D1 and D2 dopamine receptor expression is regulated by direct interaction with the chaperone protein calnexin

As for all proteins, G protein-coupled receptors (GPCRs) undergo synthesis and maturation within the endoplasmic reticulum (ER). The mechanisms involved in the biogenesis and trafficking of GPCRs from the ER to the cell surface are poorly understood, but they may involve interactions with other proteins. We have now identified the ER chaperone protein calnexin as an interacting protein for both D(1) and D(2) dopamine receptors. These protein-protein interactions were confirmed using Western blot analysis and co-immunoprecipitation experiments. To determine the influence of calnexin on receptor expression, we conducted assays in HEK293T cells using a variety of calnexin-modifying conditions. Inhibition of glycosylation either through receptor mutations or treatments with glycosylation inhibitors partially blocks the interactions with calnexin with a resulting decrease in cell surface receptor expression. Confocal fluorescence microscopy reveals the accumulation of D(1)-green fluorescent protein and D(2)-yellow fluorescent protein receptors within internal stores following treatment with calnexin inhibitors. Overexpression of calnexin also results in a marked decrease in both D(1) and D(2) receptor expression. This is likely because of an increase in ER retention because confocal microscopy revealed intracellular clustering of dopamine receptors that were co-localized with an ER marker protein. Additionally, we show that calnexin interacts with the receptors via two distinct mechanisms, glycan-dependent and glycan-independent, which may underlie the multiple effects (ER retention and surface trafficking) of calnexin on receptor expression. Our data suggest that optimal receptor-calnexin interactions critically regulate D(1) and D(2) receptor trafficking and expression at the cell surface, a mechanism likely to be of importance for many GPCRs.     

Identification of dopamine D1-D3 receptor heteromers. Indications for a role of synergistic D1-D3 receptor interactions in the striatum

The function of dopamine D(3) receptors present in the striatum has remained elusive. In the present study evidence is provided for the existence of dopamine D(1)-D(3) receptor heteromers and for an intramembrane D(1)-D(3) receptor cross-talk in living cells and in the striatum. The formation of D(1)-D(3) receptor heteromers was demonstrated by fluorescence resonance energy transfer and bioluminescence resonance energy transfer techniques in transfected mammalian cells. In membrane preparations from these cells, a synergistic D(1)-D(3) intramembrane receptor-receptor interaction was observed, by which D(3) receptor stimulation enhances D(1) receptor agonist affinity, indicating that the D(1)-D(3) intramembrane receptor-receptor interaction is a biochemical characteristic of the D(1)-D(3) receptor heteromer. The same biochemical characteristic was also observed in membrane preparations from brain striatum, demonstrating the striatal co-localization and heteromerization of D(1) and D(3) receptors. According to the synergistic D(1)-D(3) intramembrane receptor-receptor interaction, experiments in reserpinized mice showed that D(3) receptor stimulation potentiates D(1) receptor-mediated behavioral effects by a different mechanism than D(2) receptor stimulation. The present study shows that a main functional significance of the D(3) receptor is to obtain a stronger dopaminergic response in the striatal neurons that co-express the two receptors.     

Dopamine D2 receptors form higher order oligomers at physiological expression levels

G-protein-coupled receptors are generally thought to be organized as dimers; whether they form higher order oligomers is a topic of much controversy. We combined bioluminescence/fluorescence complementation and energy transfer to demonstrate that at least four dopamine D2 receptors are located in close molecular proximity in living mammalian cells, consistent with their organization as higher order oligomers at the plasma membrane. This implies the existence of multiple receptor interfaces. In addition to the symmetrical interface in the fourth transmembrane segment (TM4) we identified previously by cysteine (Cys) crosslinking, we now show that a patch of residues at the extracellular end of TM1 forms a second symmetrical interface. Crosslinking of D2 receptor with Cys substituted simultaneously into both TM1 and TM4 led to higher order species, consistent with our novel biophysical results. Remarkably, the rate and extent of crosslinking at both interfaces were unaltered over a 100-fold range of receptor expression. Thus, at physiological levels of expression, the receptor is organized in the plasma membrane into a higher order oligomeric structure.     

Sequestration of dopamine D2 receptors depends on coexpression of G-protein-coupled receptor kinases 2 or 5.

We examined the agonist-dependent sequestration/internalization of dopamine D2 receptor (the long form D2L and short form D2S), which were transiently expressed in COS-7 and HEK 293 cells with or without G-protein-coupled receptor kinases (GRK2 or GRK5). Sequestration was assessed quantitatively by loss of [3H] sulpiride-binding activity from the cell surface and by transfer of [3H] spiperone-binding activity from the membrane fraction to the light vesicle fraction in sucrose-density gradients. In COS-7 cells expressing D2 receptors alone, virtually no sequestration was observed with or without dopamine (< 4%). When GRK2 was coexpressed, 50% of D2S receptors and 36% of D2L receptors were sequestered by treatment with 10(-4) M dopamine for 2 h, whereas no sequestration was observed in cells expressing the dominant negative form of GRK2 (DN-GRK2). When GRK5 was coexpressed, 36% of D2S receptors were sequestered following the same treatment. The agonist-dependent and GRK2-dependent sequestration of D2S receptors was reduced markedly in the presence of hypertonic medium containing 0.45 M sucrose, suggesting that the sequestration follows the clathrin pathway. Internalization of D2S receptors was also assessed by immunofluorescence confocal microscopy. Translocation of D2 receptors from the cell membrane to intracellular vesicles was observed following the treatment with dopamine from HEK 293 cells only when GRK2 was coexpressed. D2S receptors expressed in HEK 293 cells were shown to be phosphorylated by GRK2 in an agonist-dependent manner. These results indicate that the sequestration of D2 receptors occurs only through a GRK-mediated pathway.     

D2/D3 dopamine receptor heterodimers exhibit unique functional properties

Evidence for heterodimerization has recently been provided for dopamine D(1) and adenosine A(1) receptors as well as for dopamine D(2) and somatostatin SSTR(5) receptors. In this paper, we have studied the possibility that D(2) and D(3) receptors interact functionally by forming receptor heterodimers. Initially, we split the two receptors at the level of the third cytoplasmic loop into two fragments. The first, containing transmembrane domains (TM) I to V and the N-terminal part of the third cytoplasmic loop, was named D(2trunk) or D(3trunk), and the second, containing the C-terminal part of the third cytoplasmic loop, TMVI and TMVII, and the C-terminal tail, was named D(2tail) or D(3tail). Then we defined the pharmacological profiles of the homologous (D(2trunk)/D(2tail) and D(3trunk)/D(3tail)) as well as of the heterologous (D(2trunk)/D(3tail) and D(3trunk)/D(2tail)) cotransfected receptor fragments. The pharmacological profile of the cross-cotransfected fragments was different from that of the native D(2) or D(3) receptors. In most cases, the D(3trunk)/D(2tail) was the one with the highest affinity for most agonists and antagonists. Moreover, we observed that all of these receptor fragments reduced the expression of the wild type dopamine D(2) and D(3) receptors, suggesting that D(2) and D(3) receptors can form complexes with these fragments and that these complexes bind [(3)H]nemonapride less efficiently or are not correctly targeted to the membrane. In a second set of experiments, we tested the ability of the split and the wild type receptors to inhibit adenylyl cyclase (AC) types V and VI. All of the native and split receptors inhibited AC-V and AC-VI, with the exception of D(3), which was unable to inhibit AC-VI. We therefore studied the ability of D(2) and D(3) to interact functionally with one another to inhibit AC-VI. We found that with D(2) alone, R-(+)-7-hydroxydypropylaminotetralin hydrobromide inhibited AC-VI with an IC(50) of 2.05 +/- 0.15 nm, while in the presence of D(2) and D(3) it inhibited AC-VI with an IC(50) of 0.083 +/- 0.011 nm. Similar results were obtained with a chimeric cyclase made from AC-V and AC-VI. Coimmunoprecipitation experiments indicate that D(2) and D(3) receptors are capable of physical interaction.     

Distribution of D(5) dopamine receptor mRNA in rat ventromedial hypothalamic nucleus

Estrogen induces lordosis through, in part, estrogen receptor (ER)-mediated synthesis of progesterone receptors (PR) in the ventromedial nucleus (VMN). In vitro, PR is activated by the neurotransmitter dopamine through D1-like receptors (1). In vivo, lordosis is induced by dopamine, an effect mediated in part by PR and D(5) dopamine receptors. The purpose of the present study was to determine mRNA distribution of D1-like receptors in the female rat brain using RT-PCR combined with punchout microdissection techniques. Employing specific primers to D(5) and D(1) dopamine receptors, we found detectable expression levels of D(5) dopamine receptor mRNA in VMN as well as the arcuate nucleus/median eminence (ArcN/ME). In contrast, D(1) dopamine receptor mRNA was detected only in VMN. By using this highly sensitive and specific RT-PCR methodology, we have confirmed the presence of D(5) dopamine receptor mRNA in an area of the brain that regulates reproductive behavior through PR. The data support the previous observation that D(5) dopamine receptors in VMN contribute to facilitation of female reproductive behavior by D1-like agonists.     

Pharmacological characterization of mammalian D1 and D2 dopamine receptors expressed in Drosophila Schneider-2 cells

Mammalian D1 and D2 dopamine receptors were stably expressed in Drosophila Schneider-2 (S2) cells and screened for their pharmacological properties. Saturable, dose-dependent, high affinity binding of the D1-selective antagonist [3H]SCH-23390 was detected only in membranes from S2 cells induced to express rat dopamine D1 receptors, while saturable, dose-dependent, high affinity binding of the D2-selective antagonist [3H]methylspiperone was detected only in membranes from S2 cells induced to express rat dopamine D2 receptors. No specific binding of either radioligand could be detected in membranes isolated from uninduced or untransfected S2 cells. Both dopamine D1 and D2 receptor subtypes displayed the appropriate stereoselective binding of enantiomers of the nonselective antagonist butaclamol. Each receptor subtype also displayed the appropriate agonist stereoselectivities. The dopamine D1 receptor bound the (+)-enantiomer of the D1-selective agonist SKF38393 with higher affinity than the (-)-enantiomer, while the dopamine D2 receptor bound the (-)-enantiomer of the D2-selective agonist norpropylapomorphine with higher affinity than the (+)-enantiomer. At both receptor subtypes, dopamine binding was best characterized as occurring to a single low affinity site. In addition, the low affinity dopamine binding was also found to be insensitive to GTPgammaS and magnesium ions. Overall, the pharmacological profiles of mammalian dopamine D1 and D2 receptors expressed in Drosophila S2 cells is comparable to those observed for these same receptors when they are expressed in mammalian cell lines. A notable distinction is that there is no evidence for the coupling of insect G proteins to mammalian dopamine receptors. These results suggest that the S2 cell insect G system may provide a convenient source of pharmacologically active mammalian D1 and D2 dopamine receptors free of promiscuous G protein contaminants.     

Dopamine D(3) receptors are down-regulated following heterologous endocytosis by a specific interaction with G protein-coupled receptor-associated sorting protein-1

The D(3) dopamine receptor is endocytosed through a heterologous mechanism mediated by phorbol esters. Here, we show that following this endocytosis the D(3) dopamine receptors fail to recycle and are instead targeted for degradation through an interaction with the G protein-coupled receptor (GPCR)-associated sorting protein-1 (GASP-1). Furthermore, we identified a specific binding motif in the C terminus common to the D(3) and D(2) that confers GASP-1 binding. shRNA knockdown of GASP-1 delayed post-endocytic degradation of both the D(2) and D(3) dopamine receptors. In addition, mutation of the D(2) and D(3) receptor C termini to resemble the D(4), which does not interact with GASP-1, not only inhibited GASP-1 binding but slowed degradation after endocytosis. Conversely, mutation of the C terminus of the D(4) to resemble that of the D(2) and D(3) facilitated GASP-1 binding and promoted post-endocytic degradation of the mutant D(4) receptor. Thus, we have identified a motif that is both necessary and sufficient to promote GASP-1 binding and receptor degradation. In addition, these data demonstrated that GASP-1 can mediate post-endocytic degradation of dopamine receptors that have been endocytosed not only as a consequence of dopamine activation but also as a consequence of activation by phorbol esters.     

Delineation of the structural basis for the activation properties of the dopamine D1 receptor subtypes.

To delineate the structural determinants involved in the constitutive activation of the D1 receptor subtypes, we have constructed chimeras between the D1A and D1B receptors. These chimeras harbored a cognate domain corresponding to transmembrane regions 6 and 7 as well as the third extracellular loop (EL3) and cytoplasmic tail, a domain referred herein to as the terminal receptor locus (TRL). A chimeric D1A receptor harboring the D1B-TRL (chimera 1) displays an increased affinity for dopamine that is indistinguishable from the wild-type D1B receptor. Likewise, a chimeric D1B receptor containing the D1A-TRL cassette (chimera 2) binds dopamine with a reduced affinity that is highly reminiscent of the dopamine affinity for the wild-type D1A receptor. Furthermore, we show that the agonist independent activity of chimera 1 is identical to the wild-type D1B receptor whereas the chimera 2 displays a low agonist independent activity that is indistinguishable from the wild-type D1A receptor. Dopamine potencies for the wild-type D1A and D1B receptor were recapitulated in cells expressing the chimera 2 or chimera 1, respectively. However, the differences observed in agonist-mediated maximal activation of adenylyl cyclase elicited by the D1A and D1B receptors remain unchanged in cells expressing the chimeric receptors. To gain further mechanistic insights into the structural determinants of the TRL involved in the activation properties of the D1 receptor subtypes, we have engineered two additional chimeric D1 receptors that contain the EL3 region of their respective cognate wild-type counterparts (hD1A-EL3B and hD1B-EL3A). In marked contrast to chimera 1 and 2, dopamine affinity and constitutive activation were partially modulated by the exchange of the EL3. Meanwhile, hD1A-EL3B and hD1B-EL3A mutant receptors display a full switch in the agonist-mediated maximal activation, which is reminiscent of their cognate wild-type counterparts. Overall, our studies suggest a fundamental role for the TRL in shaping the intramolecular interactions implicated in the constitutive activation and coupling properties of the dopamine D1 receptor subtypes.     

Distinct regulation of internalization and mitogen-activated protein kinase activation by two isoforms of the dopamine D2 receptor

Two isoforms of the dopamine D2 receptor, D2L (long) and D2S (short), differ by the insertion of a 29-amino acid specific to D2L within the putative third intracellular loop of the receptor. Here, we examined D2 receptor-mediated MAPK activation in association with receptor internalization. Overexpression of beta-arrestin 1 and 2 increased the D2S-mediated activation of MAPK, whereas it did not affect the activation of MAPK by D2L. Expression of a dominant negative beta-arrestin 2 (319-418) mutant and of a dominant negative dynamin I (K44A) mutant inhibited the activation of MAPK by D2S, but not the activation of MAPK by D2L. Treatment with inhibitors of internalization, i.e. concanavalin A and monodansylcadaverin, blocked D2S-mediated MAPK activation but not D2L-mediated activation. By confocal microscopy, we observed beta-arrestin 1 and 2, translocated to the plasma membrane and colocalized with D2L and D2S receptors upon stimulation with dopamine, and this was followed by the translocation of receptors into endocytic vesicles. Moreover, the expression of the beta-arrestin 2 (319-418) mutant blocked the internalization of both D2L and D2S. In addition, although K44A dynamin mutant expression did not alter D2L internalization, it completely blocked the internalization of D2S. The stimulation of D2L induces activation of MAPK via transactivation of the platelet-derived growth factor receptor, whereas D2S does not. Taken together, these data suggest that D2L activates MAPK signaling by mobilizing the growth factor receptor, platelet-derived growth factor receptor, whereas D2S appears to activate MAPK signaling by mobilizing clathrin-mediated endocytosis in a beta-arrestin/dynamin-dependent manner.     

Regulation of dopamine D1 receptor trafficking and desensitization by oligomerization with glutamate N-methyl-D-aspartate receptors

Activation of dopamine D1 receptors is critical for the generation of glutamate-induced long-term potentiation at corticostriatal synapses. In this study, we report that, in striatal neurons, D1 receptors are co-localized with N-methyl-d-aspartate (NMDA) receptors in the postsynaptic density and that they co-immunoprecipitate with NMDA receptor subunits from postsynaptic density preparations. Using modified bioluminescence resonance energy transfer, we demonstrate that D1 and NMDA receptor clustering reflects the existence of direct interactions. The tagged D1 receptor and NR1 subunit cotransfected in COS-7 cells generated a significant bioluminescence resonance energy transfer signal that was insensitive to agonist stimulation and that did not change in the presence of the NR2B subunit, suggesting that the D1 receptor constitutively and selectively interacts with the NR1 subunit of the NMDA channel. Oligomerization with the NR1 subunit substantially modified D1 receptor trafficking. In individually transfected HEK293 cells, NR1 was localized in the endoplasmic reticulum, whereas the D1 receptor was targeted to the plasma membrane. In cotransfected cells, both the D1 receptor and NR1 subunit were retained in cytoplasmic compartments. In the presence of the NR2B subunit, the NR1-D1 receptor complex was translocated to the plasma membrane. These data suggest that D1 and NMDA receptors are assembled within intracellular compartments as constitutive heteromeric complexes that are delivered to functional sites. Coexpression with NR1 and NR2B subunits also abolished agonist-induced D1 receptor cytoplasmic sequestration, indicating that oligomerization with the NMDA receptor could represent a novel regulatory mechanism modulating D1 receptor desensitization and cellular trafficking.     

D1 dopamine receptor stimulation increases GluR1 surface expression in nucleus accumbens neurons

The goal of this study was to understand how dopamine receptors, which are activated during psychostimulant administration, might influence glutamate-dependent forms of synaptic plasticity that are increasingly recognized as important to drug addiction. Regulation of the surface expression of the alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) receptor subunit GluR1 plays a critical role in long-term potentiation, a well-characterized form of synaptic plasticity. Primary cultures of rat nucleus accumbens neurons were used to examine whether dopamine receptor stimulation influences cell surface expression of GluR1, detected using antibody to the extracellular portion of GluR1 and fluorescence microscopy. Surface GluR1 labeling on processes of medium spiny neurons and interneurons was increased by brief (5-15 min) incubation with a D1 agonist (1 microm SKF 81297). This effect was attenuated by the D1 receptor antagonist SCH 23390 (10 microm) and reproduced by the adenylyl cyclase activator forskolin (10 microm). Labeling was decreased by glutamate (10-50 microm, 15 min). These results are the first to demonstrate modulation of AMPA receptor surface expression by a non-glutamatergic G protein-coupled receptor. Normally, this may enable ongoing regulation of AMPA receptor transmission in response to changes in the activity of dopamine projections to the nucleus accumbens. When dopamine receptors are over-stimulated during chronic drug administration, this regulation may be disrupted, leading to inappropriate plasticity in neuronal circuits governing motivation and reward.     

Molecular characterization of dopamine D2 receptor isoforms tagged with green fluorescent protein

In this study, a cleavable signal peptide fused to the enhanced green fluorescent protein (EGFP) was tagged to the extracellular N-terminus of the human dopamine D2 receptor short and long isoforms (D2S and D2L). Ligand-binding properties of EGFP-tagged receptors were essentially unchanged in comparison to their respective wild-type receptors. The dopamine-mediated activation of both EGFP-D2 isoforms generated a robust inhibition of adenylyl cyclase type 5 in intact cells. In addition, the receptor density of EGFP-D2S and EGFP-D2L in transfected human embryonic kidney 293 (HEK293) cells was not altered when compared to cells transfected with the untagged D2S and D2L. However, the receptor densities of untagged and EGFP-tagged D2L were significantly lower in comparison to those measured with D2S constructs. Moreover, the receptor density of EGFP-D2S and EGFP-D2L was differentially upregulated in cells treated with antipsychotic drugs. As assessed by confocal microscopy, both EGFP-D2 isoforms were present on the cell surface. Notably, in contrast to the predominant plasma membrane localization of EGFP-D2S, EGFP-D2L was visualized both on the plasma membrane and intracellularly before dopamine exposure. Importantly, EGFP-D2S and EGFP-D2L are robustly internalized after dopamine treatment. Overall, our data suggest a differential intracellular sorting of D2S and D2L.     

Dopamine D1 and D2 receptors in spontaneously hypertensive rat brain striatum

Since it has been reported that dopamine D2 receptors are elevated in the brain striatum of spontaneously hypertensive (SH) rats, and since both D1 and D2 receptors may interact with one another, we measured the densities of both these receptors in SH rat striatum, as well as those in the normotensive Wistar-Kyoto rat striatum. The D1 receptor density in both strains was virtually the same, 72.9 +/- 2.2 and 71.3 +/- 3.2 pmol/g, respectively (mean +/- SD). The D2 receptor densities were also almost identical, 16.3 +/- 0.6 and 16.8 +/- 1.0 pmol/g, respectively (mean +/- SD). Thus, these data do not support the concept of a dopamine receptor related role in spontaneous hypertension.     

Autocrine activation of adenosine A1 receptors blocks D1A but not D1B dopamine receptor desensitization

Adenosine is known to modulate dopamine responses in several brain areas. Here, we show that tonic activation of adenosine receptors is able to impede desensitization of D1 dopamine receptors. As measured by cAMP accumulation in transfected COS-7 cells, long-term exposure to dopamine agonists promoted desensitization of D1B receptor but not that of D1A receptor. The inability of D1A receptor to desensitize was a result of the adenosine present in culture medium acting through activation of adenosine A1 receptors. Cell incubation with either adenosine deaminase, CGS-15943, a generic adenosine receptor antagonist, or the A1 antagonist DPCPX restored the long-term desensitization time-course of D1A receptors. In Ltk cells stably expressing A1 adenosine receptors and D1A dopamine receptors, pre-treatment of cells with R(-)-PIA, a full A1 receptor agonist, did not significantly inhibit the acute increase in cAMP levels induced by D1 receptor agonists, but blocked desensitization of D1A receptors. However, simultaneous activation of A1 and D1A receptors promoted a delayed D1A receptor desensitization. This suggests that functional interaction between A1 and D1A receptors may depend on the activation kinetics of components regulating D1 receptor responses, acting differentially on D1A and D1B receptors.     

多巴胺D2受体在肿瘤治疗中的研究进展

多巴胺(Dopamine)(C6H3(OH)2-CH2-CH2-NH2)是人类中枢神经系统的重要儿茶酚胺类神经递质,通过其相应的膜受体而发挥情绪、饮食、运动、认知及外周血等的调节作用。多巴胺受体属于膜G蛋白偶联受体家族。目前发现的多巴胺受体有五种,其中D2受体基因主要分布于脑部。近年来的研究表明,多巴胺D2受体对肿瘤细胞具有抑制作用,对肿瘤的药物治疗具有重要意义。目前,D2受体激动剂已经成为大多数泌乳素瘤的首选治疗药物。本文通过文献回顾,对多巴胺受体在肿瘤的预后和治疗中的作用进行综述。     

Excessive D1 Dopamine Receptor Activation in the Dorsal Striatum Promotes Autistic-Like Behaviors

The dopamine system has been characterized in motor function, goal-directed behaviors, and rewards. Recent studies recognize various dopamine system genes as being associated with autism spectrum disorder (ASD). However, how dopamine system dysfunction induces ASD pathophysiology remains unknown. In the present study, we demonstrated that mice with increased dopamine functions in the dorsal striatum via the suppression of dopamine transporter expression in substantia nigra neurons or the optogenetic stimulation of the nigro-striatal circuitry exhibited sociability deficits and repetitive behaviors relevant to ASD pathology in animal models, while these behavioral changes were blocked by a D1 receptor antagonist. Pharmacological activation of D1 dopamine receptors in normal mice or the genetic knockout (KO) of D2 dopamine receptors also produced typical autistic-like behaviors. Moreover, the siRNA-mediated inhibition of D2 dopamine receptors in the dorsal striatum was sufficient to replicate autistic-like phenotypes in D2 KO mice. Intervention of D1 dopamine receptor functions or the signaling pathways-related D1 receptors in D2 KO mice produced anti-autistic effects. Together, our results indicate that increased dopamine function in the dorsal striatum promotes autistic-like behaviors and that the dorsal striatum is the neural correlate of ASD core symptoms.     

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.     

Dopamine D1 and D2 receptor Co-activation generates a novel phospholipase C-mediated calcium signal

Although dopamine D1 and D2 receptors belong to distinct subfamilies of dopamine receptors, several lines of evidence indicate that they are functionally linked. However, a mechanism for this linkage has not been elucidated. In this study, we demonstrate that agonist stimulation of co-expressed D1 and D2 receptors resulted in an increase of intracellular calcium levels via a signaling pathway not activated by either receptor alone or when only one of the co-expressed receptors was activated by a selective agonist. Calcium signaling by D1-D2 receptor co-activation was abolished following treatment with a phospholipase C inhibitor but not with pertussis toxin or inhibitors of protein kinase A or protein kinase C, indicating coupling to the G(q) pathway. We also show, by co-immunoprecipitation from rat brain and from cells co-expressing the receptors, that D1 and D2 receptors are part of the same heteromeric protein complex and, by immunohistochemistry, that these receptors are co-expressed and co-localized within neurons of human and rat brain. This demonstration that D1 and D2 receptors have a novel cellular function when co-activated in the same cell represents a significant step toward elucidating the mechanism of the functional link observed between these two receptors in brain.