Homologous desensitization of D(1) dopamine receptors is thought to occur through their phosphorylation leading to arrestin association which interdicts G protein coupling. In order to identify the relevant domains of receptor phosphorylation, and to determine how this leads to arrestin association, a series of mutated D(1) receptor constructs were created. In one mutant, all of the serine/threonine residues within the 3rd cytoplasmic domain were altered (3rdTOT). A second construct was created in which only three of these serines (serines 256, 258, and 259) were mutated (3rd234). Four truncation mutants were created of the carboxyl terminus (T347, T369, T394, and T404). All of these constructs were comparable with the wild-type receptor with respect to expression and adenylyl cyclase activation. In contrast, both of the 3rd loop mutants exhibited attenuated agonist-induced receptor phosphorylation that was correlated with an impaired desensitization response. Sequential truncation of the carboxyl terminus of the receptor resulted in a sequential loss of agonist-induced phosphorylation. No phosphorylation was observed with the most severely truncated T347 mutant. Surprisingly, all of the truncated receptors exhibited normal desensitization. The ability of the receptor constructs to promote arrestin association was evaluated using arrestin-green fluorescent protein translocation assays and confocal fluorescence microscopy. The 3rd234 mutant receptor was impaired in its ability to induce arrrestin translocation, whereas the T347 mutant was comparable with wild type. These data suggest a model in which arrestin directly associates with the activated 3rd cytoplasmic domain in an agonist-dependent fashion; however, under basal conditions, this is sterically prevented by the carboxyl terminus of the receptor. Receptor activation promotes the sequential phosphorylation of residues, first within the carboxyl terminus and then the 3rd cytoplasmic loop, thereby dissociating these domains and allowing arrestin to bind to the activated 3rd loop. Thus, the role of receptor phosphorylation is to allow access of arrestin to its receptor binding domain rather than to create an arrestin binding site per se (Kim, 2004).
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. The ER chaperone protein calnexin has been identified 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, assays were conducted 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, it was shown 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. These 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 (Free, 2007).
A critical event determining the functional consequences of G protein-coupled receptor (GPCR) endocytosis is the molecular sorting of internalized receptors between divergent recycling and degradative membrane pathways. The D1 dopamine receptor recycles rapidly and efficiently to the plasma membrane after agonist-induced endocytosis and is remarkably resistant to proteolytic down-regulation. Whereas the mechanism mediating agonist-induced endocytosis of D1 receptors has been investigated in some detail, little is known about how receptors are sorted after endocytosis. A sequence present in the carboxyl-terminal cytoplasmic domain of the human D1 dopamine receptor has been identified that is specifically required for the efficient recycling of endocytosed receptors back to the plasma membrane. This sequence is distinct from previously identified membrane trafficking signals and is located in a proximal portion of the carboxyl-terminal cytoplasmic domain, in contrast to previously identified GPCR recycling signals present at the distal tip. Nevertheless, fusion of this sequence to the carboxyl terminus of a chimeric mutant delta opioid neuropeptide receptor is sufficient to re-route internalized receptors from lysosomal to recycling membrane pathways, defining this sequence as a bona fide endocytic recycling signal that can function in both proximal and distal locations. These results identify a novel sorting signal controlling the endocytic trafficking itinerary of a physiologically important dopamine receptor, provide the first example of such a sorting signal functioning in a proximal portion of the carboxyl-terminal cytoplasmic domain, and suggest the existence of a diverse array of sorting signals in the GPCR superfamily that mediate subtype-specific regulation of receptors via endocytic membrane trafficking (Vargas, 2004).
There is accumulating evidence that G protein-coupled receptor signaling is regulated by localization in lipid raft microdomains. The D1 dopamine receptor (D1R) is localized in caveolae, a subset of lipid rafts. Through co-immunoprecipitation and bioluminescence resonance energy transfer assays, it has been demonstrated that this localization is mediated by an interaction between caveolin-1 and D1R in COS7 cells and an isoform selective interaction between D1R and caveolin-1alphain rat brain. The D1R interaction with caveolin-1 requires a putative caveolin binding motif identified in transmembrane domain 7. Agonist stimulation of D1R caused translocation of D1R into caveolin-1 enriched sucrose fractions which was determined to be a result of D1R endocytosis through caveolae. This was found to be PKA-independent and a kinetically slower process than clathrin mediated endocytosis. Site directed mutagenesis of the caveolin binding motif at amino acids F313 and W318 significantly attenuated caveolar endocytosis of D1R. It was also found that these caveolin binding mutants had a diminished capacity to stimulate cAMP production which was determined to be due to constitutive desensitization of these receptors. In contrast, D1Rs had an enhanced ability to maximally generate cAMP in chemically induced caveolae disrupted cells. Taken together, these data suggest that caveolae has an important role in regulating D1R turnover and signaling in brain (Kong. 2007).
After activation, most G protein coupled receptors (GPCRs) are regulated by a cascade of events involving desensitization and endocytosis. Internalized receptors can then be recycled to the plasma membrane, retained in an endosomal compartment, or targeted for degradation. The GPCR associated sorting protein, GASP, has been shown to preferentially sort a number of native GPCRs to the lysosome for degradation after endocytosis. This study shows that a mutant beta-2 adrenergic receptor and a mutant mu opioid receptor that have previously been described as lacking 'recycling signals' due to mutations in their C-termini, in fact bind to GASP and are targeted for degradation. A mutant dopamine D1 receptor, which has likewise been described as lacking a recycling signal, does not bind to GASP and is therefore not targeted for degradation. Together these results indicate that alteration of receptors in their C-termini can expose determinants with affinity for GASP binding and consequently target receptors for degradation (Thompson, 2007).
Evidence is provided for the formation of a novel phospholipase C-mediated calcium signal arising from coactivation of D1 and D2 dopamine receptors. In the present study, robust fluorescence resonance energy transfer showed that these receptors exist in close proximity indicative of D1-D2 receptor heterooligomerization. The close proximity of these receptors within the heterooligomer allowed for cross-phosphorylation of the D2 receptor by selective activation of the D1 receptor. D1-D2 receptor heterooligomers were internalized when the receptors were coactivated by dopamine or either receptor was singly activated by the D1-selective agonist (±)-6-chloro-7,8-dihydroxy-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine hydrobromide (SKF 81297) or the D2-selective agonist quinpirole. The D2 receptor expressed alone did not internalize after activation by quinpirole except when coexpressed with the D1 receptor. D1-D2 receptor heterooligomerization resulted in an altered level of steady-state cell surface expression compared with D1 and D2 homooligomers, with increased D2 and decreased D1 receptor cell surface density. Together, these results demonstrated that D1 and D2 receptors formed heterooligomeric units with unique cell surface localization, internalization, and transactivation properties that are distinct from that of D1 and D2 receptor homooligomers (So, 2005).
A heteromeric D1-D2 dopamine receptor signaling complex in brain is coupled to Gq/11 and requires agonist binding to both receptors for G protein activation and intracellular calcium release. The D1 agonist SKF83959 was identified as a specific agonist for the heteromer that activated Gq/11 by functioning as a full agonist for the D1 receptor and a high-affinity partial agonist for a pertussis toxin-resistant D2 receptor within the complex. Evidence that the D1-D2 signaling complex can be more readily detected in mice that are 8 months in age compared with animals that are 3 months old, suggesting that calcium signaling through the D1-D2 dopamine receptor complex is relevant for function in the postadolescent brain. Activation of Gq/11 through the heteromer increases levels of calcium/calmodulin-dependent protein kinase IIalpha in the nucleus accumbens, unlike activation of Gs/olf-coupled D1 receptors, indicating a mechanism by which D1-D2 dopamine receptor complexes may contribute to synaptic plasticity (Rashid, 2007).
Dopamine is an important neurotransmitter involved in learning and memory including emotional memory. The involvement of dopamine in conditioned fear has been widely documented. However, little is known about the molecular mechanisms that underlie contextual fear conditioning and memory consolidation. To address this issue, dopamine D1-deficient mice (D1-/-) and their wild-type (D1+/+) and heterozygote (D1+/-) siblings were used to assess aversive learning and memory. Two different aspects were quantified of fear responses to an environment where the mice have previously received unsignaled footshocks. Using one-trial step-through passive avoidance and conditioned freezing paradigms, mice were conditioned to receive mild inescapable footshocks then tested for acquisition, retention and extinction of conditioned fear responses 5 min after and up to 45-90 days post-training. No differences were observed among any of the genotypes in the acquisition of passive avoidance response or fear-induced freezing behavior. However, with extended testing, D1-/- mice exhibited prolonged retention and delayed extinction of conditioned fear responses in both tasks, suggesting that D1-/- mice are capable of acquiring aversive learning normally. These findings demonstrate that the dopamine D1 receptor is not important for acquisition or consolidation of aversive learning and memory but has an important role in modulating the extinction of fear memory (El-Ghundi, 2001).
Dopamine has been implicated in the modulation of diverse forms of behavioral plasticity, including appetitive learning and addiction. An important challenge is to understand how dopamine's effects at the cellular level alter the properties of neural circuits to modify behavior. In the nematode C. elegans, dopamine modulates habituation of an escape reflex triggered by body touch. In the absence of food, animals habituate more rapidly than in the presence of food; this contextual information about food availability is provided by dopaminergic mechanosensory neurons that sense the presence of bacteria. Dopamine alters habituation kinetics by selectively modulating the touch responses of the anterior-body mechanoreceptors; this modulation involves a D1-like dopamine receptor, a Gq/PLC-beta signaling pathway, and calcium release within the touch neurons. Interestingly, the body touch mechanoreceptors can themselves excite the dopamine neurons, forming a positive feedback loop capable of integrating context and experience to modulate mechanosensory attention (Kindt, 2007).
In a recently developed in vitro analog of appetitive classical conditioning of feeding in Aplysia, the unconditioned stimulus (US) was electrical stimulation of the esophageal nerve (En). This nerve is rich in dopamine (DA)-containing processes, which suggests that DA mediates reinforcement during appetitive conditioning. To test this possibility, methylergonovine was used to antagonize DA receptors. Methylergonovine (1 nM) blocked the pairing-specific increase in fictive feeding that is usually induced by in vitro classical conditioning. The present results and previous observation that methylergonovine also blocks the effects of contingent reinforcement in an in vitro analog of appetitive operant conditioning suggest that DA mediates reinforcement for appetitive associative conditioning of feeding in Aplysia (Reyes, 2005).
The involvement of dopamine (DA) in conditioned taste aversion (CTA) learning was studied with saccharin or sucrose as the conditioned stimulus (CS) and intraperitoneal lithium as the unconditioned stimulus (US). The dopamine D1 antagonist R(+)-7-chloro-8-hydroxy-3-methyl-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine hydrochloride (SCH 23390) (12.5-50 microg/kg, s.c.), given 5 min after the CS, impaired the acquisition of CTA in a paradigm consisting of three or a single CS-lithium association. SCH 23390 failed to impair CTA acquisition given 45 min after, 30 min before, or right before the CS. (-)-trans-6,7,7a,8,9,13b-hexahydro-3-chloro-2-hydroxy-N-methyl-5a-benzo-(d)-naphtho-(2,1b) azepine (SCH 39166) (12.5-50.0 microg/kg, s.c), a SCH 23390 analog that does not bind to 5HT2 receptors, also impaired CTA. No significant impairment of CTA was obtained after administration of the specific D2/D(3) antagonist raclopride (100 and 300 microg/kg, s.c.). The ability of SCH 23390 to impair CTA learning was confirmed by its ability to reduce the conditional aversive reactions to a gustatory CS (sweet chocolate) as estimated in a taste reactivity paradigm. SCH 39166 impaired CTA also when infused in the nucleus accumbens (NAc) shell 5 min after the CS. No impairment was obtained from the NAc core or from the bed nucleus stria terminalis. The results indicate that D1 receptor blockade impairs CTA learning by disrupting the formation of a short-term memory trace of the gustatory CS and that endogenous dopamine acting on D1 receptors in the NAc shell plays a role in short-term memory processes related to associative gustatory learning (Fenu, 2001; full text of article).
The effects of dopamine (DA) D1 and D2 receptor antagonists on the acquisition and expression of flavor-preferences conditioned by the sweet taste of fructose were examined. Food-restricted rats were trained over eight alternating one-bottle sessions to drink an 8% fructose solution containing one novel flavor (CS+) and a less preferred 0.2% saccharin solution containing a different flavor (CS-). Three groups of rats were treated daily with either vehicle (control group), SCH23390 (200 nmol/kg; D1 group), or raclopride (200 nmol/kg; D2 group) during training. Additional groups of vehicle-treated rats had their daily training intakes matched to that of the D1 and D2 groups. Preferences were assessed in two-bottle tests with the CS+ and CS- flavors presented in 0.2% saccharin solutions following doses of 0, 50, 200, 400, or 800 nmol/kg of either D1 or D2 antagonists. The D1 and D2 groups, unlike the control and yoked-control groups, failed to display a significant CS+ preference in the two-bottle tests following vehicle treatment. In addition, treatment with SCH23390 prior to the two-bottle tests blocked the expression of the CS+ preference in the control groups. Pretest raclopride treatment attenuated the CS+ preference at some dose levels. Raclopride also attenuated the preference for fructose in rats given two-bottle training with the CS+/fructose (CS+/F) and CS-/saccharin (CS-/S) solutions. These findings indicate that D1 and D2 antagonists block flavor-preference conditioning by sweet taste and that D1, and to a lesser extent D2, receptor antagonists attenuate the expression of a previously acquired preference (Baker, 2003).
Previous studies have shown that D1 receptor blockade disrupts and D2 receptor blockade enhances long-term potentiation. These data lead to the prediction that D1 antagonists will attenuate and D2 antagonists will potentiate at least some types of learning. The prediction is difficult to test, however, because disruptions in either D1 or D2 transmission lead to reduced locomotion, exploration, and response execution and are therefore likely to impair learning that requires behavioral responding (including exploration of an environment) during the learning episode. Under a paradigm that minimizes motor requirements, rats were trained to enter a food compartment during pellet presentation. Animals then received tone-food pairings under the influence of D1 antagonist SCH23390 (0, 0.4, 0.8, and 0.16 mg/kg) or D2 antagonist raclopride (0, 0.2, 0.4, and 0.8 mg/kg). An additional group received unpaired presentations of tone and food. On a drug-free test day 24 hr later, animals that had been under the influence of SCH23390 (like animals that had received unpaired presentations of tone and food) showed reduced head entries in response to the tone, whereas animals that had been under the influence of raclopride showed increased head entries in response to the tone compared with vehicle controls. These data demonstrate that, under a conditioned approach paradigm, D1 and D2 family receptor antagonists disrupt and promote learning, respectively, as predicted by the effects of D1 and D2 receptor blockade on neuronal plasticity (Eyny, 2003: full text of article).
Conditioned taste aversion (CTA), is a form of Pavlovian learning wherein a novel flavour is powerfully associated with subsequent feelings of illness, and is afterwards avoided. In rats, pharmacological blockade of dopamine D1 receptors has been reported to prevent the expression of a CTA to the sweet taste of sucrose or saccharine. Genetically modified mice were used to determine whether dopamine D1 receptors are necessary for the expression of a CTA. Food-deprived mice lacking the dopamine D1 receptor (D1r-/-) did not express a LiCl-induced (125 or 254 mg/kg) CTA to the sweet taste of 0.5 m sucrose, in agreement with previous pharmacological studies. However, water-deprived D1r-/- mice did express normal LiCl-induced (40, 150 and 254 mg/kg) CTA to a salty taste (0.2 m NaCl). These results suggest that activation of D1 receptors might contribute to the strength of an aversive gustatory association, but might not be required for the formation of a CTA in general (Cannon, 2005).
Recent research has implicated the nucleus accumbens (NAc) in consolidating recently acquired goal-directed appetitive memories, including spatial learning and other instrumental processes. However, an important but unresolved issue is whether this forebrain structure also contributes to the consolidation of fundamental forms of appetitive learning acquired by Pavlovian associative processes. In addition, although dopaminergic and glutamatergic influences in the NAc have been implicated in instrumental learning, it is unclear whether similar mechanisms operate during Pavlovian conditioning. To evaluate these issues, the effects of posttraining intra-NAc infusions of D1, D2, and NMDA receptor antagonists, as well as d-amphetamine, were determined on Pavlovian autoshaping in rats, which assesses learning by discriminated approach behavior to a visual conditioned stimulus predictive of food reward. Intracerebral infusions were given either immediately after each conditioning session to disrupt early memory consolidation or after a delay of 24 h. Findings indicate that immediate, but not delayed, infusions of both D1 (SCH 23390) and NMDA (AP-5) receptor antagonists significantly impair learning on this task. By contrast, amphetamine and the D2 receptor antagonist sulpiride were without significant effect. These findings provide the most direct demonstration to date that D1 and NMDA receptors in the NAc contribute to, and are necessary for, the early consolidation of appetitive Pavlovian learning (Dalley, 2005; full text of article).
Intracranial self-stimulation (ICSS) activates the neural pathways that mediate reward, including dopaminergic terminal areas such as the nucleus accumbens (NAc). However, a direct role of dopamine in ICSS-mediated reward has been questioned. Simultaneous voltammetric and electrophysiological recordings from the same electrode reveal that, at certain sites, the onset of anticipatory dopamine surges and changes in neuronal firing patterns during ICSS are coincident, whereas sites lacking dopamine changes also lack patterned firing. Intrashell microinfusion of a D1, but not a D2 receptor antagonist, blocks ICSS. An iontophoresis approach was implemented to explore the effect of dopamine antagonists on firing patterns without altering behavior. Similar to the microinfusion experiments, ICSS-related firing is selectively attenuated following D1 receptor blockade. This work establishes a temporal link between anticipatory rises of dopamine and firing patterns in the NAc shell during ICSS and suggests that they may play a similar role with natural rewards and during drug self-administration (Cheer, 2007).
Hebbian learning models require that neurons are able to both strengthen and weaken their synaptic connections. Hippocampal synaptic plasticity, in the form of long-term potentiation (LTP) and long-term depression (LTD), has been implicated in both spatial memory formation as well as novelty acquisition. In addition, the ventral tegmental area-hippocampal loop has been proposed to control the entry of information into long-term memory, whereas the dopaminergic system is believed to play an important role in information acquisition and synaptic plasticity. D1/D5 dopamine receptors are positively coupled to adenylyl cyclase and have been to modulate certain forms of synaptic plasticity, particularly in vitro. This study investigated how D1/D5 dopamine receptors modify long-lasting synaptic plasticity at CA1 synapses of adult freely moving rats and found that receptor activation lowered the threshold for the induction of both LTP and LTD. Specific types of learning are associated with specific types of hippocampal synaptic plasticity. Object-configuration learning, facilitation of late-phase LTD by object exploration, and late-phase LTP by exploration of empty space were all prevented by D1/D5 receptor antagonism. Furthermore, receptor antagonism prevented electrically induced late-LTP, whereas receptor activation facilitated induction of both LTP and LTD by patterned electrical stimulation. These findings suggest that the dopaminergic system, acting via D1/D5 receptors, gates long-term changes in synaptic strength and that these changes are a critical factor in the acquisition of novel information (Lemon, 2006: full text of article).
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