From the
Division of Pharmacology, Department of Biomedical Sciences and Biotechnology, and the Centre of Excellence on Diagnostic and Therapeutic Innovation, University of Brescia, Viale Europa 11, 25123 Brescia, Italy and the
Centre of Excellence on Neurodegenerative Diseases and the Department of Pharmacological Sciences, University of Milano, Via Balzaretti 9, 20133 Milano, Italy
Received for publication, December 23, 2002 , and in revised form, March 11, 2003.
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ABSTRACT |
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INTRODUCTION |
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At the cellular level, nigral and cortical fibers converge on the medium spiny projection neurons (4), where dopamine D1- and D2-like receptors are coexpressed to high degree with glutamate NMDA1 and non-NMDA receptor channels (5, 6, 7, 8). From a functional point of view, it is well established that dopamine modulates the firing pattern of these neurons. In particular, there is evidence that dopamine, while attenuating the responses mediated by non-NMDA receptors, potentiates those associated with activation of NMDA receptors (2). The D1 receptor appears to be involved in these interactions. In fact, activation of D1 receptors in medium spiny neurons enhances NMDA-induced whole cell currents (2, 9) and is a critical requirement for the formation of NMDA-mediated long-term potentiation at corticostriatal synapses (2, 10, 11, 12). Moreover, activation of NMDA receptors in striatal neurons triggers the translocation of cytoplasmic D1 receptors to the plasma membrane and spines (13). Within neuronal spines, D1 receptors are mainly localized in the spine shaft and, to a lesser extent, also in the spine head and in the postsynaptic density (PSD) (14, 15, 16). This cell structure is typical of the glutamatergic synapse and consists of a complex network of critical proteins involved in synaptic plasticity, many of which bind directly or indirectly to the NMDA receptor, which is an abundant component of the fraction (17, 18). The mechanisms that specifically drive D1 receptor delivery to different spine domains are still unknown. The partial overlap in the subcellular distribution of NMDA and D1 receptors and the observation that both D1 and NMDA receptor delivery to synapses is dependent on glutamate transmission (13, 19) suggest that direct protein-protein interactions might direct the trafficking of these receptors to the same subcellular domain.
In this study, we report that the dopamine D1 receptor forms a heteromeric complex with the NR1 subunit of the NMDA receptor in both purified striatal PSDs and cotransfected cells. This interaction is constitutive, occurs in the endoplasmic reticulum (ER), influences D1 receptor targeting to the cell membrane, and prevents agonist-induced D1 receptor internalization.
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EXPERIMENTAL PROCEDURES |
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PSD and Triton-insoluble Fraction PreparationStriatal PSD were isolated according to Carlin et al. (20) with minor modifications as described previously (21). Briefly, the tissue was homogenized in ice-cold 0.32 M sucrose containing 1 mM Hepes, 1 mM MgCl2,1 mM NaHCO3, 0.1 mM phenylmethylsulfonyl fluoride, and a mixture of protease inhibitors (Complete, Roche Diagnostic, Milano) at pH 7.4 (buffer A) and centrifuged at 1000 x g for 10 min. The supernatant was centrifuged at 3000 x g for 15 min. The resulting pellet (containing mitochondria and synaptosomes) was resuspended in ice-cold 0.32 M sucrose containing 1 mM Hepes, 1 mM NaHCO3, and 0.1 mM phenylmethylsulfonyl fluoride (buffer B); overlaid on a sucrose gradient (0.85 to 1.0 to 1.2 M); and centrifuged at 82,500 x g for 2 h. The fraction between 1.0 and 1.2 M was diluted with buffer B containing 1% Triton X-100, stirred at 4 °C for 15 min, and centrifuged at 82,500 x g for 30 min. The resulting pellet was resuspended, layered on a sucrose gradient (1.0 to 1.5 to 2.1 M), and centrifuged at 100,000 x g for 2 h at 4 °C. The fraction between 1.5 and 2.1 M was removed and diluted with 150 mM KCl containing 1% Triton X-100. PSD were collected by centrifugation at 100,000 x g for 30 min at 4 °C.
To isolate the Triton-insoluble fraction (TIF), tissue was homogenized in ice-cold buffer A and centrifuged at 1000 x g for 10 min. The resulting supernatant was centrifuged at 3000 x g for 15 min, and the pellet was resuspended in 1 mM Hepes and centrifuged at 100,000 x g for 1 h. The pellet was resuspended in 75 mM KCl containing 1% Triton X-100, and TIF was collected by centrifugation at 100,000 x g for 1 h. TIF was characterized by enrichment in PSD proteins as previously described (22).
Immunoprecipitation and Western BlottingTen micrograms of PSD were incubated overnight at 4 °C with antibodies against either the NR1 subunit (1 µg/ml) or the D1 receptor (1:250 dilution; mouse monoclonal) in 200 mM NaCl, 10 mM EDTA, 10 mM Na2HPO4, 0.5% Nonidet P-40, and 0.1% SDS (buffer C). Protein A-agarose beads (Santa Cruz Biotechnology) were added, and incubation was continued for 2 h at room temperature. The beads were collected and extensively washed with buffer C. The resulting proteins were resolved by SDS-PAGE, transferred onto polyvinylidene difluoride membranes, and blotted for 1 h at room temperature in Tris-buffered saline containing 0.1% Tween 20 and 5% low fat dry milk. Membranes were incubated for 2 h at room temperature with the anti-NR1 (1 µg/ml) or anti-D1 receptor (1:250 dilution) antibodies. Detection was performed by chemiluminescence (ECL, Amersham Biosciences, Milano) with horseradish peroxidase-conjugated secondary antibodies (1:1500 dilution).
Cloning, Expression, and Purification of GST Fusion ProteinsThe C-terminal regions of the D1 receptor (D1-CT-(321446)) and of the D5 receptor (D5-CT-(373477)) and two fragments of the NR1 subunit C terminus (NR1-CT-(834930) and NR1-CT-(834892)) were generated by PCR amplification, cloned into the pGEX-KG plasmid, and expressed in BL21 competent cells. Synthesis of recombinant proteins was induced by 0.1 mM isopropyl--D-thiogalactopyranoside (Sigma) for 24h. The bacteria were lysed, and the proteins were purified by incubation with glutathione-agarose beads (50% (v/v) in PBS) for 12 h at 4 °C as previously described (23).
Affinity Purification ("Pull-out")TIF proteins (35 µg) were diluted with PBS containing 0.1% SDS and incubated for 1 h at room temperature with glutathione-agarose beads saturated with GST fusion proteins. Beads were washed with PBS containing 0.1% Triton X-100, and bound proteins were resolved by SDS-PAGE and immunoblotted with anti-NR1 and anti-NR2A/B antibodies.
Generation of Bioluminescence Resonance Energy Transfer (BRET2) Fusion ConstructsThe D1 receptor and NR1a subunit coding sequences were amplified out of their original vectors using sense and antisense primers containing unique XhoI and BamHI sites and Hin- dIII and BamHI sites, respectively, and the native Pfu DNA polymerase (Stratagene, Milano) to generate stop codon-free fragments. The D1 receptor fragment was cloned in-frame into the Renilla luciferase-containing vector pRluc-N2(h) (PerkinElmer Life Sciences, Milano) to generate the plasmid D1-Rluc. The NR1a fragment was cloned in-frame into the pGFP2-N2(h) vector containing the green fluorescent protein (GFP2) (PerkinElmer Life Sciences) to generate the plasmid NR1-GFP2. The D1-Rluc receptor was tested for its efficiency in activating adenylyl cyclase in transfected COS-7 cells as previously described (24). The influence of GFP2 on glutamate-mediated 45Ca2+ influx in COS-7 cells cotransfected with NR1-GFP2 and NR2B was assessed by standard methods.
Cell Culture, Transfection, and BRET2 AssayCOS-7 cells were cultured in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum, 2 mM glutamine, 100 units/ml penicillin, and 100 µg/ml streptomycin. Semiconfluent cells were cotransfected for 3 h with D1-Rluc and NR1-GFP2 at a 1:4 DNA ratio, which was shown to give the best BRET2 signal, in the absence or presence of NR2B using the LipofectAMINE technique (Invitrogen, Milano). The total amount of DNA was kept at 10 µg. Forty-eight hours post-transfection, cells were harvested, centrifuged, and resuspended in PBS containing 0.1 mg/ml CaCl2, 0.1 mg/ml MgCl2, and 1 mg/ml D-glucose. Approximately 50,000 cells/well were distributed in a 96-well microplate (white Optiplate, PerkinElmer Life Sciences) and incubated in the absence or presence of 50 µM dopamine, 100 µM glutamate, and 10 µM glycine for 10 min at 37 °C. DeepBlueCTM coelenterazine (PerkinElmer Life Sciences) was added at a final concentration of 5 µM, and BRET2 signals were determined using a FusionTM universal microplate analyzer (PerkinElmer Life Sciences), which allows sequential integration of signals detected at 390/400 and 505/510 nm. Untransfected cells and cells transfected with D1-Rluc alone were used to define the nonspecific signals, and cells transfected with the pRluc-GFP2 control vector (PerkinElmer Life Sciences) were used as positive controls. The BRET signal was calculated as the difference in the ratio between emission at 510 and 395 nm of cotransfected Rluc and GFP2 fusion proteins and the ratio between emission at 510 and 395 nm of the Rluc fusion protein alone.
Immunofluorescence and Confocal MicroscopyHEK293 cells were maintained in high glucose Dulbecco's modified Eagle's medium containing 10% fetal bovine serum, 2 mM glutamine, 0.1 mM nonessential amino acids, 1 mM sodium pyruvate, 100 units/ml penicillin, and 100 µg/ml streptomycin. Semiconfluent cells were transfected with different combinations of D1 receptor, NR1-GFP2, and NR2B cDNAs using LipofectAMINE 2000 reagent (Invitrogen). Twenty-four hours after transfection, cells were plated onto poly-L-lysine-coated coverslips, fixed in 4% paraformaldehyde for 20 min at room temperature, and permeabilized with 0.1% Triton X-100 in PBS containing 5% bovine serum albumin and 5% normal goat serum for 10 min at room temperature. Cells were incubated overnight at 4 °C with either the rat monoclonal anti-D1 receptor antibody (1:600 dilution in PBS containing 1% normal goat serum) or the anti-PDI antibody (1:400 dilution in PBS containing 1% normal goat serum) and then for 45 min at room temperature with the Cy3-conjugated anti-goat secondary antibody (1:1000 dilution). The immunolabeled cells were recorded with a Bio-Rad laser scanning confocal microscope. Untransfected cells and omission of the primary antibodies were used as negative controls.
Sequestration AssayHEK293 cells, which spontaneously express different G protein-coupled receptor kinases and arrestin (25), were transfected with the D1 receptor in the absence or presence of NR1 and NR2B subunits using the LipofectAMINE 2000 method, plated onto poly-L-lysine-coated glass coverslips, and allowed to recover for 1 day. Cells were incubated for 1 h at 37 °C in the absence or presence of 10 µM SKF-81297 and processed as described above for confocal microscopy detection of the D1 receptor.
Membrane Preparation and [3H]SCH23390 BindingCells were rinsed, harvested, and centrifuged at 100 x g for 10 min. Cells were homogenized with a Polytron homogenizer in 5 mM Tris-HCl containing 2 mM EDTA and a mixture of protease inhibitors (pH 7.8) and centrifuged at 80 x g for 10 min to pellet unbroken cells and nuclei. The supernatant was centrifuged at 30,000 x g for 20 min at 4 °C. The resulting pellet was resuspended in 50 mM Tris-HCl containing 5 mM MgCl2, 1 mM EGTA, and the protease inhibitors (pH 7.8), layered on a 35% sucrose cushion, and centrifuged at 150,000 x g for 90 min to separate the light vesicular and heavy membrane fractions as described by Lamey et al. (26). The heavy fraction, at the bottom of the sucrose cushion, was resuspended in 50 mM Tris-HCl containing 5 mM EDTA, 1.5 mM CaCl2, 5 mM MgCl2, 5 mM KCl, and 120 mM NaCl (pH 7.4) and used for binding assay. Protein concentration was determined according to Lowry et al. (27) using DC protein assay reagent (Bio-Rad, Milano). Aliquots of membrane suspension (50 µg of protein/sample) were incubated at room temperature for 90 min with a saturating concentration (4 nM) of [3H]SCH-23390 (86 Ci/mmol; Perkin-Elmer Life Sciences). Nonspecific binding was defined with 1 µM D-butaclamol. The reaction was stopped by rapid filtration under reduced pressure through Whatman GF/C filters.
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RESULTS |
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Pull-out experiments were then performed with GST fusion proteins containing the C-terminal domains of both the D1 receptor and NR1 subunit. Striatal TIF proteins were incubated with GST fusion proteins containing the D1 receptor C-terminal tail or, as a control, the D5 receptor C terminus. D1 and D5 receptors display, in fact, particular sequence divergence within the C-terminal domain, a region that might confer subtype-selective properties (29). As shown in Fig. 2A, a 116-kDa species, detected by the anti-NR1 antibody, was pulled out from striatal TIF by GST-D1-CT-(321446) (lane 3), but not by GST-D5-CT-(373477) (lane 4) or GST alone (lane 2). By contrast, the NR2A/B subunits that were present in our TIF preparation (lane 1) did not interact with GST-D1-CT-(321446), indicating that, in striatal PSD, the D1 receptor selectively complexes with the NR1 subunit of the NMDA channel through its C-terminal tail. To identify the NR1 region responsible for this interaction, GST fusion proteins containing two different domains of the NR1 C-terminal tail were constructed. As shown in Fig. 2B, GST-NR1-CT-(834930), encoding the entire C-terminal region of the NR1a/b isoforms, was able to pull-out luciferase-tagged D1 receptors from solubilized membrane preparations obtained from transfected HEK293 cells. This activity was still present when the region downstream of the alternatively spliced C1 domain in the NR1 subunit C terminus was deleted, suggesting that both NR1a/b and NR1e/f isoforms may potentially interact with the D1 receptor.
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D1 and NMDA Receptors Constitutively Interact in Living CellsBRET is a newly developed biophysical approach that detects energy transfer between a luminescent donor and a fluorescent acceptor when they are <5080 Å apart. To evaluate whether D1 and NMDA receptors could exist as oligomers in living cells, we used an improved BRET technology (BRET2, PerkinElmer Life Sciences) that takes advantage of the properties of a particular luciferase substrate, DeepBlueC coelenterazine, which allows a spectral resolution between the Rluc and GFP2 emissions at 105 nm, a characteristic that confers high sensitivity to the assay. For this purpose, the D1 receptor was fused to Renilla luciferase, and the NR1 subunit of the NMDA receptor was fused to GFP2. The kinetic and transduction properties of these fusion receptors were superimposable with those of their wild-type counterparts (data not shown). BRET2 signals were determined in COS-7 cells simultaneously or individually expressing the D1-Rluc and NR1-GFP2 constructs. As shown in Fig. 3A, no BRET2 was observed in cells expressing only NR1-GFP2, and a negligible nonspecific signal was detected in cells expressing only the D1-Rluc construct. A significant BRET2 signal was observed in cells expressing a fusion construct covalently linking Rluc to GFP2 (pRluc-GFP2), confirming the importance of molecular proximity between the BRET partners for signal detection. Coexpression of the tagged D1 receptor and NR1 subunit yielded a BRET2 ratio that was significantly higher than that observed with cells expressing D1-Rluc alone or with cells individually expressing D1-Rluc and NR1-GFP2 and mixed before analysis. The specificity of this interaction is illustrated by the absence of significant energy transfer between the D1-Rluc construct and the pGFP2-N2(h) vector (Fig. 3A). This BRET2 ratio was unchanged when the NR2B subunit of the NMDA receptor was also expressed, suggesting that there is no competition between NR1 and NR2B for interaction with the D1 receptor. Moreover as shown in Fig. 3B, the BRET2 signal recorded in cells cotransfected with D1-Rluc, NR1-GFP2, and NR2B was insensitive to stimulation by 50 µM dopamine with or without 100 µM glutamate and 10 µM glycine. These data demonstrate a physical proximity between D1-Rluc and NR1-GFP2 that can be explained best by the formation of constitutive protein dimers.
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Oligomerization with the NMDA Receptor Regulates D1 Receptor Targeting to the Plasma MembraneTo identify the cellular compartment in which the D1 receptor and NR1 subunit are assembled, HEK293 cells transfected with the D1 receptor and NR1-GFP2 construct, either individually or simultaneously, were analyzed for receptor distribution by confocal microscopy. As shown in Fig. 4a, the D1 receptor expressed in HEK293 cells was completely targeted to the plasma membrane. By contrast, as previously reported (19, 30), when expressed alone, the NR1 subunit accumulated in the perinuclear region and in cytoplasmic compartments with a reticular staining pattern (Fig. 4d) that was identified as the ER using an antibody to PDI, a specific marker for this structure (Fig. 4e). Virtually all the intracellular NR1 staining was in fact co-localized with PDI (Fig. 4f). When the D1 receptor and NR1 subunit were coexpressed in the same cells, the D1 receptor was only partially targeted to the cell membrane (Fig. 4, h and i), with the majority of D1 receptor staining retained in cytoplasmic structures (Fig. 4h), where it was co-localized with NR1 (Fig. 4, g and i). Coexpression of the D1 receptor with both the NR1 and NR2B subunits relieved the cytoplasmic retention of the complex, allowing insertion of both the NR1 subunit (Fig. 4l) and D1 receptor (Fig. 4m) at the plasma membrane, where they were completely co-localized (Fig. 4n). These data suggest that D1 and NMDA receptors are assembled as oligomeric units in the ER and transported to the cell surface as a preformed complex.
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Oligomerization with the NMDA Receptor Abolishes Agonist-mediated D1 Receptor SequestrationA common adaptive response of G protein-coupled receptors to agonist stimulation is redistribution from the plasma membrane to cytosolic compartments. Using confocal microscopy and receptor binding in transfected HEK293 cells, which spontaneously express different G protein-coupled receptor kinases and -arrestin (25), we investigated whether interaction with NMDA receptors alters D1 receptor sequestration induced by agonist administration (26, 31). As shown in Fig. 5A, in unstimulated cells, the fluorescence distribution of the D1 receptor was exclusively localized at the plasma membrane (panel a). Exposure to 10 µM SKF-81297 for 1 h resulted in D1 receptor sequestration into cytosolic compartments, as shown by the D1 receptor fluorescence that was detectable also in the cytoplasm with a punctate appearance (panel b). In contrast, when the D1 receptor was coexpressed with NR1 and NR2B subunits, SKF-81297 failed to induce D1 receptor internalization. Under these conditions, D1 receptor immunofluorescence was in fact retained at the plasma membrane (panel c). Similar results were obtained by [3H]SCH23390 binding in the purified heavy membrane fraction. As shown in Fig. 5 (B and C), pretreatment with 10 µM SKF-81297 resulted in 20 ± 2.8% reduction of cell-surface [3H]SCH23390 binding in HEK293 cells expressing only the D1 receptor. On the other hand, exposure to 10 µM SKF-81297 did not modify cell-surface [3H]SCH-23390 binding in cells expressing both the D1 receptor and NR1 and NR2B subunits (Fig. 5, B and C). The dose-response curve and the time course of SKF-81297-induced D1 receptor sequestration in HEK293 cells expressing the D1 receptor either alone or in combination with NR1 and NR2B subunits are shown in Fig. 6. The SKF-81297-induced decrease in membrane [3H]SCH-23390 binding was dose-dependent, with an EC50 of 80 ± 2 nM in cells expressing the D1 receptor, but not in those coexpressing also the NR1 and NR2B subunits (Fig. 6A). Moreover, in cells expressing only the D1 receptor, SKF-81297-induced receptor internalization was detectable after 10 min of incubation and reached a maximum within 30 min (Fig. 6B). By contrast, in cells coexpressing the D1 receptor and the NR1 and NR2B subunits, no decrease in membrane [3H]SCH-23390 binding was detectable at any time tested. Increasing SKF-81297 incubation to 2 h did not modify [3H]SCH-23390 binding as well (data not shown). Taken together, these data suggest that interaction with the NMDA receptor immobilizes the D1 receptor at the plasma membrane, impairing the mechanisms of the receptor plasticity that normally occurs as an adaptive response to agonist stimulation.
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DISCUSSION |
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Using classical biochemical approaches, we have clearly shown that the D1 receptor is concentrated in purified striatal PSD, displaying a subcellular distribution that is consistent with the reported localization of NMDA receptors (17, 18). In addition, the D1 receptor was co-immunoprecipitated from striatal PSD with NMDA receptor subunits, suggesting that these proteins are co-clustered in this structure. Recently, energy transfer approaches such as fluorescence resonance energy transfer and BRET have been developed as the systems of choice to study protein-protein interactions (33). These techniques have the advantage of monitoring protein oligomerization in living cells without disrupting the natural environment where they are clustered, thus eliminating the possibility of artifactual aggregation that could happen during the solubilization and concentration of membrane proteins. Using BRET2, we have demonstrated that D1 and NMDA receptor clustering reflects the existence of direct protein-protein binding. In fact, the tagged D1 receptor and NR1 subunit generated a significant and specific BRET2 signal for energy transfer when cotransfected in COS-7 cells. This signal did not change when the NR2B subunit was also expressed in the same cells, suggesting that this subunit does not compete with the NR1 subunit for binding to the D1 receptor. In addition, the association of the NR1 subunit with the D1 receptor was insensitive to agonist stimulation. Taken together, these observations point to a constitutive, direct, and selective interaction of the D1 receptor with the NR1 subunit of the NMDA channel. Using specific GST fusion proteins, we have also shown that the interaction between D1 and NMDA receptors involves the binding of the D1 receptor C-terminal tail to the C-terminal sequence of the NR1a/b and NR1e/f isoforms, with no contribution from NR2 subunits. The NR1 subunit, the essential component of the NMDA receptor, gives rise to eight splice variants, with four possible C termini (34, 35). These isoforms differ in their physiological and pharmacological properties and show different regional and cellular distribution (34, 36). Our present data point to the capability of interacting with the D1 receptor as a further difference among these isoforms and suggest that the interaction between D1 and NMDA receptors might be a specific feature of certain neuronal populations. In line with our findings, it was reported, while this manuscript was in preparation, that D1 and NMDA receptors directly interact in the hippocampus (37). In particular, in this brain area, the D1 receptor apparently associates with both the NR1 and NR2A subunits, but not with the NR2B subunit. Our observation that the D1 receptor does not interact with NR2 subunits in striatal PSD may reflect the fact that NR2B is the prevalent species in this structure (36).
Oligomerization may play important roles in receptor trafficking and/or signaling. In several cases, receptors appear to fold as constitutive dimers early after biosynthesis, whereas ligand-promoted dimerization at the cell surface has been proposed for others (33). Our data obtained by BRET showing that the D1 receptor and NR1 subunit interact in the absence of the NR2B subunit and in an agonist-independent way suggest that this interaction is constitutive. The results obtained by confocal microscopy give support to this concept and indicate that the trafficking properties of the D1 receptor are substantially modified by heteromerization with the NMDA receptor. When the NR1 subunit and D1 receptor were individually transfected in HEK293 cells, NR1 was retained in the ER, whereas the D1 receptor was targeted to the plasma membrane. In cotransfected cells, both the D1 receptor and NR1 subunit were co-localized in cytoplasmic compartments, suggesting that interaction with NR1 blocks D1 receptor delivery to the plasma membrane. In the presence of the NR2B subunit, however, the NR1-D1 receptor complex was completely translocated to the plasma membrane. These observations are consistent with previous data showing that, when expressed alone in both heterologous cells and cultured hippocampal neurons, the NR1 subunit accumulates in the ER (19, 30) due to the presence of an ER retention motif in the alternatively spliced C1 domain in its C terminus (38) and that coexpression of NR2 subunits is necessary to drive the complex to the cell membrane (19, 30). Taken together, these data suggest that, in striatal medium spiny neurons, D1 and NMDA receptors are assembled within intracellular compartments as constitutive heteromeric complexes that are delivered to functional sites. Interaction with the NMDA receptor thus represents a critical mechanism to recruit the D1 receptor to the PSD. The postsynaptic specialization of corticostriatal glutamatergic synapses finely regulates the strength of synaptic transmission, thus determining the activity of medium spiny neurons. Several lines of evidence suggest that the efficacy of corticostriatal transmission is highly dependent on the concurrent activation of D1 and NMDA receptors. In particular, it has been shown that NMDA currents are potentiated by activation of D1 receptors, which is also an essential requirement for long-term potentiation generation (2, 9, 10, 11, 12). In this context, the direct interaction between D1 and NMDA receptors may be crucial to recruit the D1 receptor in the place of synaptic plasticity and to keep it in close proximity with the NMDA receptor to allow rapid cAMP/protein kinase A/DARPP32-mediated potentiation of NMDA transmission (39, 40, 41, 42).
The interaction of the D1 receptor with the NR1 subunit does not reflect simply a chaperon-like strategy to deliver the D1 receptor to the PSD, but also implies regulation of D1 receptor function by interfering with the mechanisms of receptor plasticity. A common adaptive response of G protein-coupled receptors to agonist stimulation is desensitization involving both G protein-coupled receptor kinase-mediated phosphorylation and arrestin binding and internalization (43). In line with this paradigm and with in vitro studies (26, 31), there is morphological evidence that, in striatal medium spiny neurons, extra-synaptic D1 receptors, localized in cell bodies and dendrites, respond to agonist administration by massive internalization (32). We have shown here that association with the NMDA receptor abolishes agonist-induced D1 receptor cytoplasmic sequestration, indicating that oligomerization with NMDA receptors could represent a novel regulatory mechanism modulating D1 receptor function. Taken together, these observations suggest that, within a single neuron, D1 receptor plasticity may be subjected to different regulatory mechanisms in different neuronal microdomains. In particular, agonist stimulation would induce D1 receptor sequestration in all neuronal compartments except the PSD, where this receptor is immobilized at the plasma membrane by association with the NMDA receptor. Along this line, Dumartin et al. (32) have reported that, in striatal medium spiny neurons, the localization of the perisynaptic D1 receptor in dendritic spines is apparently unmodified by agonist treatment. It is well known that agonist-induced internalization dynamically calibrates receptor availability for extracellular ligands. Disruption of D1 receptor cytoplasmic sequestration in response to agonist stimulation due to heteromerization with the NMDA receptor might represent a neuronal mechanism to preserve the optimal synaptic strength at corticostriatal synapses in the presence of alterations in the dopamine environment as occurs, for instance, during drug administration. In conclusion, our present data suggesting that, in striatal medium spiny neurons, D1 and NMDA receptors are assembled within intracellular compartments and recruited to the PSD as a constitutive heteromeric complex may provide a new rationale for a better understanding of the mechanisms that control corticostriatal synaptic transmission under both physiological and pathological conditions.
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FOOTNOTES |
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¶ To whom correspondence should be addressed. Tel.: 39-03-03-371-7518; Fax: 39-03-03-371-7529; E-mail: cmissale{at}med.unibs.it.
1 The abbreviations used are: NMDA, N-methyl-D-aspartate; PSD, postsynaptic densities; ER, endoplasmic reticulum; PDI, protein disulfide isomerase; TIF, Triton-insoluble fraction; GST, glutathione S-transferase; PBS, phosphate-buffered saline; BRET, bioluminescence resonance energy transfer; Rluc, Renilla luciferase; GFP, green fluorescent protein.
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ACKNOWLEDGMENTS |
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REFERENCES |
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