Title: Regulation of Dopamine D1 Receptor Trafficking and Desensitization by Oligomerization with Glutamate N-Methyl-D-aspartate Receptors
Abstract: 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. 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. Dopaminergic fibers originating in the substantia nigra and cortical glutamatergic neurons extensively interact in the striatum to drive the physiological functions of this structure from motor planning to reward seeking and procedural learning (1Berke J.D. Hyman S.E. Neuron. 2000; 25: 515-532Abstract Full Text Full Text PDF PubMed Scopus (983) Google Scholar, 2Nicola S.M. Surmeier J. Malenka R.C. Annu. Rev. Neurosci. 2000; 23: 185-215Crossref PubMed Scopus (747) Google Scholar). The critical importance of dopamine in this system is such that the degeneration of nigral dopaminergic neurons leads to the motor and cognitive deficits of Parkinson's disease (3Olanow C.W. Obeso A.J. Nutt J.G. Trends Neurosci. 2000; 23: S1-S126PubMed Google Scholar).At the cellular level, nigral and cortical fibers converge on the medium spiny projection neurons (4Smith A.D. Bolam J.P. Trends Neurosci. 1990; 13: 259-265Abstract Full Text PDF PubMed Scopus (859) Google Scholar), where dopamine D1- and D2-like receptors are coexpressed to high degree with glutamate NMDA 1The 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.1The 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. and non-NMDA receptor channels (5Surmeier D.J. Song W.J. Yan Z. J. 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In fact, activation of D1 receptors in medium spiny neurons enhances NMDA-induced whole cell currents (2Nicola S.M. Surmeier J. Malenka R.C. Annu. Rev. Neurosci. 2000; 23: 185-215Crossref PubMed Scopus (747) Google Scholar, 9Levine M.S. Altemus K.L. Cepeda C. Cromwell H.C. Crawford C. Ariano M.A. Drago J. Sibley D.R. Westphal H. J. Neurosci. 1996; 16: 5870-5882Crossref PubMed Google Scholar) and is a critical requirement for the formation of NMDA-mediated long-term potentiation at corticostriatal synapses (2Nicola S.M. Surmeier J. Malenka R.C. Annu. Rev. Neurosci. 2000; 23: 185-215Crossref PubMed Scopus (747) Google Scholar, 10Calabresi P. Pisani A. Mercuri N.B. Bernardi G. Trends Neurosci. 1996; 19: 19-24Abstract Full Text Full Text PDF PubMed Scopus (405) Google Scholar, 11Centonze D. Gubellini P. Picconi B. Calabresi P. Giacomini P. Bernardi G. J. Neurophysiol. 1999; 82: 3575-3579Crossref PubMed Scopus (168) Google Scholar, 12Kerr J.N.D. Wickens J.R. J. Neurophysiol. 2001; 85: 117-124Crossref PubMed Scopus (278) Google Scholar). Moreover, activation of NMDA receptors in striatal neurons triggers the translocation of cytoplasmic D1 receptors to the plasma membrane and spines (13Scott L. Kruse M.S. Forssberg H. Brismar H. Greengard P. Aperia A. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 1661-1664Crossref PubMed Scopus (128) Google Scholar). 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) (14Hersch S.M. Ciliax B.J. Gutekunst C.A. Rees H.D. Heilman C.J. Yung K.K. Bolam J.P. Ince E. Yi H. Levey A.I. J. Neurosci. 1995; 13: 2237-2248Google Scholar, 15Yung K.K. Bolam J.P. Smith A.D. Hersch S.M. Ciliax B.J. Levey A.I. Neuroscience. 1995; 65: 709-730Crossref PubMed Scopus (446) Google Scholar, 16Bergson C. Mrzljak L. Smiley J.F. Pappy M. Levenson R. Goldman-Rakic P.S. J. Neurosci. 1995; 15: 7821-7836Crossref PubMed Google Scholar). 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 (17Kennedy M.B. Science. 2000; 290: 750-754Crossref PubMed Scopus (652) Google Scholar, 18Sheng M. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 7058-7061Crossref PubMed Scopus (284) Google Scholar). 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 (13Scott L. Kruse M.S. Forssberg H. Brismar H. Greengard P. Aperia A. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 1661-1664Crossref PubMed Scopus (128) Google Scholar, 19Barria A. Malinow R. Neuron. 2002; 35: 345-353Abstract Full Text Full Text PDF PubMed Scopus (406) Google Scholar) 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.EXPERIMENTAL PROCEDURESMaterials—Human embryonic kidney cells (HEK293) were provided by Deutsche Sammlung von Mikroorganismen und Zellculturen GmbH (Braunschweg, Germany). Tissue culture media and fetal bovine serum were obtained from Euroclone Celbio (Milano, Italy). Dopamine, glutamate, d-butaclamol, SKF-81297, and the rat monoclonal anti-D1 receptor antibody (clone 1-1-F11-S.E6) were purchased from Sigma. Glycine was obtained from Tocris (Avonmouth, UK). The rabbit anti-PDI antibody was from Stressgen Biotech Corp. (Victoria, British Columbia, Canada). Cy3-labeled anti-rat and anti-rabbit secondary antibodies were from Jackson ImmunoResearch Laboratories, Inc. (West Grove, PA). The anti-NR1 and anti-NR2A/B and mouse monoclonal anti-D1 receptor antibodies were from Chemicon International (Temecula, CA). The horseradish peroxidase-conjugated anti-mouse antibody was purchased from DAKO (Milano), and the horseradish peroxidase-conjugated anti-rabbit antibody was from Santa Cruz Biotechnology (Heidelberg, Germany). Dr. Marc Caron (Duke University, Durham, NC) kindly provided D1 and D5 receptor cDNAs. Dr. Hannah Monyer (Heidelberg University) kindly provided the NR2B cDNA, and the NR1 cDNA was a gift of Dr. Shigetada Nakanishi (Kyoto University, Kyoto, Japan).PSD and Triton-insoluble Fraction Preparation—Striatal PSD were isolated according to Carlin et al. (20Carlin R.K. Grab D.J. Cohen R.S. Siekevitz P. J. Cell Biol. 1980; 86: 831-843Crossref PubMed Scopus (599) Google Scholar) with minor modifications as described previously (21Gardoni F. Caputi A. Cimino M. Pastorino L. Cattabeni F. Di Luca M. J. Neurochem. 1998; 71: 1733-1741Crossref PubMed Scopus (153) Google Scholar). 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 × g for 10 min. The supernatant was centrifuged at 3000 × 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 × 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 × 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 × 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 × 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 × g for 10 min. The resulting supernatant was centrifuged at 3000 × g for 15 min, and the pellet was resuspended in 1 mm Hepes and centrifuged at 100,000 × 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 × g for 1 h. TIF was characterized by enrichment in PSD proteins as previously described (22Caputi A. Gardoni F. Cimino M. Pastorino L. Cattabeni F. Di Luca M. Eur. J. Neurosci. 1999; 11: 141-148Crossref PubMed Scopus (28) Google Scholar).Immunoprecipitation and Western Blotting—Ten 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 Proteins—The C-terminal regions of the D1 receptor (D1-CT-(321–446)) and of the D5 receptor (D5-CT-(373–477)) and two fragments of the NR1 subunit C terminus (NR1-CT-(834–930) and NR1-CT-(834–892)) 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-β-d-thiogalactopyranoside (Sigma) for 2–4h. 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 (23Gardoni F. Schrama L.H. van Dalen J.J. Gispen W.H. Cattabeni F. Di Luca M. FEBS Lett. 1999; 456: 394-398Crossref PubMed Scopus (100) Google Scholar).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 Constructs—The 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 (24Missale C. Boroni F. Castelletti L. Dal Toso R. Gabellini N. Sigala S. Spano P. J. Biol. Chem. 1991; 266: 23392-23398Abstract Full Text PDF PubMed Google Scholar). 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 Assay—COS-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. DeepBlueC™ coelenterazine (PerkinElmer Life Sciences) was added at a final concentration of 5 μm, and BRET2 signals were determined using a Fusion™ 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 Microscopy—HEK293 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 Assay—HEK293 cells, which spontaneously express different G protein-coupled receptor kinases and arrestin (25Menard L. Ferguson S.S. Zhang J. Lin F.T. Lefkowitz R.J. Caron M.G. Barak L.S. Mol. Pharmacol. 1997; 51: 800-808Crossref PubMed Scopus (213) Google Scholar), 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 Binding—Cells were rinsed, harvested, and centrifuged at 100 × 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 × g for 10 min to pellet unbroken cells and nuclei. The supernatant was centrifuged at 30,000 × 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 × g for 90 min to separate the light vesicular and heavy membrane fractions as described by Lamey et al. (26Lamey M. Thompson M. Varghese G. Chi H. Sawzdargo M. George S.R. O'Dowd B.F. J. Biol. Chem. 2002; 277: 9415-9421Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). 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. (27Lowry O.H. Rosebrough N.J. Farr A.L. Randall R.J. J. Biol. Chem. 1951; 193: 265-275Abstract Full Text PDF PubMed Google Scholar) 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.RESULTSDopamine D1and Glutamate NMDA Receptors Are Co-clustered in Striatal PSD—Striatal PSD were isolated and analyzed for the presence of D1 receptors and other PSD-associated proteins. Fig. 1A shows the results from Western blot analysis performed with different tissue fractions with antibodies recognizing the D1 receptor, the NR1 subunit, α-Ca2+/calmodulin-dependent protein kinase II, and protein kinase Cϵ. As previously reported (28Gines S. Hillion J. Torvinen M. Le Crom S. Casado V. Canela E.I. Rondin S. Lew J.Y. Watson S. Zoli M. Agnati L.F. Vernier P. Lluis C. Ferré S. Fuxe K. Franco R. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 8606-8611Crossref PubMed Scopus (385) Google Scholar), immunoreaction with the anti-D1 receptor antibody revealed a major specific band of ∼50 kDa. This species was detectable in all tissue fractions, but was enriched in the PSD fraction. This pattern of D1 receptor distribution paralleled that of α-Ca2+/calmodulin-dependent protein kinase II and the NR1 subunit of the NMDA receptor, two known PSD constituents. The purification yield and the purity of our PSD fraction were confirmed by the absence of immunoreactivity for protein kinase Cϵ, a protein present exclusively in the presynaptic compartment. Co-immunoprecipitation studies were performed to evaluate whether D1 and NMDA receptors might interact in striatal PSD. As shown in Fig. 1B, a 50-kDa band, which was detected by the anti-D1 receptor antibody, was present in PSD proteins immunoprecipitated with the antibody raised against the NR1 subunit (lane 2). Similarly, the anti-D1 receptor antibody immunoprecipitated a 116-kDa band corresponding to NR1 (lane 3), indicating relevant complex formation between these two holoreceptor moieties in vivo. These bands did not appear when either an irrelevant antibody, such as that directed against protein kinase Cϵ (lane 4), was used or the precipitating antibody was omitted (lane 5).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 (29Missale C. Nash R. Robinson S.W. Jaber M. Caron M.G. Physiol. Rev. 1998; 78: 189-225Crossref PubMed Scopus (2700) Google Scholar). 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-(321–446) (lane 3), but not by GST-D5-CT-(373–477) (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-(321–446), 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-(834–930), 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.Fig. 2The D1 receptor and NR1 subunit interact through their C-terminal domains.A, fusion proteins of GST with the C-terminal domains of the D1 (GST-D1-CT-(321–446)) and the D5 (GST-D5-CT-(373–477)) receptors bound to glutathione-agarose beads were incubated with 35 μg of striatal TIF, and proteins that were pulled out were immunoreacted with the anti-NR1 and anti-NR2A/B antibodies. The D1 receptor C-terminal tail (lane 3), but not the D5 receptor C-terminal tail (lane 4), was able to bind the NR1 subunit, but not the NR2A/B subunits. B, fusion proteins of GST with two different fragments of the NR1 subunit C terminus (GST-NR1-CT-(834–930) and GST-NR1-CT-(834–930)) were incubated with membranes obtained from HEK293 cells expressing the D1 receptor fused to luciferase (D1-Rluc). After extensive washing, the glutathione-agarose beads containing the pulled out proteins were assayed for luciferase activity using DeepBlueC coelenterazine as a substrate. Both NR1 C-terminal fragments were able to bind the D1 receptor. Bars represent the means ± S.E. of three experiments. *, p < 0.001 versus GST (Student' t test). WB, Western blot; RLU, relative light units.View Large Image Figure ViewerDownload Hi-res image Download (PPT)D1and NMDA Receptors Constitutively Interact in Living Cells—BRET is a newly developed biophysical approach that detects energy transfer between a luminescent donor and a fluorescent acceptor when they are <50–80 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.Fig. 3Detection D1 receptor and NR1 subunit interaction by BRET2. The D1 receptor fused to luciferase (D1-Rluc) and the NR1 subunit fused to GFP2 (NR1-GFP2) were transfected either individually or simultaneously in COS-7 cells. The DeepBlueC coelenterazine substrate was added at a final concentration of 5 μm, and BRET2 signals were determined using the Fusion™ universal microplate analyzer. A, quantification of BRET2 data (means ± S.E., n = 5) from a series of control experiments with a single receptor construct (D1-Rluc, NR1-GFP2, and pRluc-GFP2) and of BRET2 data obtained when both D1-Rluc (donor) and NR1-GFP2 (acceptor) were coexpressed in the same cells (D1-Rluc/NR1-GFP2). D1-Rluc and NR1-GFP2 coexpressed in COS-7 cells