable dysregulation at the level of D2 receptor signaling.     

Dynamin and rab5 regulate GRK2-dependent internalization of dopamine D2 receptors.

Dopamine D2 receptors (D2Rs; short form, which is one of the alternative splicing variants) expressed in COS-7 cells are internalized in an agonist-dependent manner only when G protein-coupled receptor kinase 2 (GRK2) is coexpressed [Ito, K., Haga, T., Lameh, J. & Sadée, W., (1999) Eur. J. Biochem. 260, 112-119]. We have examined the effects of coexpression of dynamin, a small molecular mass GTP-binding protein, rab5A, and their mutants on the internalization of D2Rs in the presence of both dopamine (10 or 100 microM) and GRK2. The rate and extent of D2R internalization was increased or decreased by coexpression of dynamin I or a dominant-negative form of dynamin I (dynamin I K44E), respectively. The effects of coexpressing these two dynamins were more prominent at 10 microM dopamine than at 100 microM. In the presence of 10 microM dopamine, internalization of D2R was completely suppressed when dynamin I K44E was coexpressed, and the half-life (t 1/2) of D2R internalization decreased relative to cells not expressing dynamin from 82 to 29 min when dynamin I was coexpressed. Internalization of D2Rs was facilitated or suppressed by coexpression of a constitutively active form of rab5A (rab5A Q79L) or a dominant-negative form of rab5A (rab5A S34N), respectively. The t 1/2 of D2R internalization at 10 microM dopamine decreased from 82 to 16 min in cells coexpressing rab5A Q79L. The effect of coexpression of rab5A S34N was more apparent at 100 microM dopamine than at 10 microM; the t 1/2 of D2R internalization at 100 microM dopamine increased from 20 to 56 min and the proportion of internalized D2Rs after 120 min decreased from 53 to 28%. These results indicate that the internalization of D2Rs is dependent on the action of dynamin as well as GRK2, and is regulated by the action of rab5A.     

How calmodulin interacts with the adenosine A(2A) and the dopamine D(2) receptors

Receptor heteromerization is a mechanism used by G protein-coupled receptors to diversify their properties and function. We previously demonstrated that these interactions occur through salt bridge formation between epitopes of the involved receptors. Recent studies claim that calmodulin (CaM) binds to an Arg-rich epitope located in the amino-terminus of the dopamine D(2) receptor third intracellular loop. This is the same epitope involved in adenosine A(2A)-D(2) receptor heteromerization, through Coulombic interaction between the Arg residues and a phosphorylated serine (pS) located in the medial segment of the C-terminus of the A(2A) receptor. Mass spectrometric analysis indicates that an electrostatic interaction involving the D(2) receptor Arg-rich epitope and several CaM acidic epitopes are mainly responsible for the D(2) receptor-CaM binding. CaM could also form multiple noncovalent complexes by means of electrostatic interactions with an epitope localized in the proximal segment of the C-terminus of the A(2A) receptor. Ca(2+) disrupted the binding of CaM to the D(2) but not to the A(2A) receptor epitope, and CaM disrupted the electrostatic interactions between the D(2) receptor epitope and the more distal A(2A) receptor epitope. A model is introduced with the possible functional implications of A(2A)-D(2)-CaM interactions. These in vitro findings imply a possible regulatory role for CaM in receptor heteromers formation.     

TM2D genes regulate Notch signaling and neuronal function in Drosophila

TM2 domain containing (TM2D) proteins are conserved in metazoans and encoded by three separate genes in each model organism species that has been sequenced. Rare variants in TM2D3 are associated with Alzheimer’s disease (AD) and its fly ortholog almondex is required for embryonic Notch signaling. However, the functions of this gene family remain elusive. We knocked-out all three TM2D genes (almondex, CG11103/amaretto, CG10795/biscotti) in Drosophila and found that they share the same maternal-effect neurogenic defect. Triple null animals are not phenotypically worse than single nulls, suggesting these genes function together. Overexpression of the most conserved region of the TM2D proteins acts as a potent inhibitor of Notch signaling at the γ-secretase cleavage step. Lastly, Almondex is detected in the brain and its loss causes shortened lifespan accompanied by progressive motor and electrophysiological defects. The functional links between all three TM2D genes are likely to be evolutionarily conserved, suggesting that this entire gene family may be involved in AD.     

Structural insights into the human D1 and D2 dopamine receptor signaling complexes

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Differential regulation of dopamine D1 and D2 signaling by nicotine in neostriatal neurons

Nicotine, acting on nicotinic acetylcholine receptors (nAChRs) expressed at pre-synaptic dopaminergic terminals, has been shown to stimulate the release of dopamine in the neostriatum. However, the molecular consequences of pre-synaptic nAChR activation in post-synaptic neostriatal neurons are not clearly understood. Here, we investigated the effect of nAChR activation on dopaminergic signaling in medium spiny neurons by measuring phosphorylated DARPP-32 (dopamine- and cAMP-regulated phosphoprotein of Mr 32 kDa) at Thr34 (the PKA-site) in mouse neostriatal slices. Nicotine produced dose-dependent responses, with a low concentration (1 microm) causing a sustained decrease in DARPP-32 Thr34 phosphorylation and a high concentration (100 microm) causing a transient increase in DARPP-32 Thr34 phosphorylation. Depending on the concentration of nicotine, either dopamine D2 or D1 receptor signaling was predominantly activated. Nicotine at a low concentration (1 microm) activated dopamine D2 receptor signaling in striatopallidal/indirect pathway neurons, likely by activating alpha4beta2* nAChRs at dopaminergic terminals. Nicotine at a high concentration (100 microm) activated dopamine D1 receptor signaling in striatonigral/direct pathway neurons, likely by activating (i) alpha4beta2* nAChRs at dopaminergic terminals and (ii) alpha7 nAChRs at glutamatergic terminals, which, by stimulating the release of glutamate, activated NMDA/AMPA receptors at dopaminergic terminals. The differential effects of low and high nicotine concentrations on D2- and D1-dependent signaling pathways in striatal neurons may contribute to dose-dependent actions of this drug of abuse.     

Activation of adenosine A1 and A2A receptors modulates dopamine D2 receptor-induced responses in stably transfected human neuroblastoma cells

Adenosine can influence dopaminergic neurotransmission in the basal ganglia via postsynaptic interaction between adenosine A2A and dopamine D2 receptors. We have used a human neuroblastoma cell line (SH-SY5Y) that was found to express constitutively moderate levels of adenosine A1 and A2A receptors (approximately 100 fmol/mg of protein) to investigate the interactions of A2A/D2 receptors, at a cellular level. After transfection with human D2L receptor cDNA, SH-SY5Y cells expressed between 500 and 1,100 fmol of D2 receptors/mg of protein. In membrane preparations, stimulation of adenosine A2A receptors decreased the affinity of dopamine D2 receptors for dopamine. In intact cells, the calcium concentration elevation induced by KCI treatment was moderate, and dopamine had no effect on either resting intracellular free Ca2+ concentration ([Ca2+]i) or KCI-induced responses. In contrast, pretreatment with adenosine deaminase for 2 days dramatically increased the elevation of [Ca2+]i evoked by KCI, which then was totally reversed by dopamine. The effects induced by 48-h adenosine inactivation were mimicked by application of adenosine A1 antagonists and could not be further reversed by acute activation of either A1 or A2A receptors. Acute application of the selective A2 receptor agonist CGS-21680 counteracted the D2 receptor-induced [Ca2+]i responses. The present study shows that SH-SY5Y cells are endowed with functional adenosine A2A and A1 receptors and that A2A receptors exert an antagonistic acute effect on dopamine D2 receptor-mediated functions. In contrast, A1 receptors induce a tonic modulatory role on these dopamine functions.     

D2-receptor-linked signaling pathways regulate the expression of hepatic CYP2E1

This study investigated the role of catecholamine-related signaling pathways in the regulation of hepatic cytochrome P450 (CYP2E1). Central and peripheral catecholamine depletion with reserpine down-regulated CYP2E1. On the other hand, selective peripheral catecholamine depletion with guanethidine increased CYP2E1 apoprotein levels. Enrichment of peripheral catecholamines with adrenaline suppressed p-nitrophenol hydroxylase activity (PNP). PNP activity was also markedly suppressed by l-DOPA. Stimulation of D(2)-receptors with bromocriptine up-regulated CYP2E1, as assessed by enzyme activity and protein levels, whereas blockade of D(2)-dopaminergic receptors with sulpiride down-regulated this isozyme. These findings indicate that central and peripheral catecholamines have different effects on CYP2E1. Central catecholamines appear related to the up-regulation, whereas the role of peripheral catecholamines is clearly related to the type and location of adrenoceptors involved. D(2)-receptor-linked signaling pathways have an up-regulating effect on CYP2E1, while D(1)-receptor pathways may down-regulate this isozyme. It is worth noting that the widespread environmental pollutant benzo(alpha)pyrene (B(alpha)P) altered the modulating effect of catecholaminergic systems on CYP2E1 regulation. In particular, whereas stimulation or blockade of adrenoceptors had no effect on constitutive PNP activity, exposure to B(alpha)P modified the impact of central and peripheral catecholamines and alpha(2)-adrenoceptors on CYP2E1 expression. It appears that under the influence of B(alpha)P, alpha(2)-adrenergic receptor-linked signaling pathways increased CYP2E1 apoprotein levels. Given that a wide range of xenobiotics and clinically used drugs are activated by CYP2E1 to toxic metabolites, including the production of reactive oxygen species (ROS), it is possible that therapies challenging dopaminergic receptor- and/or alpha(2)-adrenoceptor-linked signaling pathways may alter the expression of CYP2E1, thus affecting the progress and development of several pathologies.     

L-Stepholidine rescues memory deficit and synaptic plasticity in models of Alzheimer's disease via activating dopamine D1 receptor/PKA signaling pathway

It is accepted that amyloid β-derived diffusible ligands (ADDLs) have a prominent role in triggering the early cognitive deficits that constitute Alzheimer''s disease (AD). However, there is still no effective treatment for preventing or reversing the progression of the disease. Targeting α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptor trafficking and its regulation is a new strategy for AD early treatment. Here we investigate the effect and mechanism of L-Stepholidine (L-SPD), which elicits dopamine D1-type receptor agonistic activity, while acting as D2-type receptor antagonist on cognition and synaptic plasticity in amyloid precursor protein (APP) and presenilin 1 (PS1) double-transgenic (APP/PS1) mice, and hippocampal cultures or slices treated with ADDLs. L-SPD could improve the hippocampus-dependent memory, surface expression of glutamate receptor A (GluA1)-containing AMPA receptors and spine density in hippocampus of APP/PS1 transgenic mice. L-SPD not only rescued decreased phosphorylation and surface expression of GluA1 in hippocampal cultures but also protected the long-term potentiation in hippocampal slices induced by ADDLs. Protein kinase A (PKA) agonist Sp-cAMPS or D1-type receptor agonist {"type":"entrez-protein","attrs":{"text":"SKF81297","term_id":"1156277425"}}SKF81297 had similar effects, whereas PKA antagonist Rp-cAMPS or D1-type receptor antagonist {"type":"entrez-protein","attrs":{"text":"SCH23390","term_id":"1052733334","term_text":"SCH23390"}}SCH23390 abolished the effect of L-SPD on GluA1 trafficking. This was mediated mainly by PKA, which could phosphorylate serine residue at 845 of the GluA1. L-SPD may be explored as a potential therapeutic drug for AD through a mechanism that improves AMPA receptor trafficking and synaptic plasticity via activating D1/PKA signaling pathway.Alzheimer''s disease (AD) is an age-dependent neurodegenerative disorder, which likely begins with deficits in synaptic transmission in brain regions such as the hippocampus. Now it is accepted that amyloid β-derived diffusible ligands (ADDLs) have a key role in synapse dysfunction and early memory loss in AD.^{1, 2} Excitatory synaptic transmission is tightly regulated by α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptors. Regulation of AMPA receptor trafficking in and out of excitatory synapses is important for controlling the strength of excitatory synapses in long-term potentiation (LTP), long-term depression and other forms of synaptic plasticity.^{3, 4, 5} Regulation of AMPA receptor trafficking depends on receptor subunit composition. In the hippocampus, most AMPA receptors are either glutamate receptor A (GluA)1/GluA2 (~80%) or GluA2/GluA3 (~20%) hetero-oligomers.^{6, 7} GluA1-containing receptors are delivered in an activity-dependent manner during LTP.⁸ In hippocampal CA1 pyramidal neurons, LTP involves insertion of AMPA receptors into excitatory synapses. GluA1-containing AMPA receptor phosphorylation regulates AMPA receptor exocytosis and subsequent insertion at synapses.^{3, 9}Recent studies have shown that Aβ oligomers could alter AMPA receptor trafficking by modulating relevant protein kinases or protein phosphatases.¹⁰ However, the mechanisms through which ADDLs modify AMPA receptor trafficking and synaptic plasticity, as well as cognitive deficits, are not fully known.Protein kinase A (PKA) activation strongly increases exocytosis of AMPA receptors by direct phosphorylation of AMPA receptors.¹¹ Dopamine receptors have been grouped into two families, D1 type and D2 type. The D1 type comprises D1 and D5 receptor subtypes, which couple to Gα_s/cyclic AMP (cAMP), leading to activation of PKA, whereas the D2 type comprises D2, D3 and D4 receptor subtypes, which couple to Gα_i/cAMP, leading to inactivation of PKA. Our previous works have demonstrated that brief activation of D1-type receptor accelerates GluA1 externalization at extrasynaptic sites through a PKA-dependent pathway in hippocampal pyramidal neurons.¹² Another study has shown that activation of D1-type dopamine receptors protects neurons from synapse dysfunction induced by amyloid-β oligomers.¹³ Thus, L-Stepholidine (L-SPD), one of the active ingredients of the Chinese herb Stephania intermedia belonging to the tetrahydroprotoberberines (THPBs), which elicits dopamine D1-type receptor agonistic activity, while acting as D2-type receptor antagonist,^{14, 15} may have potential therapeutic effect on AD by improving AMPA receptor trafficking and synaptic plasticity.In the present study, we demonstrated for the first time that L-SPD improved the impaired memory that was correlated with decreased phosphorylation and surface expression of GluA1-containing AMPA receptors, as well as spine loss in amyloid precursor protein (APP) and presenilin 1 (PS1) double-transgenic (APP/PS1) mice. In addition, L-SPD prevented ADDL-induced dysfunction of AMPA receptor trafficking in cultured hippocampal neurons and the inhibition of LTP in hippocampal slices through activation of D1/PKA signaling pathway.     

Dopamine signaling in food addiction: role of dopamine D2 receptors

Dopamine (DA) regulates emotional and motivational behavior through the mesolimbic dopaminergic pathway. Changes in DA signaling in mesolimbic neurotransmission are widely believed to modify reward-related behaviors and are therefore closely associated with drug addiction. Recent evidence now suggests that as with drug addiction, obesity with compulsive eating behaviors involves reward circuitry of the brain, particularly the circuitry involving dopaminergic neural substrates. Increasing amounts of data from human imaging studies, together with genetic analysis, have demonstrated that obese people and drug addicts tend to show altered expression of DA D2 receptors in specific brain areas, and that similar brain areas are activated by food-related and drug-related cues. This review focuses on the functions of the DA system, with specific focus on the physiological interpretation and the role of DA D2 receptor signaling in food addiction. [BMB Reports 2013; 46(11): 519-526]     

Selective disruption of dopamine D2-receptors/beta-arrestin2 signaling by mood stabilizers

Abstract

Mood stabilizers are a heterogeneous class of drugs having antidepressant and anti-manic effects in bipolar disorders, depression and schizophrenia. Despite wide clinical applications, the mechanisms underlying their shared actions and therapeutic specificity are unknown. Here, we examine the effects of the structurally unrelated mood stabilizers lamotrigine, lithium and valproate on G protein and beta-arrestin-dependent components of dopamine D2 receptor signaling and assess their contribution to the behavioral effects of these drugs. When administered chronically to mice lacking either D2 receptors or beta-arrestin 2, lamotrigine, lithium and valproate failed to affect Akt/GSK3 signaling as they do in normal littermates. This lack of effect on signaling resulted in a loss of responsiveness to mood stabilizers in tests assessing “antimanic” or “antidepressant”-like behavioral drug effects. This shows that mood stabilizers lamotrigine, lithium and valproate can exert behavioral effects in mice by disrupting the beta-arrestin 2-mediated regulation of Akt/GSK3 signaling by D2 dopamine receptors, thereby suggesting a shared mechanism for mood stabilizer selectivity.     

D2 dopamine receptors interact directly with N-type calcium channels and regulate channel surface expression levels

N-type channels are located on dendrites and at pre-synaptic nerve terminals where they play a fundamental role in neurotransmitter release. They are potently regulated by the activation of a number of different types of pertussis toxin (PTX)-sensitive G alpha(i/o) coupled receptors, which results in voltage-dependent inhibition of channel activity via G betagamma subunits. Using heterologous expression in HEK 293T cells, we show via whole cell patch clamp recordings that D2 receptors mediate both G betagamma (i.e., voltage-dependent) and voltage-independent inhibition of channel activity. Furthermore, using co-immunoprecipitation and pull down assays involving the intracellular regions of each protein, we show that D2 receptors and N-type channels form physical signaling complexes. Finally, we use confocal microscopy to demonstrate that D2 receptors regulate N-type channel trafficking to affect the number of calcium channels available at the plasma membrane. Taken together, these data provide evidence for multiple voltage-dependent and voltage-independent mechanisms by which D2 receptor subtypes influence N-type channel activity.     

D2 dopamine receptors interact directly with N-type calcium channels and regulate channel surface expression levels

N-type channels are located on dendrites and at pre-synaptic nerve terminals where they play a fundamental role in neurotransmitter release. They are potently regulated by the activation of a number of different types of pertussis toxin (PTX)-sensitive Gαi/o coupled receptors, which results in voltage-dependent inhibition of channel activity via Gβγ subunits. Using heterologous expression in HEK 293T cells, we show via whole cell patch clamp recordings that D2 receptors mediate both Gβγ (i.e. voltage-dependent) and voltage-independent inhibition of channel activity. Furthermore, using co-immunoprecipitation and pull down assays involving the intracellular regions of each protein, we show that D2 receptors and N-type channels form physical signaling complexes. Finally, we use confocal microscopy to demonstrate that D2 receptors regulate N-type channel trafficking to affect the number of calcium channels available at the plasma membrane. Taken together, these data provide evidence for multiple voltage-dependent and voltage-independent mechanisms by which D2 receptor subtypes influence N-type channel activity.     

An antagonistic interaction between A2B adenosine and D2 dopamine receptors modulates the function of rat carotid body chemoreceptor cells

We have previously demonstrated that adenosine controls the release of catecholamines (CA) from carotid body (CB) acting on A2B receptors. Here, we have tested the hypothesis that the control is exerted via an interaction between adenosine A2B and dopamine D2 receptors present in chemoreceptor cells. Experiments were performed in vitro in CB from 3 months rats. The effect of A2B adenosine and D2 dopamine agonists and antagonists applied alone or in combination were studied on basal (20%O2) and hypoxia (10%O2)-evoked release of CA and cAMP content of CB. We have found that adenosine A2 agonists and D2 antagonists dose-dependently increased basal and evoked release CA from the CB while A2 antagonists and D2 agonists had an inhibitory action. The existence of A2B-D2 receptor interaction was established because the inhibitory action of A2 antagonists was abolished by D2 antagonists, and the stimulatory action of A2 agonists was abolished by D2 agonists. Further, A2 agonists increased and D2 agonist decreased cAMP content in the CB; their co-application eliminated the response. The present results provide direct pharmacological evidence that an antagonistic interaction between A2B adenosine and D2 dopamine receptors exist in rat CB and would explain the dopamine-adenosine interactions on ventilation previously observed.     

Effect of free radicals on adenosine A(2A) and dopamine D2 receptors in the striatum of young adult and aged rats

Parkinson's disease (PD) is a prevalent age-related motor dysfunction resulting from the hyperactivity of the indirect striatal pathway, which is controlled in an antagonistic manner by inhibitory dopamine D2 and facilitatory adenosine A(2A) receptors. Thus, dopamine precursors like l-DOPA are the standard therapy and A(2A) antagonists are now tested as anti-parkinsonians. Increased free radicals levels occur on aging and are proposed to be a contributing factor for PD. We now tested if free radicals affected A(2A) and D2 receptors in striatal membranes of young adult (2 months) and old (24 months) rats. The A(2A) receptor antagonist [3H]SCH 58261 bound to striatal membranes with a KD of 0.9 nM and a Bmax of 953 fmol/mg protein in young rats and with a KD of 0.8 nM and a Bmax of 725 fmol/mg protein in aged rats (24% decrease). The D2 receptor antagonist [3H]raclopride bound to striatal membranes with a KD of 4.0 nM and a Bmax of 598 fmol/mg protein in young rats and with a KD of 4.3 nM and a Bmax of 368 fmol/mg protein in aged rats (38% decrease). Exposure of striatal membranes to a free radical generation system (2 mM FeSO4 and 4 mM ascorbate) caused a similar decrease of [3H]SCH 58261 (35%) and [3H]raclopride (37%) binding in young adult rats but caused a greater decrease of [3H]SCH 58261 (49%) than of [3H]raclopride (20%) binding in aged rats. Thus, in aged rats, there is an unbalance of A(2A)/D2 receptor density favouring A(2A) receptors, which is restored on exposure to free radicals. This supports the hypothesis that the effectiveness of A(2A) receptor antagonists as anti-parkinsonians, demonstrated in young adult animals, may not be affected by a modified A(2A)/D2 receptor density in aged individuals suffering from exposure to increased free radical levels, as occurs in PD.     

Hallucinogenic 5-HT2AR agonists LSD and DOI enhance dopamine D2R protomer recognition and signaling of D2-5-HT2A heteroreceptor complexes

Dopamine D2_LR-serotonin 5-HT2AR heteromers were demonstrated in HEK293 cells after cotransfection of the two receptors and shown to have bidirectional receptor–receptor interactions. In the current study the existence of D2_L-5-HT2A heteroreceptor complexes was demonstrated also in discrete regions of the ventral and dorsal striatum with in situ proximity ligation assays (PLA). The hallucinogenic 5-HT2AR agonists LSD and DOI but not the standard 5-HT2AR agonist TCB2 and 5-HT significantly increased the density of D2like antagonist ³H-raclopride binding sites and significantly reduced the pK_iH values of the high affinity D2R agonist binding sites in ³H-raclopride/DA competition experiments. Similar results were obtained in HEK293 cells and in ventral striatum. The effects of the hallucinogenic 5-HT2AR agonists on D2R density and affinity were blocked by the 5-HT2A antagonist ketanserin. In a forskolin-induced CRE-luciferase reporter gene assay using cotransfected but not D2R singly transfected HEK293 cells DOI and LSD but not TCB2 significantly enhanced the D2_LR agonist quinpirole induced inhibition of CRE-luciferase activity. Haloperidol blocked the effects of both quinpirole alone and the enhancing actions of DOI and LSD while ketanserin only blocked the enhancing actions of DOI and LSD. The mechanism for the allosteric enhancement of the D2R protomer recognition and signalling observed is likely mediated by a biased agonist action of the hallucinogenic 5-HT2AR agonists at the orthosteric site of the 5-HT2AR protomer. This mechanism may contribute to the psychotic actions of LSD and DOI and the D2-5-HT2A heteroreceptor complex may thus be a target for the psychotic actions of hallunicogenic 5-HT2A agonists.