Cortical dopamine D5 receptors regulate neuronal circuit oscillatory activity and memory in rats.

INTRODUCTION: The dopamine D5 receptor (D5R) shows high expression in cortical regions, yet the role of the receptor in learning and memory remains poorly understood. This study evaluated the impact of prefrontal cortical (PFC) D5R knockdown in rats on learning and memory and assessed the role of the D5R in the regulation of neuronal oscillatory activity and glycogen synthase kinase-3 (GSK-3ß), processes integral to cognitive function. MATERIALS AND METHODS: Using an adeno-associated viral (AAV) vector, male rats were infused with shRNA to the D5R bilaterally into the PFC. Local field potential recordings were taken from freely moving animals and spectral power and coherence were evaluated in, and between, the PFC, orbitofrontal cortex (OFC), hippocampus (HIP), and thalamus. Animals were then assessed in object recognition, object location, and object in place tasks. The activity of PFC GSK-3ß, a downstream effector of the D5R, was evaluated. RESULTS: AAV-mediated knockdown of the D5R in the PFC induced learning and memory deficits. These changes were accompanied by elevations in PFC, OFC, and HIP theta spectral power and PFC-OFC coherence, reduced PFC-thalamus gamma coherence, and increased PFC GSK-3ß activity. CONCLUSION: This work demonstrates a role for PFC D5Rs in the regulation of neuronal oscillatory activity and learning and memory. As elevated GSK-3ß activity has been implicated in numerous disorders of cognitive dysfunction, this work also highlights the potential of the D5R as a novel therapeutic target via suppression of GSK-3ß.

Dopamine promotes the progression of AML via activating NLRP3 inflammasome and IL-1ß.

Mental stress has been shown to activate sympathetic adrenergic system to produce dopamine and finally promote the progression of cancer. Dopamine can also regulate the immune system through secreting kinds of cytokines. However, what role does dopamine play in acute myeloid leukemia (AML) remains unclear. Here, we investigated the effects and mechanisms of dopamine in NLRP3 inflammasome activation and cellular viability of acute myeloid leukemia U937 cells. Our results showed that dopamine enhanced the viability of U937 cells and activated the NLRP3 inflammasome in U937 cells. To further explore the mechanism of dopamine on U937 cells, we examined the expression level of dopamine receptors (DRs). We found that the mRNA expression level of DR5 in U937 cells was significantly higher than other dopamine receptors. Furthermore, we treated U937 cells with DR1/2/3/5 antagonist before dopamine, and it manifestly reversed the NLRP3 inflammasome activation and the viability-enhancing effect in U937 cells induced by dopamine. Anti-IL-1ß antibody also could partly reversed the viability-enhancing effect by dopamine. We concluded that dopamine could enhance the viability of U937 cells through DR1/5 receptor pathway and activate NLRP3 inflammasome.

Characterization of the Functional Cross-Talk between Surface GABAA and Dopamine D5 Receptors.

The γ-aminobutyric acid type A receptor (GABAAR) plays a major role in fast inhibitory synaptic transmission and is highly regulated by the neuromodulator dopamine. In this aspect, most of the attention has been focused on the classical intracellular signaling cascades following dopamine G-protein-coupled receptor activation. Interestingly, the GABAAR and dopamine D5 receptor (D5R) have been shown to physically interact in the hippocampus, but whether a functional cross-talk occurs is still debated. In the present study, we use a combination of imaging and single nanoparticle tracking in live hippocampal neurons to provide evidence that GABAARs and D5Rs form dynamic surface clusters. Disrupting the GABAAR-D5R interaction with a competing peptide leads to an increase in the diffusion coefficient and the explored area of both receptors, and a drop in immobile synaptic GABAARs. By means of patch-clamp recordings, we show that this fast lateral redistribution of surface GABAARs correlates with a robust depression in the evoked GABAergic currents. Strikingly, it also shifts in time the expression of long-term potentiation at glutamatergic synapses. Together, our data both set the plasma membrane as the primary stage of a functional interplay between GABAAR and D5R, and uncover a non-canonical role in regulating synaptic transmission.

Metabolic and inflammatory reprogramming of macrophages by ONC201 translates in a pro-inflammatory environment even in presence of glioblastoma cells.

Tumor-associated macrophages facilitate tumor progression and resistance to therapy. Their capacity for metabolic and inflammatory reprogramming represents an attractive therapeutic target. ONC201/TIC10 is an anticancer molecule that antagonizes the dopamine receptor D2 and affects mitochondria integrity in tumor cells. We examined whether ONC201 induces a metabolic and pro-inflammatory switch in primary human monocyte-derived macrophages that reactivates their antitumor activities, thus enhancing the onco-toxicity of ONC201. Contrary to glioblastoma cells, macrophages exhibited a low ratio of dopamine receptors D2/D5 gene expression and were resistant to ONC201 cytotoxicity. Macrophages responded to ONC201 with a severe loss of mitochondria integrity, a switch to glycolytic ATP production, alterations in glutamate transport, and a shift towards a pro-inflammatory profile. Treatment of macrophages-glioblastoma cells co-cultures with ONC201 induced similar alterations in glutamatergic and inflammatory gene expression profiles of macrophages. It induced as well metabolic changes and a pro-inflammatory switch of the co-culture milieu. However, these changes did not translate into increased onco-toxicity. This study provides the first evidence that ONC201 affects macrophage immunometabolism and leads to a pro-inflammatory tumor environment. This speaks in favor of implementing ONC201 in combinatorial therapies and warrants further investigation of the mechanisms of action of ONC201 in macrophages and other immune cells.

Hypermethylation of DRD5 Promoter Is a Biomarker Across 12 Cancer Types.

Aberrant DNA methylation is thought to be an early event in cancer development. Thus, identification of DNA methylation-based markers may provide valuable evidence in clinical decision-making. In this study, a DNA methylation dataset from 514 normal-tumor pairs is used to explore possible shared differentially methylated regions (DMRs) across 12 cancer types. Results showed that DMR in Dopamine receptor D5 (DRD5) promoter may be serviced as a good candidate biomarker across different cancer types. We further validated the extended DMR (292bp) in DRD5 promoter using SEQUENOM MassARRAY platform. Detection of DRD5 promoter dynamic methylation will allow rapid risk assessment at diagnosis, for suspicious tumor with the tissue biopsies.

Dopamine Receptor D5 is a Modulator of Tumor Response to Dopamine Receptor D2 Antagonism.

PURPOSE: Dopamine receptor D2 (DRD2) is a G protein-coupled receptor antagonized by ONC201, an anticancer small molecule in clinical trials for high-grade gliomas and other malignancies. DRD5 is a dopamine receptor family member that opposes DRD2 signaling. We investigated the expression of these dopamine receptors in cancer and their influence on tumor cell sensitivity to ONC201. EXPERIMENTAL DESIGN: The Cancer Genome Atlas was used to determine DRD2/DRD5 expression broadly across human cancers. Cell viability assays were performed with ONC201 in >1,000 Genomic of Drug Sensitivity in Cancer and NCI60 cell lines. IHC staining of DRD2/DRD5 was performed on tissue microarrays and archival tumor tissues of glioblastoma patients treated with ONC201. Whole exome sequencing was performed in RKO cells with and without acquired ONC201 resistance. Wild-type and mutant DRD5 constructs were generated for overexpression studies. RESULTS: DRD2 overexpression broadly occurs across tumor types and is associated with a poor prognosis. Whole exome sequencing of cancer cells with acquired resistance to ONC201 revealed a de novo Q366R mutation in the DRD5 gene. Expression of Q366R DRD5 was sufficient to induce tumor cell apoptosis, consistent with a gain-of-function. DRD5 overexpression in glioblastoma cells enhanced DRD2/DRD5 heterodimers and DRD5 expression was inversely correlated with innate tumor cell sensitivity to ONC201. Investigation of archival tumor samples from patients with recurrent glioblastoma treated with ONC201 revealed that low DRD5 expression was associated with relatively superior clinical outcomes. CONCLUSIONS: These results implicate DRD5 as a negative regulator of DRD2 signaling and tumor sensitivity to ONC201 DRD2 antagonism.

A Study of Short- and Long-term mRNA Levels of the Retn, Iapp, and Drd5 Genes in Obese Mice Induced with High-fat Diet.

BACKGROUND/AIM: Adipocyte gene expression is altered in obese individuals through multiple metabolic and biochemical pathways. In this study, we aimed to examine the expression of resistin (Retn), amylin (Iapp), and dopamine receptor domain 5 (Drd5) genes previously suggested to contribute to the pathogenesis of obesity, albeit controversially. We also aimed to determine the effects on short and long-term mRNA levels of these genes in obese mice, induced with high-fat diet (HFD). MATERIALS AND METHODS: Two obesity models were created in our study: group T1 (20 mice) was fed with HFD (60% fat) for 3 months, and group T2 (20 mice) was fed with HFD (60% fat) for 6 months. The control group T0 (20 mice) was fed with a diet of 10% kcal fat supplement for 6 months. At the end of the experiment, their adipose tissues were dissected surgically. Tissue samples of each group were pooled for RNA isolation, cDNA synthesis was carried out and the mRNA levels were examined by quantitative real-time polymerase chain reaction. Serum resistin levels were measured using multiplex bead (luminex) technology for validation. RESULTS: In T2 mice, the mRNA expression of Retn showed a moderate up-regulation (fold change=8.32; p=0.0019) in the adipose tissues. Iapp expression was also significantly up-regulated (fold change=9.78; p=0.012). Moreover, a 6.36-fold up-regulation for Drd5 was observed in the adipose tissues of T2 mice (p<0.001). At the same time, serum levels of resistin were found to be high in T1 and T2 mice compared to the control group (p<0.001 and p=0.024, respectively). CONCLUSION: Our study demonstrated that the mRNA levels of the genetic markers considered to play a role in adipogenesis were different in short- and long-term obesity models formed in C57BL/6J mice using HFD.

Role of Dopamine Receptors in the Anticancer Activity of ONC201.

ONC201/TIC10 is a first-in-class small molecule inducer of TRAIL that causes early activation of the integrated stress response. Its promising safety profile and broad-spectrum efficacy in vitro have been confirmed in Phase I/II trials in several advanced malignancies. Binding and reporter assays have shown that ONC201 is a selective antagonist of the dopamine D2-like receptors, specifically, DRD2 and DRD3. We hypothesized that ONC201's interaction with DRD2 plays a role in ONC201's anticancer effects. Using cBioportal and quantitative reverse-transcription polymerase chain reaction analyses, we confirmed that DRD2 is expressed in different cancer cell types in a cell type-specific manner. On the other hand, DRD3 was generally not detectable. Overexpressing DRD2 in cells with low DRD2 levels increased ONC201-induced PARP cleavage, which was preceded and correlated with an increase in ONC201-induced CHOP mRNA expression. On the other hand, knocking out DRD2 using CRISPR/Cas9 in three cancer cell lines was not sufficient to abrogate ONC201's anticancer effects. Although ONC201's anticancer activity was not dependent on DRD2 expression in the cancer cell types tested, we assessed the cytotoxic potential of DRD2 blockade. Transient DRD2 knockdown in HCT116 cells activated the integrated stress response and reduced cell number. Pharmacological antagonism of DRD2 significantly reduced cell viability. Thus, we demonstrate in this study that disrupting dopamine receptor expression and activity can have cytotoxic effects that may at least be in part due to the activation of the integrated stress response. On the other hand, ONC201's anticancer activity goes beyond its ability to antagonize DRD2, potentially due to ONC201's ability to activate other pathways that are independent of DRD2. Nevertheless, blocking the dopamine D1-like receptor DRD5 via siRNA or the use of a pharmacological antagonist promoted ONC201-induced anticancer activity.

Activation of DRD5 (dopamine receptor D5) inhibits tumor growth by autophagic cell death.

Dopamine agonists such as bromocriptine and cabergoline have been successfully used in the treatment of pituitary prolactinomas and other neuroendocrine tumors. However, their therapeutic mechanisms are not fully understood. In this study we demonstrated that DRD5 (dopamine receptor D5) agonists were potent inhibitors of pituitary tumor growth. We further found that DRD5 activation increased production of reactive oxygen species (ROS), inhibited the MTOR pathway, induced macroautophagy/autophagy, and led to autophagic cell death (ACD) in vitro and in vivo. In addition, DRD5 protein was highly expressed in the majority of human pituitary adenomas, and treatment of different human pituitary tumor cell cultures with the DRD5 agonist SKF83959 resulted in growth suppression, and the efficacy was correlated with the expression levels of DRD5 in the tumors. Furthermore, we found that DRD5 was expressed in other human cancer cells such as glioblastomas, colon cancer, and gastric cancer. DRD5 activation in these cell lines suppressed their growth, inhibited MTOR activity, and induced autophagy. Finally, in vivo SKF83959 also inhibited human gastric cancer cell growth in nude mice. Our studies revealed novel mechanisms for the tumor suppressive effects of DRD5 agonists, and suggested a potential use of DRD5 agonists as a novel therapeutic approach in the treatment of different human tumors and cancers.

Induction of dopamine D1 and D5 receptors in R28 cells by light exposures.

Dopamine is known to play an important role in the pathophysiological process of myopia development relevant to the ambient lighting, but it is still poorly understood about how lighting regulates dopamine and its interaction with dopamine receptors to mediate the pathogenic signal transduction leading to alterations of ocular globe and the pathogenesis of myopia. Many studies have highlighted changes of ocular dopamine amount in response to different lighting conditions, but little attention has been paid to the dopamine receptors during these processes. Here we examined the effects of different lighting exposures on the expression of dopamine receptors in rat R28 retinal precursor cells. R28 cells normally grown in dark were exposed to a low (10 lux) or high (500 lux) intensity of a source of LED white light (5000 K-6000 K) for 12 h and total RNA was isolated either immediately or after certain time continuous growing in dark. Both conventional and real-time RT-PCR were performed to determine the expression of all five different dopamine receptors in cells after treatments. While the transcripts of dopamine D2, D3, and D4 receptors were not detected in the total RNA preparations of all the cells, those of D1 and D5 receptors (DRD1 and DRD5) were induced by lighting in contrast to the dark control. Elevated levels of DRD1 and DRD5 mRNA returned back close to the original levels once the cells were maintained in dark after light exposures. Immunofluorescence microscopy using a specific antibody confirmed an increase in the immunoreactivity of DRD1 in the cells exposed to 500 lux lighting versus dark control. Notably, treatments of R28 cells with nanomolar dosages of dopamine (0-500 nM) directly downregulated expression of both DRD1 and DRD5, whereas haloperidol (0-50 nM), a DRD2 antagonist, significantly induced expression of DRD1. These results suggest that dopamine receptors in the retinal cells might actively respond to the environmental lighting to act as an important player in the activation of the dopaminergic system in the ocular structures relevant to the lighting-induced pathogenic development of myopia.

Dopamine receptors D3 and D5 regulate CD4(+)T-cell activation and differentiation by modulating ERK activation and cAMP production.

Dopamine receptors have been described in T-cells, however their signalling pathways coupled remain unknown. Since cAMP and ERKs play key roles regulating T-cell physiology, we aim to determine whether cAMP and ERK1/2-phosphorylation are modulated by dopamine receptor 3 (D3R) and D5R, and how this modulation affects CD4(+) T-cell activation and differentiation. Our pharmacologic and genetic evidence shows that D3R-stimulation reduced cAMP levels and ERK2-phosphorylation, consequently increasing CD4(+) T-cell activation and Th1-differentiation, respectively. Moreover, D5R expression reinforced TCR-triggered ERK1/2-phosphorylation and T-cell activation. In conclusion, these findings demonstrate how D3R and D5R modulate key signalling pathways affecting CD4(+) T-cell activation and Th1-differentiation.

Identification of G protein-biased agonists that fail to recruit ß-arrestin or promote internalization of the D1 dopamine receptor.

The D1 dopamine receptor (D1R) has been implicated in numerous neuropsychiatric disorders, and D1R-selective ligands have potential as therapeutic agents. Previous studies have identified substituted benzazepines as D1R-selective agonists, but the in vivo effects of these compounds have not correlated well with their in vitro pharmacological activities. A series of substituted benzazepines, and structurally dissimilar D1R-selective agonists, were tested for their functional effects on D1R-mediated cAMP accumulation, D1R-promoted ß-arrestin recruitment, and D1R internalization using live cell functional assays. All compounds tested elicited an increase in the level of cAMP accumulation, albeit with a range of efficacies. However, when the compounds were evaluated for ß-arrestin recruitment, a subset of substituted benzazepines, SKF83959, SKF38393, SKF82957, SKF77434, and SKF75670, failed to activate this pathway, whereas the others showed similar activation efficacies as seen with cAMP accumulation. When tested as antagonists, the five biased compounds all inhibited dopamine-stimulated ß-arrestin recruitment. Further, D1R internalization assays revealed a corroborating pattern of activity in that the G protein-biased compounds failed to promote D1R internalization. Interestingly, the biased signaling was unique for the D1R, as the same compounds were agonists of the related D5 dopamine receptor (D5R), but revealed no signaling bias. We have identified a group of substituted benzazepine ligands that are agonists at D1R-mediated G protein signaling, but antagonists of D1R recruitment of ß-arrestin, and also devoid of agonist-induced receptor endocytosis. These data may be useful for interpreting the contrasting effects of these compounds in vitro versus in vivo, and also for the understanding of pathway-selective signaling of the D1R.

D1-like dopamine receptors downregulate Na+-K+-ATPase activity and increase cAMP production in the posterior gills of the blue crab Callinectes sapidus.

Dopamine-mediated regulation of Na(+)-K(+)-ATPase activity in the posterior gills of some crustaceans has been reported to be involved in osmoregulation. The dopamine receptors of invertebrates are classified into three groups based on their structure and pharmacology: D1- and D2-like receptors and a distinct invertebrate receptor subtype (INDR). We tested the hypothesis that a D1-like receptor is expressed in the blue crab Callinectes sapidus and regulates Na(+)-K(+)-ATPase activity. RT-PCR, using degenerate primers, showed the presence of D1ßR mRNA in the posterior gill. The blue crab posterior gills showed positive immunostaining for a dopamine D5 receptor (D5R or D1ßR) antibody in the basolateral membrane and cytoplasm. Confocal microscopy showed colocalization of Na(+)-K(+)-ATPase and D1ßR in the basolateral membrane. To determine the effect of D1-like receptor stimulation on Na(+)-K(+)-ATPase activity, intact crabs acclimated to low salinity for 6 days were given an intracardiac infusion of the D1-like receptor agonist fenoldopam, with or without the D1-like receptor antagonist SCH23390. Fenoldopam increased cAMP production twofold and decreased Na(+)-K(+)-ATPase activity by 50% in the posterior gills. This effect was blocked by coinfusion with SCH23390, which had no effect on Na(+)-K(+)-ATPase activity by itself. Fenoldopam minimally decreased D1ßR protein expression (10%) but did not affect Na(+)-K(+)-ATPase α-subunit protein expression. This study shows the presence of functional D1ßR in the posterior gills of euryhaline crabs chronically exposed to low salinity and highlights the evolutionarily conserved function of the dopamine receptors on sodium homeostasis.

Differential roles of the dopamine 1-class receptors, D1R and D5R, in hippocampal dependent memory.

Activation of the hippocampal dopamine 1-class receptors (D1R and D5R) are implicated in contextual fear conditioning (CFC). However, the specific role of the D1R versus D5R in hippocampal dependent CFC has not been investigated. Generation of D1R- and D5R-specific in situ hybridization probes showed that D1R and D5R mRNA expression was greatest in the dentate gyrus (DG) of the hippocampus. To identify the role of each receptor in CFC we generated spatially restricted KO mice that lack either the D1R or D5R in DG granule cells. DG D1R KOs displayed significant fear memory deficits, whereas DG D5R KOs did not. Furthermore, D1R KOs but not D5R KOs, exhibited generalized fear between two similar but different contexts. In the familiar home cage context, c-Fos expression was relatively low in the DG of control mice, and it increased upon exposure to a novel context. This level of c-Fos expression in the DG did not further increase when a footshock was delivered in the novel context. In DG D1R KOs, DG c-Fos levels in the home cage was higher than that of the control mice, but it did not further increase upon exposure to a novel context and remained at the same level upon a shock delivery. In contrast, the levels of DG c-Fos expression was unaffected by the deletion of DG D5R neither in the home cage nor upon a shock delivery. These results suggest that DG D1Rs, but not D5Rs, contribute to the formation of distinct contextual representations of novel environments.

Psychosis-proneness correlates with expression levels of dopaminergic genes.

Psychosis-proneness or schizotypy is a personality organisation mirroring individual risk for schizophrenia-development. Believed to be a fully dimensional construct sharing considerable geno- and phenotypal variance with clinical schizophrenia, it has become an increasingly promising tool for basic psychosis-research. Although many studies show genetic commonalities between schizotypy and schizophrenia, changes in regulation of gene expression have never been examined in schizotypy before. We therefore extracted RNA from the blood, a valid surrogate for brain tissue, of a large sample of 67 healthy male volunteers and correlated the activities of all genes relevant for dopaminergic neurotransmission with the positive schizotypy-scale of the O-LIFE. We found significant negative correlations regarding the expression of the genes COMT, MAOB, DRD4, DRD5 and FOS, indicating that increased schizotypy coincides with higher levels of dopaminergic dysregulation on the mRNA-level. Considering the advantages of this method, we suggest that it be applied more often in fundamental psychosis-research.

Dopamine receptor gene (DRD1-DRD5) expression changes as stress factors associated with breast cancer.

Breast cancer is the most common cancer among females worldwide and a most prevalent malignancy in Iranian women. Chronic stress may make an important contribution to cancer, especially in the breast. Numerous studies showed roles of neurotransmitters in the occurrence and progression of cancers which are mediated by their various types of receptors. This study was conducted to evaluate alterations in the expression profile of dopamine receptor genes in peripheral blood mononuclear cells (PBMC) as stress factors in breast cancer patients and the human breast cancer cell line (MCF-7). Peripheral blood samples were obtained from 30 patients and 30 healthy individuals. Total mRNA was extracted from PBMC and MCF-7 cells and RT-PCR was performed to confirm the presence of five dopamine receptors (DRD1-DRD5). Expression changes of dopamine receptor genes were evaluated by real time PCR. We observed that DRD2-DRD4 in PBMCs of breast cancer patients were increased compared to healthy individuals. In addition, all dopamine receptor subtypes but DRD1 were expressed in MCF-7 cells. Therefore, alterations of these receptors as stress factorsshould be assessed for selecting appropriate drugs such as D2-like agonists for treatment of breast cancer after performing complimentary tests. Determining the expression profile of dopamine receptor genes thus seems promising.

Genetic variation in dopamine-related gene expression influences motor skill learning in mice.

Several neurodevelopmental disorders with a strong genetic basis, including attention-deficit/hyperactivity disorder, autism spectrum disorders and developmental coordination disorder, involve deficits in fine motor skills. This phenotype may depend on heritable variation in components of the dopamine (DA) system, which is known to play a critical role in motor skill learning. In this study, we took advantage of two inbred strains of mice (BALB/c and C57BL/6) that differ markedly in the number of midbrain DA neurons in order to investigate the influence of such naturally occurring genetic variation on the acquisition and performance of fine motor skills. Gene expression analysis of midbrain, frontal cortex and striatum showed significant differences in the expression of presynaptic and postsynaptic dopaminergic (DAergic) markers (e.g. tyrosine hydroxylase, DA transporter, DA D4 receptor, DA D5 receptor and DARPP-32) between these two strains. BALB/c mice had lower learning rate and performance scores in a complex skilled reaching task when compared with C57BL/6 mice. A negative correlation was found between the motor learning rate and level of DARPP-32 mRNA expression in the frontal cortex contralateral to the trained forelimb. The rate of motor learning was also negatively correlated with the levels of DARPP-32 and DA D1 receptor mRNAs in the striatum. Our results suggest that genetically driven variation in frontostriatal DAergic neurotransmission is a major contributor to individual differences in motor skill learning. Moreover, these findings implicate the D1R/cAMP/DARPP-32 signaling pathway in those neurodevelopmental disorders that are associated with fine motor skill deficits.

Dopamine receptors modulate cytotoxicity of natural killer cells via cAMP-PKA-CREB signaling pathway.

Dopamine (DA), a neurotransmitter in the nervous system, has been shown to modulate immune function. We have previously reported that five subtypes of DA receptors, including D1R, D2R, D3R, D4R and D5R, are expressed in T lymphocytes and they are involved in regulation of T cells. However, roles of these DA receptor subtypes and their coupled signal-transduction pathway in modulation of natural killer (NK) cells still remain to be clarified. The spleen of mice was harvested and NK cells were isolated and purified by negative selection using magnetic activated cell sorting. After NK cells were incubated with various drugs for 4 h, flow cytometry measured cytotoxicity of NK cells against YAC-1 lymphoma cells. NK cells expressed the five subtypes of DA receptors at mRNA and protein levels. Activation of D1-like receptors (including D1R and D5R) with agonist SKF38393 enhanced NK cell cytotoxicity, but activation of D2-like receptors (including D2R, D3R and D4R) with agonist quinpirole attenuated NK cells. Simultaneously, SKF38393 elevated D1R and D5R expression, cAMP content, and phosphorylated cAMP-response element-binding (CREB) level in NK cells, while quinpirole reduced D3R and D4R expression, cAMP content, and phosphorylated CREB level in NK cells. These effects of SKF38393 were blocked by SCH23390, an antagonist of D1-like receptors, and quinpirole effects were abolished by haloperidol, an antagonist of D2-like receptors. In support these results, H89, an inhibitor of phosphokinase A (PKA), prevented the SKF38393-dependent enhancement of NK cells and forskolin, an activator of adenylyl cyclase (AC), counteracted the quinpirole-dependent suppression of NK cells. These findings show that DA receptor subtypes are involved in modulation of NK cells and suggest that D1-like receptors facilitate NK cells by stimulating D1R/D5R-cAMP-PKA-CREB signaling pathway and D2-like receptors suppress NK cells by inhibiting D3R/D4R-cAMP-PKA-CREB signaling pathway. The results may provide more targets of therapeutic strategy for neuroimmune diseases.

Functional analysis of human D1 and D5 dopaminergic G protein-coupled receptors: lessons from mutagenesis of a conserved serine residue in the cytosolic end of transmembrane region 6.

In mammals, dopamine G protein-coupled receptors (GPCR) are segregated into two categories: D1-like (D1R and D5R) and D2-like (D2R(short), D2R(long), D3R, and D4R) subtypes. D1R and D5R are primarily coupled to stimulatory heterotrimeric GTP-binding proteins (Gs/olf) leading to activation of adenylyl cyclase and production of intracellular cAMP. D1R and D5R share high level of amino acid identity in transmembrane (TM) regions. Yet these two GPCR subtypes display distinct ligand binding and G protein coupling properties. In fact, our studies suggest that functional properties reported for constitutively active mutants of GPCRs (e.g., increased basal activity, higher agonist affinity and intrinsic activity) are also observed in cells expressing wild type D5R when compared with wild type D1R. Herein, we describe an experimental method based on mutagenesis and transfection of human embryonic kidney 293 (HEK293) cells to explore the molecular mechanisms regulating ligand affinity, agonist-independent and dependent activity of D1R and D5R. We will demonstrate how to mutate one conserved residue in the cytosolic end of TM6 of D1R (Ser263) and D5R (Ser287) by modifying two or three nucleotides in the cDNA of human D1-like receptors. Genetically modified D1R and D5R cDNAs are prepared using a polymerase chain reaction method, propagated in E. coli, purified and mutations confirmed by DNA sequencing. Receptor expression constructs are transfected into HEK293 cells cultured in vitro at 37°C in 5% CO(2) environment and used in radioligand binding and whole cAMP assays. In this study, we will test the effect of S263A/G/D and S287A/G/D mutations on ligand binding and DA-dependent activation of D1R and D5R.

Sorting nexin 1 loss results in D5 dopamine receptor dysfunction in human renal proximal tubule cells and hypertension in mice.

The peripheral dopaminergic system plays a crucial role in blood pressure regulation through its actions on renal hemodynamics and epithelial ion transport. The dopamine D5 receptor (D(5)R) interacts with sorting nexin 1 (SNX1), a protein involved in receptor retrieval from the trans-Golgi network. In this report, we elucidated the spatial, temporal, and functional significance of this interaction in human renal proximal tubule cells and HEK293 cells stably expressing human D(5)R and in mice. Silencing of SNX1 expression via RNAi resulted in the failure of D(5)R to internalize and bind GTP, blunting of the agonist-induced increase in cAMP production and decrease in sodium transport, and up-regulation of angiotensin II receptor expression, of which expression was previously shown to be negatively regulated by D(5)R. Moreover, siRNA-mediated depletion of renal SNX1 in C57BL/6J and BALB/cJ mice resulted in increased blood pressure and blunted natriuretic response to agonist in salt-loaded BALB/cJ mice. These data demonstrate a crucial role for SNX1 in D(5)R trafficking and that SNX1 depletion results in D(5)R dysfunction and thus may represent a novel mechanism for the pathogenesis of essential hypertension.