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Previous Article | Next Article
The Journal of Neuroscience, February 15, 2001, 21(4):1228-1237
Assembly with the NR1 Subunit Is Required for Surface Expression of NR3A-Containing NMDA Receptors
Isabel Pérez-Otaño1, Christine T. Schulteis1, Anis Contractor1, Stuart A. Lipton2, James S. Trimmer3, Nikolaus J. Sucher4, and Stephen F. Heinemann1 1 Molecular Neurobiology Laboratory, The Salk Institute for Biological Studies, La Jolla, California 92037, 2 Center for Neuroscience and Aging, The Burnham Institute, La Jolla, California 92037, 3 Department of Biochemistry and Cell Biology, State University of New York, Stony Brook, New York 11794, and 4 Department of Biology, Hong Kong University of Science and Technology, Hong Kong, China
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
Functional NMDA receptors are heteromultimeric complexes of the NR1 subunit in combination with at least one of the four NR2 subunits (A-D). Coexpression of NR3A, an additional subunit of the NMDA receptor family, modifies NMDA-mediated responses. It is unclear whether NR3A interacts directly with NR1 and/or NR2 subunits and how such association might regulate the intracellular trafficking and membrane expression of NR3A. Here we show that NR3A coassembles with NR1-1a and NR2A to form a receptor complex with distinct single-channel properties and a reduced relative calcium permeability. NR3A associates independently with both NR1-1a and NR2A in the endoplasmic reticulum, but only heteromeric complexes containing the NR1-1a NMDA receptor subunit are targeted to the plasma membrane. Homomeric NR3A complexes or complexes composed of NR2A and NR3A were not detected on the cell surface and are retained in the endoplasmic reticulum. Coexpression of NR1-1a facilitates the surface expression of NR3A-containing receptors, reduces the accumulation of NR3A subunits in the endoplasmic reticulum, and induces the appearance of intracellular clusters where both subunits are colocalized. Our data demonstrate a role for subunit oligomerization and specifically assembly with the NR1 subunit in the trafficking and plasma membrane targeting of the receptor complex.
Key words: NMDA receptor; glutamate; calcium permeability; single channel; surface expression; assembly; NR3A
INTRODUCTION
Glutamate receptors of the NMDA subtype are involved in a number of physiological and pathological processes in the brain, including synaptic plasticity, refinement of synaptic connections during development, and excitotoxicity (Choi, 1988
; Constantine-Paton et al., 1990
Coassembly between the various NR1 and NR2 subunits generates functionally distinct types of NMDA receptors. Incorporation of different NR1 splice variants into NMDA receptor complexes influences receptor properties such as modulation by zinc, polyamines, and protein kinase C, as well as binding to intracellular proteins (Durand et al., 1993
An additional NMDA receptor subunit, NR3A, has been identified in mammalian brains. Coexpression of NR3A with both NR1 and NR2 in Xenopus oocytes reduces whole-cell currents and results in the appearance of NMDA-gated channels with a smaller unitary conductance, compared with receptors assembled from NR1 and NR2 subunits (Das et al., 1998
To address these issues, we have analyzed the ability of NR3A to interact with other NMDA receptor subunits and the role that such assembly plays in the trafficking of functional NR3A-containing receptors. We present biochemical evidence that NR3A first assembles with the other NMDA receptor subunits in the endoplasmic reticulum (ER) and can independently associate with either NR1 or NR2 subunits. However, only heteromeric complexes including the NR1-1a subunit are targeted to and properly inserted at the plasma membrane. In the absence of NR1-1a, NR3A subunits remain in the ER and are unable to access the cell surface. These results suggest that subunit assembly and subsequent ER export of properly assembled complexes constitute a key mechanism to control the plasma membrane targeting of NMDA receptors with defined subunit composition.
MATERIALS AND METHODS
Construction of NMDA receptor expression vectors. cDNAs encoding the rat NR1-1a and NR2A NMDA receptor subunits were subcloned into the pcDNA1-Amp mammalian expression vector (Invitrogen, Carlsbad, CA). For expression in mammalian cells, the rat NR3A cDNA was subcloned into pCINeo (Promega, Madison, WI). To allow the visualization of the NR3A subunit, the enchanced green fluorescent protein (EGFP) (Clontech, Palo Alto, CA) was inserted in-frame at the N terminus of NR3A after the predicted signal peptide (residue 33).
Electrophysiological recordings. Calcium phosphate coprecipitation was used to transfect human embryonic kidney 293 (HEK293) cells transiently with plasmids containing the rat NMDA subunits and a plasmid carrying the coding sequence for the CD8 surface antigen (generously supplied by Dr. B. Seed, Massachusetts General Hospital, Boston, MA). For all experiments, NR1-1a and NR2A were coexpressed with the NR3A subunit at a ratio of 1:2:2. Two to 3 d after transfection, cells were labeled with anti-CD8 antibody-coated beads (Dynal, Great Neck, NY), transferred to a recording chamber, and continuously perfused with HEPES-buffered extracellular solution, pH 7.4, containing (in mM): NaCl 135, KCl 5, CaCl2 1, sucrose 20, glucose 10, and HEPES 5. All chemicals were from Sigma (St. Louis, MO) unless otherwise stated. Whole-cell recordings were made from cells labeled with CD8-coated beads. The pipette solution for whole-cell recording contained (in mM): gluconolactone 140, NaOH 140, HEPES 10, EGTA 11, and NaCl 10. NMDA (100 µM; Research Biochemicals, Natick, MA) and glycine (20 µM) were bath applied in an external solution containing either 1 or 10 mM Ca2+. Current-voltage (I-V) relationships were constructed by performing voltage ramps generated by pClamp6 software (Axon Instruments, Foster City, CA). Each trial consisted of three 4 sec ramps from
100 to +40 mV. Ramps were performed before, during, and after drug application, and each set was averaged. The net I-V plot was constructed by subtracting the averages of the trials before and after drug application from the average during drug application. After first correcting for the liquid junction potentials (Neher, 1992
where E1 and E2 are reversal potentials measured in the presence of the Ca2+ concentrations Ca2+1 and Ca2+2, R is the Gas constant, T is the absolute temperature, and F is the Faraday constant.
Single-channel recordings from HEK293 cells. For single-channel recordings, HEK293 cells were perfused with HEPES-buffered extracellular solution containing 1 mM Ca2+. Sylgard (Dow Corning, Midland, MI) resin-coated electrodes with a tip resistance of 15-20 M
were used to make outside-out patch-clamp recordings, and channel openings were induced by application of NMDA and glycine into the bath.
Single-channel data analysis. Data records were stored on digital audiotape, filtered at 1-2 kHz, and continuously sampled at 10-20 kHz onto a computer using a 1401 plus interface (Cambridge Electronic Design, Cambridge, UK). The records were analyzed using SCAN, a time course fitting program, kindly provided by David Colquhoun (University College London, London, UK), and distributions were constructed of the current amplitudes and open times (Colquhoun and Sigworth, 1995
Immunoprecipitation and immunoblotting. NMDA receptor subunits were heterologously expressed in HEK293T cells [formerly TsA201 cells (Dubridge et al., 1987
Immunoprecipitated proteins were subjected to SDS-PAGE on 7.5% separating gels with 4% stacking gels. Proteins were transferred to Immobilon P membranes (Millipore, Bedford, MA) and immunoblotted as described previously (Stern-Bach et al., 1994
Metabolic labeling experiments. At 36-48 hr after transfection, transfected HEK293T cells were incubated for 30 min in methionine- and cysteine-free DMEM (Mediatech, Herndon, VA) and subsequently pulsed for 20 min with 200 µCi/ml 35S-labeled methionine (EasyTag Express Protein Labeling Mix; DuPont NEN, Boston, MA). Cells were then washed in PBS and chased in complete media, which in some cases was supplemented with 5 mM methionine. After the chase, the cells were washed in ice-cold PBS and lysed by the addition of cold RIPA buffer (10 mM phosphate buffer, pH 7.4, 150 mM NaCl, 1% deoxycholate, 1% NP-40, and 0.1% SDS). Insoluble material was removed by a 15 min spin at 15,000 rpm, and the resulting samples were subjected to immunoprecipitation, SDS-PAGE (as described above), and autoradiography.
Brefeldin A (BFA) and nocodazole treatment was performed as described previously (Nagaya and Papazian, 1997
Cell surface biotinylation. At 48 hr after transfection, transfected HEK293T cells were washed in borate buffer (100 mM NaCl, and 10 mM borate, pH 8.8) and incubated in borate buffer with 0.05 mg/ml sulfo-NHS-biotin (Pierce, Rockford, IL). After 10 min, fresh reagent was added, and the cells were incubated an additional 10 min. The reaction was quenched with 15 mM NH4Cl. The cells were washed in 100 mM NaCl and 50 mM Tris-HCl, pH 7.5 and lysed in 1% deoxycholate, and insoluble material was removed by centrifugation at 15,000 rpm for 15 min. Biotinylated protein was precipitated in a 2 hr incubation in streptavidin-agarose beads (Sigma). The beads were washed extensively and resuspended in Laemmli SDS sample buffer before electrophoresis and immunoblotting.
Cell surface localization studies using GFP-tagged NR3A. Different combinations of NR1-1a, NR2A, and GFP-tagged NR3A were expressed in HEK293T cells. At 48 hr after transfection, cells were incubated with the cell surface marker Concanavalin A conjugated to tetramethylrhodamine (100 µg/ml; Molecular Probes, Eugene, OR) for 5 min at 37°C, washed twice with ice-cold PBS, and fixed with 4% paraformaldehyde for 5 min. Coverslips were mounted in SlowFade mounting medium (Molecular Probes). The cells were then observed with an LSM 510 Zeiss laser-scanning confocal microscope using a 40× oil immersion lens and a pinhole of 112. GFP fluorescence was excited using a 488 nm argon/krypton laser, and emitted fluorescence was detected with a 515-540 nm bandpass filter. For detection of tetramethylrhodamine-conjugated Concanavalin A, a 568 nm argon/krypton laser was used for excitation, and fluorescence was detected with a 590 nm bandpass filter. There was no bleed through of fluorescence between the channels under the conditions used for these experiments. All observations were made blind. The acquired images were analyzed using Zeiss KS300 software, and masks of the overlapping red and green fluorescence were generated. Fluorescence pixels of the total green fluorescence (total receptor expression) and of green fluorescence in the colocalization mask (receptor at the cell surface) were measured. The relative percentage of the receptor on the cell surface was estimated as the ratio of green fluorescence pixels at the colocalization mask and total green fluorescence.
Immunocytochemistry. At 48 hr after transfection, immunofluorescence was performed in nonpermeabilized HEK293T cells to detect surface expression of NMDA receptors. The cells were fixed with ice-cold 4% paraformaldehyde in PBS for 10 min. After blocking with 5% bovine serum albumin in PBS for 30 min, cells were incubated with the monoclonal anti-NR1 antibody, which reacts with the extracellular loop between transmembrane domains III and IV (1:500 dilution; PharMingen), and rabbit (1:3000; Clontech) or chicken (1:3000; Chemicon) anti-GFP antibody overnight at 4°C. After washing, the cells were incubated with indocarbocyanine (Cy3)-conjugated goat anti-mouse (1: 600 dilution; Jackson ImmunoResearch, West Grove, PA) and FITC-conjugated goat anti-rabbit (1:600 dilution; Vector Laboratories, Burlingame, CA) or Texas Red-conjugated goat anti-chicken (1:1000 dilution; Chemicon) secondary antibodies for 1 hr at room temperature. For immunostaining with rabbit anti-calreticulin (1:200) and mouse anti-Grp78 (1:200; Stressgen%20Biotechnologies">Stressgen Biotechnologies, Victoria, British Columbia, Canada) antibodies, cells were permeabilized with 0.1% Triton X-100 in PBS for 10 min before addition of the primary antibody. Biotinylated anti-rabbit secondary antibody (1:1000; Vector Laboratories) followed by avidin-Texas Red (1:1000; Vector Laboratories) or Cy3-conjugated anti-mouse antibody (1:600; Jackson ImmunoResearch) was used. Cells were then washed and mounted, and images were acquired using a Zeiss confocal microscope.
RESULTS
Coexpression of NR3A with NR1-1a and NR2A in HEK293 cells results in the appearance of two types of NMDA receptors
To test the ability of HEK293 cells to process functional NMDA receptors incorporating the NR3A subunit, we analyzed the electrophysiological properties of recombinant NMDA receptors in transiently transfected cells. NR3A did not form functional channels when expressed alone in HEK293 cells, as shown previously in Xenopus oocytes (Ciabarra et al., 1995
To determine whether the NR3A subunit was included in heteromeric complexes containing NR1-1a and NR2A subunits, we measured the single-channel properties of NMDA receptors in outside-out patches pulled from cells transfected with cDNAs encoding the NR1-1a, NR2A, and NR3A subunits. Two distinct channel types were observed after application of NMDA (0.5-5 µM) and glycine (0.5 µM) in patches from cells expressing all three subunits. These channels could be differentiated by their characteristic conductance states (Fig. 1A). One of the channel populations had a conductance of 47.9 ± 2.1 pS (mean ± SEM; n = 4;
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Figure 1. Functional properties of NR1-1a/NR2A/NR3A channels in HEK293 cells. A, Single-channel openings induced by application of 5 µM NMDA and 0.5 µM glycine to an outside-out patch held at , area; 1.9 msec, 12%; 12 msec, 88%). C, Open period histogram of the high conductance (47 pS) NR1-1a/NR2A channel. The histogram is fitted with the sum of two exponential components (
NMDA receptors containing the NR3A subunit display a low relative Ca2+ permeability
One critical feature of NMDA receptors is their high relative Ca2+ permeability. Because a previous report indicated that NMDA receptors containing NR3A expressed in oocytes may be less permeable to Ca2+ than heteromeric combinations of NR1 and NR2 subunits (Das et al., 1998
NR3A specifically coimmunoprecipitates with NR1-1a and NR2A
To test whether this second type of NMDA receptor channel was a result of NR3A forming stable complexes with other NMDA receptor subunits, we performed coimmunoprecipitation experiments. NR3A was coexpressed with NR1-1a, NR2A, or NR1-1a and NR2A in HEK293T cells. For immunodetection of the NR3A subunit, we used a monoclonal antibody directed against an extracellular epitope of NR3A located between the predicted transmembrane domains III and IV. Immunoblot analysis of crude lysates from cells transfected with NR3A resulted in a single band of ~130 kDa. This band was absent in mock-transfected cells and cells transfected with only NR1-1a and NR2A, demonstrating the specificity of the NR3A antibody (Fig. 2A, top blot). After coexpression with NR1-1a or NR1-1a and NR2A, a second, fainter band of higher molecular weight (~140 kDa) was observed. This upper band corresponds to a difference in N-linked glycosylation of the NR3A protein in the presence of NR1-1a, because treatment with N-glycosidase F makes both bands collapse into a single band of ~115 kDa, in agreement with the expected molecular size of the NR3A polypeptide (Fig. 2C).
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Figure 2. NR3A coimmunoprecipitates with the NMDA receptor subunits NR1-1a and NR2A. A, Crude lysates from mock-transfected HEK293T cells or cells transfected with the combinations of NMDA subunits indicated above each lane were immunoblotted with anti-NR3A, anti-NR1, or anti-NR2A/B antibodies to verify expression of the corresponding subunits after transfection. B, NMDAR subunits were immunoprecipitated from HEK293T cell lysates using anti-NR1 and anti-NR2A/B antibodies. Immunoprecipitates were immunoblotted for NR1, NR2A, and NR3A as indicated. Asterisks mark the bands corresponding to NR1-1a and NR3A. Additional bands derive from nonspecific binding to the secondary antibody because they are present when the primary antibody is excluded (data not shown). C, NR3A is differentially glycosylated in the presence of the NR1-1a subunit. Lysates of HEK293T cells transfected with NR1-1a/NR3A or NR1-1a/NR2A/NR3A were immunoprecipitated with anti-NR3A antibodies, treated with N-glycosidase F (N-Glyc F), or mock digested as indicated and analyzed by immunoblotting with anti-NR3A antibody. Arrowheads indicate the position of the glycosylated (top arrowheads) and deglycosylated (bottom arrowhead) forms of NR3A. Both differentially glycosylated forms behaved similarly in coimmunoprecipitation and biotinylation experiments. D, The specificity of coimmunoprecipitation was tested by coexpression of NR3A with non-NMDA glutamate receptors. NR3A was cotransfected with GluR1 and GluR2, and lysates were immunoprecipitated with anti-NR3A antibody. Immunoprecipitates were probed with anti-GluR1 or anti-GluR2/3 antibodies. Lanes labeled crude contain 2% of the cell lysate used for immunoprecipitation and demonstrate expression of NR3A, GluR1, and GluR2. The bands specific for NR3A, GluR1, and GluR2 are indicated. Positions of molecular size markers in kilodaltons are shown on the left. IP, Immunoprecipitate.
The NR1-1a and NR2A subunits were immunoprecipitated from transfected cell lysates using anti-NR1 and anti-NR2A/B antibodies. NR3A coimmunoprecipitated with both NR1-1a (116 kDa) and NR2A (180 kDa) when all three subunits were coexpressed (Fig. 2B). Coimmunoprecipitation was also observed when either NR1-1a or NR2A was expressed with NR3A, demonstrating that both NR1 and NR2 subunits are able to associate independently with NR3A. In control experiments, NR3A, when expressed by itself, did not immunoprecipitate with NR1 or NR2A/B antibodies. This excludes the possibility that NR3A could be pulled down nonspecifically by these antibodies (Fig. 2B).
The anti-NR3A antibody did not coimmunoprecipitate the AMPA receptor subunits GluR1 or GluR2 when coexpressed with NR3A in HEK293T cells (Fig. 2D). These results demonstrate subfamily-specific assembly of NR3A with the NR1-1a and NR2A NMDA receptor subunits in mammalian cells.
Association of NR3A with NR1-1a and NR2A occurs in the endoplasmic reticulum
To identify the intracellular compartment in which NR3A first associates with NR1 and NR2A subunits, metabolically labeled, transfected cells were treated with BFA and nocodazole. This drug combination inhibits protein transport from the ER to the Golgi apparatus (Lippincott-Schwartz et al., 1990
NR3A association with the other NMDA receptor subunits was then evaluated by coimmunoprecipitation. Figure 3A shows the results of one such experiment in which NR2A and NR3A proteins coimmunoprecipitate with NR1-1a subunits in both the presence and absence of BFA/nocodazole. Immunoprecipitation with anti-NR2A and anti-NR3A antibodies allowed clear identification of the NR2A and NR3A protein bands in the autoradiogram. However, these antibodies were unable to coimmunoprecipitate detectable levels of the other associating receptor subunits under the conditions of solubilization used for the pulse-chase experiments. The effectiveness of BFA/nocodazole treatment was verified in parallel experiments with the Shaker B K+ channel protein, which demonstrated the expected shift in molecular mass after BFA/nocodazole treatment (Fig. 3B) (Nagaya and Papazian, 1997
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Figure 3. NR3A assembles with NR1-1a and NR2A in the endoplasmic reticulum. Transfected HEK293T cells were metabolically labeled and treated with either BFA/nocodazole, to block ER-to-Golgi transport, or DMSO vehicle. A, Autoradiograms of transfected cell lysates immunoprecipitated with antibodies against (IP ) NR1 (lanes labeled 1), NR2A/B (lanes labeled 2), or NR3A (lanes labeled 3) are shown. All three receptor subunits were immunoprecipitated by the anti-NR1 antibody in both the drug treatment and vehicle controls, demonstrating that blockade of protein transport did not prevent incorporation of NR3A into NMDA receptor complexes (NR1-1a/NR2A/NR3A). NR1-1a and NR3A subunits are also shown to assemble in the ER. The NR2A/B and NR3A antibodies were less efficient at coimmunoprecipitation under these solubilization conditions but demonstrate the identity of the NR2A and NR3A protein bands, respectively. Small decreases in the molecular mass of NR1-1a and NR3A after treatment with BFA/nocodazole suggest that some post-translational processing of the protein takes place in the Golgi apparatus. Additional bands between NR2A and NR3A and below NR1-1a are likely to be either degradation products or precursors of NR2A and NR1-1a, respectively. B, The Shaker B potassium channel subunit was expressed and immunoprecipitated in a parallel experiment. The failure of the Shaker protein to convert from the immature, ER-resident, form (double asterisks) to the mature form (single asterisk) in the presence of BFA/nocodazole verified the effectiveness of the drug treatment in blocking ER-to-Golgi transport. Representative experiments are shown; n = 3. Positions of molecular size markers in kilodaltons are shown on the left. noc, nocodazole; Sh, Shaker.
NR3A requires the presence of NR1-1a for cell surface expression
Proper assembly is often a prerequisite for expression of ion channels on the cell surface (Green and Millar, 1995
Membrane proteins were biotinylated using a membrane-impermeant biotinylation agent and were recovered by streptavidin precipitation (Fig. 4A, lanes labeled 2). As shown in Figure 4A, top blot, NR3A was not detected at the cell surface when expressed alone or in combination with the NR2A subunit. However, coexpression of NR3A with NR1-1a or both NR1-1a and NR2A resulted in the appearance of NR3A in the plasma membrane fraction. The higher molecular weight band, which appears only when NR3A is cotransfected with the NR1-1a subunit, was enriched in the plasma membrane fraction, although both forms of NR3A can reach the cell surface. As expected, NR1-1a and NR2A were present at the cell surface when all three subunits were expressed (Fig. 4A, bottom blots). Immunoblot analysis indicated that coexpression did not induce changes in NR3A protein expression levels that could account for this difference in surface labeling (see Fig. 4A, lanes labeled 1). Control experiments showed that the ER resident protein calreticulin was absent in the streptavidin-precipitated material, confirming that only cell surface proteins were recovered (Fig. 4A). These data show that NR3A is not targeted to the plasma membrane without coexpression of the NR1 subunit.
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Figure 4. NR3A is present at the cell surface only when coexpressed with the NR1-1a subunit. A, HEK293T cells were transfected with different combinations of NMDA receptor subunits and incubated for 15 min with sulfo-NHS-biotin. After solubilization, biotinylated protein was recovered by streptavidin precipitation. The streptavidin fractions (lanes labeled 2), representing the membrane proteins, and aliquots of the lysate before (lanes labeled 1) and after (lanes labeled 3) streptavidin precipitation were analyzed by immunoblotting using anti-NR1, anti-NR2A/B, anti-NR3A, and anti-calreticulin antibodies. An excess amount of protein was loaded in the lanes labeled 2 to ensure detection of any NR3A or calreticulin at the cell surface. The subunit combinations used for transfection are indicated above each blot, and the positions of molecular size markers in kilodaltons are shown on the left. A representative experiment is shown; n = 3. B, C, Surface localization of GFP-tagged NR3A. B, Left, Schematic drawing of expected transmembrane (TM) topology of NR3A-GFP is shown. Right, Protein immunoblots of HEK293T cells transfected with NR3A or NR3AGFP and probed with anti-NR3A antibody show an increase in NR3A molecular weight that corresponds to the molecular mass of GFP (27 kDa). No lower molecular weight bands were observed. C, Cells transfected with GFP-tagged NR3A alone or in combination with the other NMDA receptor subunits were immunostained in nonpermeabilizing (NP) conditions with anti-GFP antibody followed by a Texas Red-conjugated secondary antibody and imaged with filters for GFP and Texas Red. All four panels show raw superimposed confocal images combining NP anti-GFP antibody staining (red) and native GFP fluorescence from NR3A-GFP (green). Yellow corresponds to the overlap of GFP immunostaining and GFP fluorescence and reflects NR3A-GFP expressed at the cell surface. Because the intensity of red immunostaining was brighter than was green GFP fluorescence, regions of overlapping can appear red-yellow. When expressed alone, NR3A-GFP exhibits a perinuclear and reticular fluorescence pattern, and no surface staining is observed. Cotransfection of NR1-1a/NR2A leads to the appearance of patches of fluorescence at the plasma membrane. Scale bar, 10 µm.
To visualize the subcellular localization of the NR3A subunit and how this distribution is affected by the presence of NR1, we tagged NR3A with GFP by inserting GFP at the N terminus of the receptor, immediately after the signal peptide sequence (Fig. 4B). Transfection of the NR3A-GFP construct gave a single protein band of the expected molecular weight when analyzed by immunoblot with anti-NR3A (Fig. 4B) and anti-GFP antibodies (data not shown). Voltage-clamp studies in oocytes showed that introduction of the GFP tag does not alter the functional characteristics of NR3A (J. Piña-Crespo and I. Pérez-Otaño, unpublished observations).
According to the predicted transmembrane topology of glutamate receptors, the GFP tag should be located on the extracellular surface of the mature receptor (Fig. 4B) (Hollmann et al., 1994
To measure the relative amount of NR3A located at the cell surface, we analyzed the degree of colocalization of NR3A-GFP fluorescence with the cell surface marker Concanavalin A conjugated to the red fluorochrome tetramethylrhodamine. As shown in Figure 5A, cotransfection of NR1-1a and NR2A increased the amount of NR3A-GFP at the cell surface as monitored by the overlapping fluorescence pattern. With this method, a small fraction of the NR3A-GFP fluorescence was estimated to be at the plasma membrane (5.2 ± 0.6%; n = 37 cells) when NR3A-GFP was expressed alone. Coexpression of NR1-1a increased the percentage of NR3A that resides on the cell surface to 35.1 ± 3.6% (n = 39). The amount of NR3A-GFP located at the surface of the cell was significantly lower when both NR1-1a and NR2A were coexpressed in comparison with cells expressing NR1-1a and NR3A-GFP (19.6 ± 2.0%; n = 35; p < 0.001; Fig. 5B). This may reflect a limiting number of NR1 subunits available to complex with NR3A because of the formation of NMDA channels composed of NR1-1a/NR2A.
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Figure 5. Measurement of the relative amount of NR3A-GFP at the surface in the presence of the NR1-1a subunit. Transfected HEK293T cells were incubated with tetramethylrhodamine-conjugated Concanavalin A (red fluorescence) to label the surface of the cell. A, NR3A-GFP does not colocalize with tetramethylrhodamine-conjugated Concanavalin A when expressed by itself, but significant overlap (yellow) is seen when NR3A-GFP is cotransfected with NR1-1a/NR2A. Superimposed images of GFP and rhodamine channels are shown. B, Measurement of relative cell surface expression of NR3A-GFP fluorescence in the presence or absence of the other NMDA receptor subunits is shown. Each vertical bar represents the mean of 35-39 cells from three independent experiments; *p < 0.001 (vs NR3AGFP group), ANOVA followed by Student-Newman-Keuls multiple comparisons test. Scale bars, 10 µm.
NR3A is retained in the endoplasmic reticulum in the absence of the NR1-1a subunit
Without the NR1 subunit, NR3A complexes are not inserted at the plasma membrane. The inability of NR3A to traffic to the cell surface could be caused by retention at a quality control checkpoint in the biosynthetic pathway (Ellgaard et al., 1999
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Figure 6. NR3A-GFP is retained in the ER in the absence of the NR1 subunit. Immunofluorescence with anti-calreticulin antibody (red; left column), NR3A-GFP fluorescence (green; GFP-channel, middle column), and merged confocal fluorescence images (right column) indicate colocalization of NR3A-GFP with calreticulin (ER marker) in the absence of NR1-1a. Scale bars, 10 µm.
NR1 colocalizes with the NR3 subunit at intracellular and plasma membrane sites
Our experiments demonstrate that coexpression with NR1-1a reduces the ER retention and facilitates the surface expression of NR3A subunits. To determine whether NR3A-GFP colocalizes with NR1-1a at the plasma membrane, we double labeled surface receptors in nonpermeabilized cells using antibodies against the NR1 subunit and GFP (to intensify selectively the surface green fluorescence of NR3A-GFP). Figure 7B shows overlap of NR1-1a and NR3A-GFP at the plasma membrane when they are coexpressed in HEK293T cells. Both subunits were colocalized in elongated plasma membrane patches, with only occasional clustering observed.
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Figure 7. NR3A-GFP colocalizes with the NR1-1a subunit at intracellular and plasma membrane sites. HEK293T cells were transfected with the indicated combinations of NMDA receptor subunits. Immunofluorescence to NR1 (left column) and GFP (middle column) was performed in permeabilized (A) and nonpermeabilized (B) cells to analyze patterns of NR1-1a/NR3A-GFP colocalization. Confocal microscopy sections are shown. Areas of overlapping appear yellow (right column). Arrowheads point to membrane patches where both subunits are found. Scale bars, 10 µm.
Extensive intracellular colocalization of NR1-1a and NR3A-GFP was also observed in detergent permeabilized cells (Fig. 7A). Confocal analysis of the subcellular colocalization revealed that coexpression with NR1-1a alters the reticular pattern of NR3A-GFP fluorescence (compare with Fig. 4C, top panels) and results in the appearance of intracellular receptor-rich domains, which displayed positive immunostaining for both subunits: small punctate clusters (mean diameter = 0.81 ± 0.15 µm; n = 17) and irregular, plaque-like structures (2.03 ± 0.03 µm; n = 31). The coincident patterns of intracellular and surface distribution of NR1-1a and NR3A-GFP, together with the changes in subcellular localization and surface expression of NR3A induced by coexpression of the NR1-1a subunit, indicate that NR3A subunits must coassemble with NR1 to allow the receptor complex to exit the ER and be inserted at the plasma membrane.
DISCUSSION
We have investigated the intracellular trafficking and surface expression of the NR3A NMDA receptor subunit in the presence and absence of NR1-1a and NR2A. We show that NR3A-containing complexes are assembled in the ER before they can be transported to later stages of the biosynthetic pathway and inserted into the plasma membrane. Coexpression with NR1-1a but not NR2A subunits is necessary for the ER export and surface expression of NR3A-containing receptors. Although complexes containing only NR1-1a and NR3A can progress through the secretory pathway and are found at the plasma membrane, the presence of the NR2A subunit in the complex is required for receptor function.
Functional properties of recombinant NMDA receptors containing the NR3A subunit
NR3A coassembles with NR1-1a and NR2A NMDA receptor subunits to form an NMDA-activated channel. NMDA channels incorporating the NR3A subunit are inserted into the plasma membrane with the NR3A N-terminal GFP tag exposed at the extracellular surface, consistent with NR3A having the putative transmembrane topology of glutamate receptor subunits (Hollmann et al., 1994
One feature that was thought to differentiate NMDA receptors from other members of the glutamate receptor family is their high relative Ca2+ permeability (Monyer et al., 1992
Ca2+ flux through NMDA receptors is thought to initiate intracellular signaling events that trigger certain forms of synaptic plasticity, such as long-term potentiation and depression (Malenka and Nicoll, 1999
Role of the NR1 subunit in the cell surface targeting of NMDA receptors
The cellular mechanisms that control the functional expression of low Ca2+-permeable, NR3A-containing receptors have not been studied. Our results reveal that the trafficking of NMDA receptors containing NR3A to the cell surface depends on their subunit composition, specifically on the presence of the NR1 subunit in the receptor complex. NR3A can associate independently with both NR1-1a and NR2A, which provides a biochemical substrate for multiple subunit combinations. However, NR2A/NR3A heteromers were not detected at the cell surface and were retained in the ER. In contrast, formation of NR1-1a/NR2A/NR3A complexes did result in functional NR3A-containing receptors at the cell surface. This may occur because of some defect in the folding or assembly of the receptor complex in the absence of the NR1 subunit, causing the receptor to be subject to quality control in the ER and preventing its transport to the plasma membrane (Ellgaard et al., 1999
Previous studies had suggested that NR1 requires the coexpression of NR2 subunits to be targeted to the cell surface (McIlhinney et al., 1996
The signaling machinery that regulates the progression of membrane proteins through the biosynthetic pathway is largely unknown. Subunit hetero-oligomerization has been shown to be required for surface expression of a growing list of ion channels, including GABAA receptors, acetylcholine receptors, voltage-gated potassium channels, and ATP-sensitive potassium channels (KATP) (Paulson et al., 1991
Additional mechanisms not operating in HEK293T cells may be involved in the localization of the receptor in neurons. The NR3A subunit is enriched in brain postsynaptic densities, but the mechanisms that mediate its localization to postsynaptic sites have not been investigated (Das et al., 1998
FOOTNOTES Received July 11, 2000; revised Nov. 22, 2000; accepted Nov. 28, 2000.
This research was supported by a fellowship from the government of Navarra, Spain (I.P.-O.), an International Traveling Research Fellowship from The Wellcome Trust (A.C.), a National Research Service Award (C.T.S.), National Institutes of Health Grant R01 NS28709, grants from the McKnight and Adler Foundations (S.F.H) and the Research Grants Council Hong Kong (N.J.S.), and National Institutes of Health Grants R01 NS34383 to J.S.T. and P01 HD29587 and R01 EY05477 to S.A.L. We thank Kevin Sevarino for the NR3A cDNA, David Colquhoun for providing single-channel analysis software, Shigetada Nakanishi for the rat NR2A plasmid, and Diane M. Papazian for the Shaker B potassium channel cDNA and antibody. We also thank Tim Green, Juan Piña-Crespo, John Wesseling, and Alasdair Gibb for helpful discussions and Quynh-Chi Phan for technical assistance.
Correspondence should be addressed to Dr. Isabel Pérez-Otaño, Molecular Neurobiology Laboratory, The Salk Institute for Biological Studies, 10010 North Torrey Pines Road, La Jolla, CA 92037. E-mail: otano{at}salk.edu.
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