The tolerance property of human D3 dopamine receptor is determined by specific amino acid residues in the second cytoplasmic loop

The D2 and D3 dopamine receptor subtypes are structurally homologous and couple to the same signal transduction pathways. Nevertheless, their evolutionary conservation suggests that the two subtypes might exhibit unique signaling characteristics. We previously determined that D3 but not D2S dopamine receptor exhibits a tolerance property in which the D3 receptor-activated G-protein coupled inward rectifier potassium currents progressively decreases upon repeated agonist stimulation. In this paper, using AtT-20 neuroendocrine cells stably expressing either human D3 or D2S receptor, we show that the tolerance property is also observed in the D3 receptor-adenylyl cyclase and D3 receptor-mitogen-activated protein kinase signaling pathways. We have previously shown that the second cytoplasmic loop of D3 receptor is required for tolerance. Here, using site-directed mutagenesis, we identified the specific amino acids in the D3 second cytoplasmic loop involved in the tolerance property. The results show that substitution of a non-conserved cysteine residue at position 147 with positively-charged lysine or arginine residues abolishes tolerance. Interestingly, the cysteine 147 residue is embedded in a putative phosphorylation site adjacent to two serine residues. Mutation of these serine residues to alanine also attenuates tolerance. Taken together, these structural studies suggest a role for phosphorylation in D3 receptor tolerance property.     

Examination of the involvement of protein kinase A in D2 dopamine receptor antagonist-induced immediate early gene expression

Immediate early genes (IEGs) are induced by different signaling pathways. It has been proposed that D2 dopamine receptor blockade induces IEG expression through activation of protein kinase A (PKA), although few studies have examined this issue in vivo. We infused the PKA inhibitor H-89 into the striatum of male rats, followed 30 min later by systemic administration of eticlopride. Eticlopride-induced c-fos and zif268 mRNA expression in striatum was not blocked by H-89. In addition, eticlopride did not produce measurable levels of PKA activity in striatum, whereas the cAMP activator Sp-8-Br-cAMPs increased levels of activated PKA. Neither the adenosine A2a receptor agonist CGS 21680 nor the phosphodiesterase-4 inhibitor rolipram, each of which should increase PKA activation, potentiated eticlopride-induced IEG expression. To test whether other signaling pathways are involved in eticlopride-mediated gene induction, we also infused inhibitors of the mitogen-activated and calcium/calmodulin-dependent protein kinases into animals and then treated them with eticlopride. The data suggest that eticlopride-induced IEG expression is not solely dependent on these kinases either. These data suggest that PKA activation may not be necessary for induction of IEGs by D2 dopamine receptor antagonists and that other intracellular signaling pathways may be involved.     

Modulation of motor behavior by dopamine and the D1-like dopamine receptor AmDOP2 in the honey bee

Determining the specific molecular pathways through which dopamine affects behavior has been complicated by the presence of multiple dopamine receptor subtypes that couple to different second messenger pathways. The observation of freely moving adult bees in an arena was used to investigate the role of dopamine signaling in regulating the behavior of the honey bee. Dopamine or the dopamine receptor antagonist flupenthixol was injected into the hemolymph of worker honey bees. Significant differences between treated and control bees were seen for all behaviors (walking, stopped, upside down, grooming, flying and fanning), and behavioral shifts were dependent on drug dosage and time after injection. To examine the role of dopamine signaling through a specific dopamine receptor in the brain, RNA interference was used to reduce expression levels of a D1-like receptor, AmDOP2. Injection of Amdop2 dsRNA into the mushroom bodies reduced the levels of Amdop2 mRNA and produced significant changes in the amount of time honey bees spent performing specific behaviors with reductions in time spent walking offset by increases in grooming or time spent stopped. Taken together these results establish that dopamine plays an important role in regulating motor behavior of the honey bee.     

D1 dopamine receptor mediates dopamine-induced cytotoxicity via the ERK signal cascade

Postsynaptic striatal neurodegeneration occurs through unknown mechanisms, but it is linked to high extracellular levels of synaptic dopamine. Dopamine-mediated cytotoxicity of striatal neurons occurs through two distinct pathways: autoxidation and the D1 dopamine receptor-linked signaling pathway. Here we investigated the mitogen-activated protein kinase (MAPK) signaling pathways activated upon the acute stimulation of D1 dopamine receptors. In SK-N-MC neuroblastoma cells, endogenously expressing D1 dopamine receptors, dopamine caused activation of phosphorylated (p-)ERK1/2 and of the stress-signaling kinases, p-JNK and p-p38 MAPK, in a time- and dose-dependent manner. Selective stimulation of D1 receptors with the agonist SKF R-38393 caused p-ERK1/2, but not p-JNK or p-p38 MAPK activation, in a manner sensitive to the receptor-selective antagonist SCH 23390, protein kinase A inhibition (KT5720), and MEK1/2 inhibition (U0126 or PD98059). Activation of ERK by D1 dopamine receptors resulted in oxidative stress and cytotoxicity. In cells transfected with a catalytically defective mutant of MEK1, the upstream ERK-specific kinase, both dopamine- and SKF R-38393-mediated cytotoxicity was markedly attenuated, confirming the participation of the ERK signaling pathway. Cell fractionation studies showed that only a small amount of p-ERK1/2 was translocated to the nucleus, with the majority retained in the cytoplasm. From coimmunoprecipitation studies, p-ERK was found to form stable heterotrimeric complexes with the D1 dopamine receptor and beta-arrestin2. In cells transfected with the dominant negative mutant of beta-arrestin2, the formation of such complexes was substantially inhibited. These data provide novel mechanistic insights into the role of ERK in the cytotoxicity mediated upon activation of the D1 dopamine receptor.     

Opposing effects of phorbol-12-myristate-13-acetate, an activator of protein kinase C, on the signaling of structurally related human dopamine D1 and D5 receptors

The ‘cross‐talk’ between different types of neurotransmitters through second messenger pathways represents a major regulatory mechanism in neuronal function. We investigated the effects of activation of protein kinase C (PKC) on cAMP‐dependent signaling by structurally related human D1‐like dopaminergic receptors. Human embryonic kidney 293 (HEK293) cells expressing D1 or D5 receptors were pretreated with phorbol‐12‐myristate‐13‐acetate (PMA), a potent activator of PKC, followed by analysis of dopamine‐mediated receptor activation using whole cell cAMP assays. Unpredictably, PKC activation had completely opposite effects on D1 and D5 receptor signaling. PMA dramatically augmented agonist‐evoked D1 receptor signaling, whereas constitutive and dopamine‐mediated D5 receptor activation were rapidly blunted. RT–PCR and immunoblotting analyses showed that phorbol ester‐regulated PKC isozymes (conventional: α, βI, βII, γ; novel: δ, ?, η, θ) and protein kinase D (PKCµ) are expressed in HEK293 cells. PMA appears to mediate these contrasting effects through the activation of Ca²⁺‐independent novel PKC isoforms as revealed by specific inhibitors, bisindolylmaleimide I, Gö6976, and Gö6983. The finding that cross‐talk between PKC and cAMP pathways can produce such opposite outcomes following the activation of structurally similar D1‐like receptor subtypes is novel and further strengthens the view that D1 and D5 receptors serve distinct functions in the mammalian nervous and endocrine systems.     

Dopamine D2 receptor isoforms expressed in AtT20 cells differentially couple to G proteins to acutely inhibit high voltage-activated calcium channels

The dopamine D2 receptor belongs to the serpentine superfamily of receptors, which have seven transmembrane segments and activate G proteins. D2 receptors are known to be linked, through Galpha(o)- and Galpha(i)-containing G proteins, to several signaling pathways in neuronal and secretory cells, including inhibition of adenylyl cyclase and high voltage-activated Ca2+ channels (HVA-CCs). The dopamine D2 receptor exists in two alternatively spliced isoforms, "long" and "short" (D2L, and D2S, respectively), which have identical ligand binding sites but differ by 29 amino acids in the third intracellular loop, the proposed site for G protein interaction. This has led to the speculation that the two isoforms may interact with different G proteins. We have transfected the AtT20 cell line with either D2L (KCL line) or D2S (KCS line) to facilitate experimentation on the individual isoforms. Both lines show dopamine agonist-dependent inhibition of Q-type HVA-CCs. We combined G protein antisense knock-down studies with multiwavelength fluorescence video microscopy to measure changes in HVA-CC inhibition to investigate the possibility of differential G protein coupling to this inhibition. The initial, rapid, K+ depolarization-induced increase in intracellular Ca2+ concentration is due to influx through HVA-CCs. Our studies reveal that both D2 isoforms couple to Galpha(o) to partially inhibit this influx. However, D2L also couples to Galpha(i)3, whereas D2S couples to Galpha(i)2. These data support the hypothesis of differential coupling of D2 receptor isoforms to G proteins.     

D1 dopamine receptor signaling is modulated by the R7 RGS protein EAT-16 and the R7 binding protein RSBP-1 in Caenoerhabditis elegans motor neurons

Dopamine signaling modulates voluntary movement and reward-driven behaviors by acting through G protein-coupled receptors in striatal neurons, and defects in dopamine signaling underlie Parkinson's disease and drug addiction. Despite the importance of understanding how dopamine modifies the activity of striatal neurons to control basal ganglia output, the molecular mechanisms that control dopamine signaling remain largely unclear. Dopamine signaling also controls locomotion behavior in Caenorhabditis elegans. To better understand how dopamine acts in the brain we performed a large-scale dsRNA interference screen in C. elegans for genes required for endogenous dopamine signaling and identified six genes (eat-16, rsbp-1, unc-43, flp-1, grk-1, and cat-1) required for dopamine-mediated behavior. We then used a combination of mutant analysis and cell-specific transgenic rescue experiments to investigate the functional interaction between the proteins encoded by two of these genes, eat-16 and rsbp-1, within single cell types and to examine their role in the modulation of dopamine receptor signaling. We found that EAT-16 and RSBP-1 act together to modulate dopamine signaling and that while they are coexpressed with both D1-like and D2-like dopamine receptors, they do not modulate D2 receptor signaling. Instead, EAT-16 and RSBP-1 act together to selectively inhibit D1 dopamine receptor signaling in cholinergic motor neurons to modulate locomotion behavior.     

Agonist-specific transactivation of phosphoinositide 3-kinase signaling pathway mediated by the dopamine D2 receptor

Bromocriptine, acting through the dopamine D2 receptor, provides robust protection against apoptosis induced by oxidative stress in PC12-D2R and immortalized nigral dopamine cells. We now report the characterization of the D2 receptor signaling pathways mediating the cytoprotection. Bromocriptine caused protein kinase B (Akt) activation in PC12-D2R cells and the inhibition of either phosphoinositide (PI) 3-kinase, epidermal growth factor receptor (EGFR), or c-Src eliminated the Akt activation and the cytoprotective effects of bromocriptine against oxidative stress. Co-immunoprecipitation studies showed that the D2 receptor forms a complex with the EGFR and c-Src that was augmented by bromocriptine, suggesting a cross-talk between these proteins in mediating the activation of Akt. EGFR repression by inhibitor or by RNA interference eliminated the activation of Akt by bromocriptine. D2 receptor stimulation by bromocriptine induced c-Src tyrosine 418 phosphorylation and EGFR phosphorylation specifically at tyrosine 845, a known substrate of Src kinase. Furthermore, Src tyrosine kinase inhibitor or dominant negative Src interfered with Akt translocation and phosphorylation. Thus, the predominant signaling cascade mediating cytoprotection by the D2 receptor involves c-Src/EGFR transactivation by D2 receptor, activating PI 3-kinase and Akt. We also found that the agonist pramipexole failed to stimulate activation of Akt in PC12-D2R cells, providing an explanation for our previous observations that, despite efficiently activating G-protein signaling, this agonist had little cytoprotective activity in this experimental system. These results support the hypothesis that specific dopamine agonists stabilize distinct conformations of the D2 receptor that differ in their coupling to G-proteins and to a cytoprotective c-Src/EGFR-mediated PI-3 kinase/Akt pathway.     

Dopamine D2 Receptor Relies upon PPM/PP2C Protein Phosphatases to Dephosphorylate Huntingtin Protein

Striatal dopamine D2 receptor (D2R) relies upon G protein- and β-arrestin-dependent signaling pathways to convey its action on motor control and behavior. Considering that D2R activation inhibits Akt in the striatum and that huntingtin physiological functions are affected by Akt phosphorylation, we sought to investigate whether D2R-mediated signaling could regulate huntingtin phosphorylation. We demonstrate that D2R activation decreases huntingtin phosphorylation on its Akt site. This dephosphorylation event depends upon the Gα_i-dependent engagement of specific members of the protein phosphatase metallo-dependent (PPM/PP2C) family and is independent of β-arrestin 2. These observations identify the PPM/PP2C family as a mediator of G protein-coupled receptor signaling and thereby suggest a novel mechanism of dopaminergic signaling.     

Calcium-related signaling pathways contributed to dopamine-induced cortical neuron apoptosis

Accumulating pathological evidence showing layer-specific neuronal reduction, dendrite deficits and brain volume loss have implicated an apoptotic process in schizophrenia, but the exact mechanism remains elusive. Dopamine hyperactivity at D2 receptor sites was considered as an important mechanism in schizophrenia pathogenesis. Recently, a newly identified D1 and D2 receptor heterooligomer activated by the specific agonist SKF83959 has been shown to stimulate phospholipase C-related intracellular calcium release in the brain. In this study, we intend to test the hypothesis that overstimulation of this calcium-related signaling pathway by high concentration of dopamine and SKF83959 is capable of inducing cortical neuronal apoptosis through calcium disturbance. Our experimental results demonstrated that 10-100μM dopamine and 10-50μM SKF83959 treatments for 72h were able to induce cortical neuronal apoptosis via the D1 and D2 receptor heterooligomer mediated calcium overload and mitochondria dysfunction-dependent pathways. Meanwhile, we found that although 24h dopamine and SKF83959 treatments have not produced major apoptosis, they induced significant neuronal dendrite retraction as well as reduction of neurotrophic molecules such as phosphorylated AKT, ERK and Bcl-2 through PLC-sensitive pathways. Taken together, prolonged stimulation of dopamine and SKF83959 in cortical neurons can reduce dendrite extension at early stage and induce neuronal apoptosis later on through PLC-calcium related pathways, which might provide important apoptotic mechanisms for schizophrenia pathogenesis.     

Novel Dopamine D2 Receptor Signaling through Proteins Interacting with the Third Cytoplasmic Loop

The diverse activities of dopamine D2-like receptors, including D2, D3, and D4 receptors, are mediated by proteins that interact with the third cytoplasmic loop and regulate receptor signaling, receptor trafficking, and apoptosis. Such interacting proteins include calmodulin, the N-methyl-d-aspartate receptor 2B subunit, calcium/calmodulin-dependent protein kinase II, prostate apoptosis response-4, and β-arrestins, which regulate receptor signaling and the pharmacological action through D2 receptor. The gene encoding the D2 receptor gives rise to two isoforms, termed the dopamine D2 receptor long isoform (D2L) and the dopamine D2 receptor short isoform; the latter lacks 29 amino acids of the D2L receptor within the third cytoplasmic loop. In this review, we first focus on novel functions of the hetero-oligomeric D1/D2 and D2/adenosine A_2A receptors. We next discuss novel signaling through proteins interacting with the D2 receptor third cytoplasmic loop and define the function of a novel binding protein, heart-type fatty acid binding protein, which interacts with the D2L third cytoplasmic loop.     

Regulation of NMDA receptors by the tyrosine kinase Fyn

The phosphorylation and trafficking of N-methyl-d-aspartate (NMDA) receptors are tightly regulated by the Src family tyrosine kinase Fyn, through dynamic interactions with various scaffolding proteins in the NMDA receptor complex. Fyn acts as a point of convergence for many signaling pathways that upregulate GluN2B-containing NMDA receptors. In the following review, we focus on Fyn signaling downstream of different G-protein-coupled receptors: the dopamine D1 receptor, and receptors cognate to the pituitary adenylate cyclase-activating polypeptide. The net result of activation of each of these signaling pathways is upregulation of GluN2B-containing NMDA receptors. The NMDA receptor is a major target of ethanol in the brain, and accumulating evidence suggests that Fyn mediates the effects of ethanol by regulating the phosphorylation of GluN2B NMDA receptor subunits. Furthermore, Fyn has been shown to regulate alcohol withdrawal and acute tolerance to ethanol through a GluN2B-dependent mechanism. In addition to its effects on NMDA receptor function, Fyn also modifies the threshold for synaptic plasticity at CA1 synapses, an effect that probably contributes to the effects of Fyn on spatial and contextual fear learning.     

Interaction with dopamine D2 receptor enhances expression of transient receptor potential channel 1 at the cell surface

Receptor signaling is mediated by direct protein interaction with various types of cytoskeletal, adapter, effector, and additional receptor molecules. In brain tissue and in cultured neurons, activation of dopamine D2 receptors (D2Rs) has been found to impact cellular calcium signaling. Using a yeast two-hybrid approach, we have uncovered a direct physical interaction between the D2R and the transient receptor potential channel (TRPC) subtypes 1, 4 and 5. The TRPC/D2R interaction was further validated by GST-pulldown assays and coimmunoprecipitation from mammalian brain. Ultrastructural analysis of TRPC1 and D2R expression indicates colocalization of the two proteins within the cell body and dendrites of cortical neurons. In cultured cells, expression of D2Rs was found to increase expression of TRPC1 at the cell surface by 50%. These findings shed new light on the constituents of the D2R signalplex, and support the involvement of D2Rs in cellular calcium signaling pathways via a novel link to TRPC channels.     

Interaction with dopamine D2 receptor enhances expression of transient receptor potential channel 1 at the cell surface

Receptor signaling is mediated by direct protein interaction with various types of cytoskeletal, adapter, effector, and additional receptor molecules. In brain tissue and in cultured neurons, activation of dopamine D2 receptors (D2Rs) has been found to impact cellular calcium signaling. Using a yeast two-hybrid approach, we have uncovered a direct physical interaction between the D2R and the transient receptor potential channel (TRPC) subtypes 1, 4 and 5. The TRPC/D2R interaction was further validated by GST-pulldown assays and coimmunoprecipitation from mammalian brain. Ultrastructural analysis of TRPC1 and D2R expression indicates colocalization of the two proteins within the cell body and dendrites of cortical neurons. In cultured cells, expression of D2Rs was found to increase expression of TRPC1 at the cell surface by 50%. These findings shed new light on the constituents of the D2R signalplex, and support the involvement of D2Rs in cellular calcium signaling pathways via a novel link to TRPC channels.     

Effects of the Dopamine D2 Allosteric Modulator,PAOPA, on the Expression of GRK2, Arrestin-3, ERK1/2, and on Receptor Internalization

The activity of G protein-coupled receptors (GPCRs) is intricately regulated by a range of intracellular proteins, including G protein-coupled kinases (GRKs) and arrestins. Understanding the effects of ligands on these signaling pathways could provide insights into disease pathophysiologies and treatment. The dopamine D2 receptor is a GPCR strongly implicated in the pathophysiology of a range of neurological and neuropsychiatric disorders, particularly schizophrenia. Previous studies from our lab have shown the preclinical efficacy of a novel allosteric drug, 3(R)- [(2(S)-pyrrolidinylcarbonyl)amino]-2-oxo-1-pyrrolidineacetamide (PAOPA), in attenuating schizophrenia-like behavioural abnormalities in rodent models of the disease. As an allosteric modulator, PAOPA binds to a site on the D2 receptor, which is distinct from the endogenous ligand-binding site, in order to modulate the binding of the D2 receptor ligand, dopamine. The exact signaling pathways affected by this allosteric modulator are currently unknown. The objectives of this study were to decipher the in vivo effects, in rats, of chronic PAOPA administration on D2 receptor regulatory and downstream molecules, including GRK2, arrestin-3 and extracellular receptor kinase (ERK) 1/2. Additionally, an in vitro cellular model was also used to study PAOPA’s effects on D2 receptor internalization. Results from western immunoblots showed that chronic PAOPA treatment increased the striatal expression of GRK2 by 41%, arrestin-3 by 34%, phospho-ERK1 by 51% and phospho-ERK2 by 36%. Results also showed that the addition of PAOPA to agonist treatment in cells increased D2 receptor internalization by 33%. This study provides the foundational evidence of putative signaling pathways, and changes in receptor localization, affected by treatment with PAOPA. It improves our understanding on the diverse mechanisms of action of allosteric modulators, while advancing PAOPA’s development into a novel drug for the improved treatment of schizophrenia.     

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.     

Dopamine receptor interacting proteins (DRIPs) of dopamine D1-like receptors in the central nervous system

Dopamine is a major neurotransmitter in the mammalian central nervous system (CNS) that regulates neuroendocrine functions, locomotor activity, cognition and emotion. The dopamine system has been extensively studied because dysfunction of this system is linked to various pathological conditions including Parkinson's disease, schizophrenia, Tourette's syndrome, and drug addiction. Accordingly, intense efforts to delineate the full complement of signaling pathways mediated by individual receptor subtypes have been pursued. Dopamine D1-like receptors are of particular interest because they are the most abundant dopamine receptors in CNS. Recent work suggests that dopamine signaling could be regulated via dopamine receptor interacting proteins (DRIPs). Unraveling these DRIPs involved in the dopamine system may provide a better understanding of the mechanisms underlying CNS disorders related to dopamine system dysfunction and may help identify novel therapeutic targets.     

Evidence that calmodulin binding to the dopamine D2 receptor enhances receptor signaling

The Ca2+ sensor calmodulin (CaM) regulates numerous proteins involved in G protein-coupled receptor (GPCR) signaling. CaM binds directly to some GPCRs, including the dopamine D2 receptor. We confirmed that the third intracellular loop of the D2 receptor is a direct contact point for CaM binding using coimmunoprecipitation and a polyHis pull-down assay, and we determined that the D2-like receptor agonist 7-OH-DPAT increased the colocalization of the D2 receptor and endogenous CaM in both 293 cells and in primary neostriatal cultures. The N-terminal three or four residues of D2-IC3 were required for the binding of CaM; mutation of three of these residues in the full-length receptor (I210C/K211C/I212C) decreased the coprecipitation of the D2 receptor and CaM and also significantly decreased D2 receptor signaling, without altering the coupling of the receptor to G proteins. Taken together, these findings suggest that binding of CaM to the dopamine D2 receptor enhances D2 receptor signaling.     

The Caenorhabditis elegans D2-like dopamine receptor DOP-2 physically interacts with GPA-14, a Gαi subunit

ABSTRACT: Dopaminergic inputs are sensed on the cell surface by the seven-transmembrane dopamine receptors that belong to a superfamily of G-protein-coupled receptors (GPCRs). Dopamine receptors are classified as D1-like or D2-like receptors based on their homology and pharmacological profiles. In addition to well established G-protein coupled mechanism of dopamine receptors in mammalian system they can also interact with other signaling pathways. In C. elegans four dopamine receptors (dop-1, dop-2, dop-3 and dop-4) have been reported and they have been implicated in a wide array of behavioral and physiological processes. We performed this study to assign the signaling pathway for DOP-2, a D2-like dopamine receptor using a split-ubiquitin based yeast two-hybrid screening of a C. elegans cDNA library with a novel dop-2 variant (DOP-2XL) as bait. Our yeast two-hybrid screening resulted in identification of gpa-14, as one of the positively interacting partners. gpa-14 is a Gα coding sequence and shows expression overlap with dop-2 in C. elegans ADE deirid neurons. In-vitro pull down assays demonstrated physical coupling between dopamine receptor DOP-2XL and GPA-14. Further, we sought to determine the DOP-2 region necessary for GPA-14 coupling. We generated truncated DOP-2XL constructs and performed pair-wise yeast two-hybrid assay with GPA-14 followed by in-vitro interaction studies and here we report that the third intracellular loop is the key domain responsible for DOP-2 and GPA-14 coupling. Our results show that the extra-long C. elegans D2-like receptor is coupled to gpa-14 that has no mammalian homolog but shows close similarity to inhibitory G-proteins. Supplementing earlier investigations, our results demonstrate the importance of an invertebrate D2-like receptor's third intracellular loop in its G-protein interaction.     

Coexpressed D1- and D2-like dopamine receptors antagonistically modulate acetylcholine release in Caenorhabditis elegans

Dopamine acts through two classes of G protein-coupled receptor (D1-like and D2-like) to modulate neuron activity in the brain. While subtypes of D1- and D2-like receptors are coexpressed in many neurons of the mammalian brain, it is unclear how signaling by these coexpressed receptors interacts to modulate the activity of the neuron in which they are expressed. D1- and D2-like dopamine receptors are also coexpressed in the cholinergic ventral-cord motor neurons of Caenorhabditis elegans. To begin to understand how coexpressed dopamine receptors interact to modulate neuron activity, we performed a genetic screen in C. elegans and isolated mutants defective in dopamine response. These mutants were also defective in behaviors mediated by endogenous dopamine signaling, including basal slowing and swimming-induced paralysis. We used transgene rescue experiments to show that defects in these dopamine-specific behaviors were caused by abnormal signaling in the cholinergic motor neurons. To investigate the interaction between the D1- and D2-like receptors specifically in these cholinergic motor neurons, we measured the sensitivity of dopamine-signaling mutants and transgenic animals to the acetylcholinesterase inhibitor aldicarb. We found that D2 signaling inhibited acetylcholine release from the cholinergic motor neurons while D1 signaling stimulated release from these same cells. Thus, coexpressed D1- and D2-like dopamine receptors act antagonistically in vivo to modulate acetylcholine release from the cholinergic motor neurons of C. elegans.