![Introduction of the C1 domain into the C terminal of GluR1 attenuates functional expression. A , Currents evoked by the application of glutamate (10 m M for 100 msec) were recorded from HEK293 cells expressing either wild-type homomeric GluR1 AMPA receptors [ GluR1(o) ; top trace ] or GluR1 with the NR1 C1 domain inserted within the cytoplasmic C terminus [ GluR1(o)–C1 ; bottom trace ]. Representative traces for both receptor types are shown. The gray horizontal bar indicates the time of glutamate application. Cells were voltage clamped at Ϫ 70 mV. B , The mean peak current amplitude was significantly larger for GluR1(o) receptors compared with GluR1(o)–C1 receptors (767 Ϯ 287 vs 17 Ϯ 10 pA; p Ͻ 0.05 in an unpaired t test; n ϭ 14 and 10, respectively).](https://www.researchgate.net/profile/Thomas-Blanpied/publication/12022995/figure/fig2/AS:281919452401669@1444226243771/Introduction-of-the-C1-domain-into-the-C-terminal-of-GluR1-attenuates-functional_Q320.jpg)
Content uploaded by Thomas Blanpied
Author content
All content in this area was uploaded by Thomas Blanpied on Jun 01, 2014
Content may be subject to copyright.
Available via license: CC BY-NC-SA 4.0
Content may be subject to copyright.
An NMDA Receptor ER Retention Signal Regulated by
Phosphorylation and Alternative Splicing
Derek B. Scott,
1,2
Thomas A. Blanpied,
2
Geoffrey T. Swanson,
3
Chi Zhang,
2
and Michael D. Ehlers
1,2
1
Program in Cell and Molecular Biology and
2
Department of Neurobiology, Duke University Medical Center, Durham,
North Carolina 27710, and
3
Molecular Neurobiology Laboratory, The Salk Institute, La Jolla, California 92037
Formation of mature excitatory synapses requires the assembly
and delivery of NMDA receptors to the neuronal plasma mem-
brane. A key step in the trafficking of NMDA receptors to
synapses is the exit of newly assembled receptors from the
endoplasmic reticulum (ER). Here we report the identification of
an RXR-type ER retention/retrieval motif in the C-terminal tail of
the NMDA receptor subunit NR1 that regulates receptor surface
expression in heterologous cells and in neurons. In addition, we
show that PKC phosphorylation and an alternatively spliced
consensus type I PDZ-binding domain suppress ER retention.
These results demonstrate a novel quality control function for
alternatively spliced C-terminal domains of NR1 and implicate
both phosphorylation and potential PDZ-mediated interactions
in the trafficking of NMDA receptors through early stages of the
secretory pathway.
Key words: ER retention; NMDA receptors; NR1 subunit; RXR
motif; quality control mechanisms; intracellular trafficking
NMDA receptors are glutamate-gated ion channels that play a
central role in synapse formation, synaptic plasticity, and neuro-
logical disease (Mori and Mishina, 1995; Ozawa et al., 1998;
Malenka and Nicoll, 1999). These receptors are composed of
heteromeric combinations of NR1 and NR2 subunits that co-
translationally assemble in the endoplasmic reticulum (ER) to
form functional channels (Monyer et al., 1992; McIlhinney et al.,
1998; Ozawa et al., 1998). After being assembled, NMDA recep-
tors are selectively targeted to the postsynaptic membrane oppo-
site glutamatergic terminals (O’Brien et al., 1998) and appear at
nascent synapses within 1–2 hr of initial contact by an active
presynaptic terminal (Friedman et al., 2000). At more mature
synapses, new NMDA receptors are delivered to the postsynaptic
membrane in response to experience-dependent synaptic activa-
tion (Quinlan et al., 1999), and synaptic accumulation of NMDA
receptors is reciprocally regulated by chronic changes in activity
(Rao and Craig, 1997; Liao et al., 1999; Watt et al., 2000). The
synaptic localization of NMDA receptors arises, in part, via
protein–protein interactions that anchor or stabilize NMDA re-
ceptors in the postsynaptic density (O’Brien et al., 1998; Sheng
and Lee, 2000). However, before becoming localized at synapses,
newly synthesized NMDA receptors must assemble, mature, and
be transported through the ER–Golgi secretory pathway (McIl-
hinney et al., 1998), a set of processes that remains poorly
understood.
The plasma membrane delivery of multimeric protein com-
plexes requires proper folding and assembly of their constituent
subunits. In many cases, the masking of specific ER retention/
retrieval motifs during receptor assembly regulates the forward
trafficking of ion channels and other membrane proteins through
the secretory pathway (Zerangue et al., 1999; Bichet et al., 2000;
Margeta-Mitrovic et al., 2000). For example, RXR-type ER re-
tention motifs found in each of the ATP-sensitive potassium
channel (K
ATP
) subunits Kir6.1, Kir6.2, and SUR1 are sequen-
tially masked during subunit oligomerization, thus assuring that
only correctly assembled channels reach the plasma membrane
(Zerangue et al., 1999). A similar RXR motif present in GABA
B
receptor GB1 subunits is masked by assembly with GB2, ensuring
heterodimerization (Margeta-Mitrovic et al., 2000). Also, surface
expression of Ca
2⫹
channels is facilitated by

subunits, which
mask ER retention domains in
␣
subunits (Bichet et al., 2000).
Like these membrane proteins, NMDA receptors possess intra-
cellular regulatory domains that influence intracellular trafficking
of mature channels. In particular, mRNA splicing of the NR1 C
terminal affects the surface expression (Okabe et al., 1999) and
intracellular localization (Ehlers et al., 1995) of NMDA
receptors.
Recent studies suggest that specific quality control mechanisms
exist for recognizing and retaining unassembled NMDA receptor
subunits and releasing only properly assembled receptors from
the ER. When expressed alone in heterologous cells, NR1 and
NR2 subunits are retained in the ER and do not efficiently reach
the plasma membrane (Ehlers et al., 1995; McIlhinney et al.,
1996; Okabe et al., 1999). In neurons, NR1 subunits exist in two
populations: a very stable plasma membrane pool and a short-
lived intracellular pool that likely represents receptors that have
failed to assemble with NR2 subunits (Hall and Soderling, 1997;
Huh and Wenthold, 1999). Such rapid degradation of unas-
sembled NR1 subunits ensures that only functional, properly
assembled NR1/NR2 heteromeric channels reach the plasma
membrane. Despite these initial findings, little is known about the
molecular determinants of NMDA receptor assembly and quality
Received Dec. 8, 2000; revised Feb. 2, 2001; accepted Feb. 5, 2001.
This work was supported by a Ruth K. Broad fellowship (D.B.S.) and grants from
the National Institutes of Health (RO1 NS39402), the Spinal Cord Research Foun-
dation, the McKnight Foundation, the Klingenstein Fund, the Triangle Community
Foundation, the Alfred P. Sloan Foundation, the American Health Assistance
Foundation, the North Carolina Biotechnology Center, and the National Alliance
for Research on Schizophrenia and Depression (M.D.E.). We thank Dr. Pierre
Cosson for providing the Tac construct. In addition, we thank Y. Mu for making
constructs, C. Nicchitta for supplying the Trap
␣
antibody, and K. Courtney for
supplying the mannosidase II antibody. Special thanks to S. Heinemann for support
of the electrophysiology experiments.
Correspondence should be addressed to Dr. Michael D. Ehlers, Department of
Neurobiology, Duke University Medical Center, Durham, NC 27710. E-mail:
ehlers@neuro.duke.edu.
Copyright © 2001 Society for Neuroscience 0270-6474/01/210001-10$15.00/0
The Journal of Neuroscience, May 1, 2001, 21(9):3063–3072
control or the mechanisms that regulate the delivery of mature
channels to the neuronal plasma membrane.
In this study, we report the identification of a novel RXR-type
ER retention/retrieval motif present in the alternatively spliced
C1 domain of the NMDA receptor subunit NR1. This RXR
sequence functions as an ER retention motif in heterologous cells
and in neurons. Furthermore, ER retention mediated by the
RXR motif is suppressed by the presence of an adjacent alterna-
tively spliced domain containing a consensus type I PDZ-binding
sequence and by PKC phosphorylation of specific serine residues
in the C1 domain of NR1. Together, these results identify ER
retention, PKC phosphorylation, and alternative splicing of NR1
as novel mechanisms regulating the intracellular trafficking and
plasma membrane delivery of NMDA receptors.
MATERIALS AND METHODS
Cell culture. COS cells were grown in DMEM (Sigma, St. Louis, MO)
supplemented with 10% bovine serum, 1 m
M sodium pyruvate, and 50
U/ml penicillin and streptomycin. Human embryonic kidney 293
(HEK293) cells were grown in MEM (Sigma) supplemented with 10%
bovine serum, 1 m
M sodium pyruvate, and 50 U/ml penicillin and strep-
tomycin. Rat1 cells were grown in DMEM supplemented with 5%
bovine serum, 1 m
M sodium pyruvate, 100
M nonessential amino acids,
and 50 U/ml penicillin and streptomycin. Primary cultures of hippocam-
pal neurons were obtained from 1-d-old rat pups. Area CA1 was isolated
and dissociated with trypsin, and cells were plated at 60,000 cells/cm
2
in
Neurobasal medium (Sigma) supplemented with B27, glutamax I, 5%
bovine serum, and 1
g/ml gentamycin. FUDR (10
M) was added 1–3
d after plating, and cells were fed twice weekly thereafter. Hippocampal
neurons and COS cells were grown on coverslips coated with poly-
D-
lysine (Sigma). All cells were grown at 37°C and in 5% CO
2
.
Generation of Tac–NR1 receptors. Tac receptors fused with intracellu-
lar NR1 C-terminal domains were generated by first amplifying NR1
C-terminal domains with the following sets of primers: C0 (forward,
5⬘-CCCAAGCTTCCGAGATCGCCTACAAGCGAC-3⬘; reverse,
5⬘-CCCAAGCTTGGATCCTCACTGCAGGTTCTTCCTCCAC-3⬘),
C1(forward, 5⬘-CCCAAGCTTATAGAAAGAGTGGTAGAGC-3⬘;
reverse, 5⬘-CCCAAGCTTGGATCCTCACGTGTCTTTGGAGGA
CCTAC-3⬘),C2(forward, 5⬘-CCCAAGCTTCCAGCACCGGGGGTG-
GACGC-3⬘; reverse, 5⬘GCTCTAGATCAGCTCTCCCTATGAC-3⬘),
C2⬘ (forward, 5⬘-CCCAAGCTTCCCAGTACCATCCCACTGAT-3⬘;
reverse, 5⬘-GCTCTAGATCACACCACGGTGCTGACCGAGGG-3⬘),
NR1a/NR1c/allmutant NR1a (forward, 5⬘-CCCAAGCTTCCGAGAT
CCCTACAAGCGAC-3⬘; reverse, 5⬘-CCCAAGCTTGGATCCTCAG
CTCTCCCTATGAC-3⬘), NR1e (forward, 5⬘-CCCAAGCTTC-
CGAGATCCGGTACAAGCGAC-3⬘; reverse, 5⬘-CCCAAGCTTGGAT
CCTCACACCACGGTGCTGACCGAGGG-3⬘), NR1g (forward, 5⬘-
CCCAAGCTTCCGAGATCCGGTACAAGCGAC-3⬘; reverse, 5⬘-
GCTCTAGATCACACCACGGTGCTGACCGAGGG-3⬘), and NR1e
⌬VSTVV (forward, 5⬘-CCCAAGCTTCCGAGATCCGGTACAAGC-
GAC-3⬘; reverse, 5⬘-CCCAAGCTTGGATCCTCACGAGGGATCTG-
AGAGGTTGAGCGG-3⬘). After digestion with HindIII plus XbaI (C2,
C2⬘, and NR1g) or HindIII (C0, C1, NR1a/NR1c/NR1e, NR1a point
mutants, and NR1e ⌬VSTVV), PCR fragments were ligated into linear-
ized Tac pCDM8 expression vector (kindly provided by Dr. P. Cosson,
Center Medical Universitaire, Geneva, Switzerland). Tac–NR1a point
mutants were first generated in full-length NR1a subunits using the quick
change site-directed mutagenesis kit (Stratagene, La Jolla, CA), follow-
ing the manufacturer’s instructions, and then fused with Tac as described
above. GluR1(o)–C1 was generated as described (Ehlers et al., 1995). All
constructs were verified by sequencing.
Transfections. COS, HEK293, and Rat1 cells were transfected using
the Superfect Transfection Reagent (Qiagen, Valencia, CA) following
the manufacturer’s suggested protocol for transient transfection of ad-
herent cells. Seven- to 14-d-old cultured hippocampal neurons were
transfected using the LipofectAMINE 2000 Transfection Reagent (Life
Technologies, Gaithersburg, MD). Briefly, 1–2
g of DNA in 50
lof
OptiMEM (Life Technologies) was mixed with 0.5
l of Lipo-
fectAMINE 2000 in 100
l of OptiMEM and incubated at room tem-
perature for 20 min. The transfection cocktail was then added directly to
neurons plated onto coverslips in 2 ml of culture media and incubated at
37°C and in 5% CO
2
. Expression in all cell types was analyzed 24–48 hr
after transfection.
Antibodies. Monoclonal anti-Tac antibody (Covance, Princeton, NJ)
was used as follows: 1:500 (immunofluorescence of heterologous cells),
1:2500 (neurons), and 1:5000 [fluorescence-activated cell (FAC) sorter].
Polyclonal anti-Tac antibody (Santa Cruz Biotechnology, Santa Cruz,
CA) was used as follows: 1:100 (immunofluorescence of heterologous
cells) and 1:1000 (Western blots). Polyclonal anti-C1 (1747), anti-C2
(1683), anti-C2⬘ (1233), and anti-Trap
␣
(kindly provided by Dr. C.
Nicchitta, Duke University, Durham, NC) and monoclonal anti-BiP
(Transduction Laboratories, San Diego, CA) antibodies were all used at
1:100. Monoclonal anti-mannosidase II (Covance) was used at 1:1000. All
secondary antibodies conjugated to indocarbocyanine (Cy3), FITC, or
phosphatidylethanolamine (PE) (Jackson ImmunoResearch, West
Grove, PA) were used at 1:100.
Immunofluorescence. For surface labeling of heterologous cells, trans-
fected cells were incubated live with anti-Tac antibodies in DMEM
supplemented with 5% serum for 1 hr at 4°C. Cells were washed with
PBS, fixed on ice with 4% paraformaldehyde and 4% sucrose for 20 min,
washed three times with PBS, and permeabilized with 0.2% Triton X-100
for 5 min at room temperature. Intracellular expression was then deter-
mined by washing cells with PBS and incubating cells with the appropri-
ate antibody in DMEM supplemented with 5% serum at room temper-
ature for 2 hr. After three washes with PBS, cells were incubated with the
appropriate secondary antibodies in DMEM supplemented with 5%
serum for 1 hr at room temperature. Surface and intracellular expression
was captured on an epifluorescent microscope (Nikon, Melville, NY)
using a cooled CCD camera (Princeton Instruments, Monmouth, NJ) and
analyzed with Metamorph imaging software (Universal Imaging Com-
pany, West Chester, PA). Colocalization images were visualized and
captured with a confocal microscope (LSM410; Zeiss, Thornwood, NY).
Immunofluorescent localization of receptors in 7- to 14-d-old cultured
hippocampal neurons was achieved as described above, but with two
notable exceptions. First, live neurons were incubated with a monoclonal
anti-Tac antibody in extracellular buffer (120 m
M NaCl, 3 mM KCl, 10
m
M HEPES, 2 mM CaCl
2
,2mM MgCl
2
, and 10 mM glucose, pH 7.35)
plus 5% donkey serum for 15 min at 37°C or 30 min at room temperature
and then fixed and incubated with a Cy3-conjugated anti-mouse second-
ary antibody. Second, to identify intracellular expression, neurons were
permeabilized and incubated with a monoclonal anti-Tac antibody in
10% donkey serum followed by incubation with a FITC-conjugated
anti-mouse secondary antibody. For quantification of surface labeling
using flow-assisted cytometry, live transfected HEK293 cells grown in 6-
or 12-well tissue culture-treated plates (Corning, Corning, NY) were
incubated with a monoclonal anti-Tac antibody in DMEM supplemented
with 5% serum for 1 hr at 4°C. Cells were then washed with PBS and
incubated with a PE-conjugated anti-mouse antibody in PBS for 1 hr at
4°C, after which cells were washed with PBS, gently detached from the
bottom of the plate with 500
l of PBS plus 5 mM EDTA, and added to
200
l of 4% paraformaldehyde in 12 ⫻ 75 mm round-bottom test tubes
(VWR Scientific, South Plainfield, NJ). Surface expression was quanti-
fied 12–24 hr later using the Becton Dickinson FACscan sorter at the
Duke University Flow Facility. Light scattering was used to gate live
cells, and the background fluorescence was determined using HEK293
cells transfected with Tac but incubated without the primary antibody.
The average fluorescence intensity was calculated for cells registering a
fluorescence signal above a background level determined by measuring
the fluorescent intensity of cells transfected with Tac but incubated only
with the secondary antibody (Tac-primary).
Electrophysiology. HEK293 cells were maintained in DMEM plus 10%
fetal calf serum and were calcium phosphate-transfected with 0.5–1
gof
rat GluR1(o) or GluR1(o)–C1 cDNA and 0.2
g of human CD8 antigen
cDNA per glass coverslip for 5–12 hr at 37°C and in 5% CO
2
. Electro-
physiological recordings were made2dafter transfection. To visualize
transfected cells, coverslips were incubated with polystyrene beads
coated with anti-CD8 antibody (Dynal, Lake Success, NY) before trans-
ferring them to the recording bath chamber. Rapid agonist application
was performed by lifting cells into a laminar solution stream that was
displaced by a piezobimorph under the control of pClamp 8 software
(Axon Instruments, Foster City, CA). Data were acquired and analyzed
using pClamp 8 software and Origin 6.0 (OriginLab Corporation,
Northampton, MA). The internal pipette solution was composed of 110
m
M CsF, 30 mM CsCl, 4 mM NaCl, 0.5 mM CaCl
2
,10mM HEPES, and
5m
M EGTA, adjusted to pH 7.3 with CsOH. The external bath solution
contained 150 m
M NaCl, 2.8 mM KCl, 2 mM CaCl
2
, 1.0 mM MgCl
2
, and
3064 J. Neurosci., May 1, 2001, 21(9):3063–3072 Scott et al. • ER Retention of NMDA Receptors
10 mM HEPES; pH was adjusted to 7.3 with NaOH. L-Glutamate was
purchased from Sigma.
In vitro degl ycosylation. Transfected COS cells washed once with TBS
were scraped off the bottom of a 60 mm plate in 600
l of lysis buffer
(PBS, pH 8.0, plus 2 mM EDTA, 0.1 mM PMSF, 1
g/ml pepstatin A, 1
g/ml chemostatin, and 1
g/ml leupeptin). After brief sonication and
centrifugation (60,000 rpm for 15 min at 4°C), cell membranes were
resuspended in 50
l of lysis buffer plus 1% SDS and boiled for 5 min
before adding 250
l of 1% octylglucoside and incubating at 4°C over-
night. Samples were then divided into thirds and treated with either 6 U
of peptide-N-glycosidase F (PNGase F; Boehringer Mannheim, India-
napolis, IN), 0.1 U of endoglycosidase H (endo H; Boehringer Mann-
heim), or no enzyme at all and incubated at 37°C for 5 hr. After
incubations, cell membranes were stored at ⫺70°C. After thawing, add-
ing sample buffer, and boiling for 5 min, membrane proteins were
resolved on a 7.5% SDS-PAGE gel and visualized using immunoblot
analysis with an anti-Tac polyclonal antibody.
PKC activation e xperiments. COS7 cells transfected with either Tac–
NR1a or Tac–NR1a SS896–7AA were treated with 100 nM phorbol
12-myristate 13-acetate (PMA) for 30 min, washed with warm media,
and then allowed to recover at 37°C for 2–3 hr. Cells were then examined
for surface and intracellular expression as described above using anti-Tac
and anti-C2 antibodies. Quantification of surface expression was accom-
plished by counting the number of cells showing intracellular expression
of Tac–NR1 receptors (anti-C2 positives) and then determining how
many of those also showed surface expression of Tac–NR1 receptors
(anti-Tac positive). All cell counts were done in a blind manner.
RESULTS
Identification of trafficking signals in the C terminal
domain of NR1
The intracellular C terminal domain of the NMDA receptor
subunit NR1 undergoes differential mRNA splicing to create
eight different subtypes of NR1 subunits, NR1a–h (Fig. 1A).
When expressed alone in heterologous cells, these subtypes ex-
hibit differential subcellular localization (Ehlers et al., 1995) and
are differentially expressed at the cell surface (Okabe et al., 1999;
Standley et al., 2000) (data not shown). To determine which
domains of NR1 are responsible for regulating NR1 surface
expression, we constructed chimeric receptor molecules consist-
ing of the human interleukin-2 receptor
␣
subunit (Tac) tagged
with portions of the intracellular C terminal domain of the NR1
subunit (Leonard et al., 1983; Tan et al., 1998; Craven and Bredt,
2000). After expressing these Tac–NR1 constructs in COS7 cells,
we visualized receptors that trafficked to the plasma membrane
using live immunofluorescence (Fig. 1B). Tac itself showed
strong surface expression, as did Tac–C0, Tac–C2, Tac–C2⬘,
and Tac–C0–C2 (Tac–NR1c). In contrast, the C1-containing
receptors Tac–C1 and Tac–C0–C1–C2 (Tac–NR1a) were not
detectable on the cell surface despite intense intracellular label-
ing (Fig. 1B).
To quantify surface expression of Tac–NR1 receptors, flow-
assisted cytometry was performed on transfected HEK293 cells
surface labeled with anti-Tac antibody and fluorophore-
conjugated secondary antibody. Cells expressing Tac had be-
tween a 16- and 31-fold higher surface fluorescence compared
with that of Tac–C1 and Tac–NR1a (Fig. 1C). In addition,
chimeric receptors without the C1 domain had an average surface
fluorescence 9- to 25-fold higher than that of Tac–C1 or Tac–
NR1a (Fig. 1C). In all cases, total protein expression for various
Tac–NR1 receptors was similar as revealed by immunoblot (data
not shown) and by staining of permeabilized cells (Fig. 1B, right).
To ensure that regulation of surface expression by the C1
domain was not specific for Tac receptors expressed in COS7 or
HEK293 cells, we first transfected 7- to 14-d-old cultured hip-
pocampal neurons with Tac chimeras and analyzed their surface
expression. As in heterologous cells, Tac–C1 and Tac–NR1a
were retained intracellularly and did not reach the plasma mem-
brane when expressed in hippocampal neurons. In contrast, Tac,
Tac–C0, Tac–C2, Tac–C2⬘, and Tac–NR1c were all efficiently
trafficked to the neuronal plasma membrane (Fig. 1D). Second,
we asked whether the C1 domain could regulate the assembly and
plasma membrane trafficking of multimeric glutamate receptors.
For these experiments, we inserted the C1 domain into a corre-
sponding site in the intracellular C terminal domain of the
AMPA receptor GluR1(o) subunit, which by itself can form
functional homomeric channels (Ozawa et al., 1998), and mea-
sured glutamate-induced currents from transfected HEK293 cells
(Fig. 2A). Cells expressing GluR1(o) exhibited robust responses
to 10 m
M glutamate (767 ⫾ 287 pA) consistent with homomeric
GluR1 channels, whereas cells expressing GluR1(o)–C1 pro-
duced almost no response to glutamate (17 ⫾ 10 pA) (Fig. 2B). In
all cases, total protein levels of GluR1(o) and GluR1(o)–C1 were
indistinguishable (data not shown), indicating that addition of the
C1 domain prevented the trafficking of functional GluR1 homo-
meric AMPA receptors to the plasma membrane. Together, these
data demonstrate that the C1 domain is sufficient to prohibit the
surface expression of Tac receptors and glutamate receptors in
both heterologous cells and neurons.
The C1 domain contains an RXR-type ER
retention/retrieval motif
To determine whether the C1 domain of NR1 prevents surface
expression by retaining receptors in the ER, we first examined the
subcellular localization of Tac–C1 and Tac–NR1a using confocal
microscopy. After transfection of COS7 or Rat1 cells, both
Tac–C1 and Tac–NR1a colocalized extensively with the ER
resident proteins Trap
␣
(Fig. 3A) and BiP (Fig. 3B) (Sanders and
Schekman, 1992; Hartmann and Prehn, 1994) but not with the
medial Golgi protein mannosidase II (Burke et al., 1982) (Fig.
3C). In agreement with a failure of Tac–C1 and Tac–NR1a to
reach the medial Golgi, these receptors remained sensitive to
endo H (Fig. 3D), an enzyme that preferentially hydrolyzes the
high mannose N-glycans present on immature secretory proteins
in the ER (Trimble and Maley, 1984). In contrast, Tac, Tac–C0,
Tac–C2, Tac–C2⬘, and Tac–NR1c were resistant to endo H (Fig.
3D) indicating that these receptors had been processed in the
Golgi, a result consistent with their robust surface expression
(Fig. 1B,C). Note that Tac receptors are normally present as both
a mature, high-molecular weight, endo H-resistant species and an
immature endo H-sensitive species that migrates at a lower mo-
lecular weight (Leonard et al., 1983) (Fig. 3D, top panel). The lack
of a mature form of either Tac–C1 or Tac–NR1a further suggests
that these receptors are unable to exit the ER. However, to ensure
that endo H treatment was selectively deglycosylating Tac–C1
and Tac–NR1a, we incubated chimeric receptors with a less-
selective glycosidase, PNGase F, which cleaves almost all types of
asparagine-bound N-glycans (Tarentino et al., 1985). After incu-
bation with PNGase F, all Tac–NR1 receptors exhibited in-
creased electrophoretic mobility, indicating PNGase F sensitivity
(Fig. 3D). These experiments provide strong biochemical and
immunocytochemical evidence of selective ER retention of C1
domain-containing receptors.
Sequence comparisons between the C1 domain of NR1 and the
K
ATP
subunits Kir6.1 and Kir6.2 revealed that the C1 domain
shares homology around the newly identified RXR ER retention/
retrieval motif of the K
ATP
channel (Fig. 4A) (Zerangue et al.,
1999). To determine which residues are necessary for ER reten-
Scott et al. • ER Retention of NMDA Receptors J. Neurosci., May 1, 2001, 21(9):3063–3072 3065
tion of Tac–NR1 receptors, we constructed a series of Tac–NR1a
point mutants and examined their effect on ER retention. Dis-
ruption of the putative RXR ER retention/retrieval motif
(KRRR892–895AAAA) released Tac–NR1a receptors from the
ER in both COS7 cells (Fig. 4B, left) and hippocampal neurons
(Fig. 4B, right). Quantification of surface expression using flow-
assisted cytometry of HEK293 cells transfected with the Tac–
NR1a KRRR892–895AAAA mutant showed that the mutant
had sixfold greater surface fluorescence than had Tac–NR1a
(Fig. 4C). These results indicate that the C1 domain of NR1
contains an RXR ER retention/retrieval motif.
PKC phosphorylation of NR1 releases receptors from
the ER
The C1 domain contains the major phosphorylation sites of NR1
(Tingley et al., 1993), and PKC phosphorylation within the C1
domain regulates the intracellular localization of NR1 subunits
(Ehlers et al., 1995). Because these phosphorylation sites are in
close proximity to the RXR ER retention motif, we hypothesized
that phosphorylation might block ER retention and promote
plasma membrane delivery of NMDA receptors. Surprisingly,
mutations that prevent (S890A) or mimic (S890E) phosphoryla-
tion of serine 890, a known PKC phosphorylation site (Tingley et
al., 1997), had no effect on surface expression of Tac–NR1a (Fig.
5A,B). In addition, mutations that prevent phosphorylation of
serines 896 and 897 (SS896–7AA), known PKC and PKA sites
(Tingley et al., 1997), respectively, also had no effect on surface
expression (Fig. 5A,B). However, Tac–NR1a receptors carrying
negative-charge mutations that mimic phosphorylation at serines
896 and 897 (SS896–7EE) exhibited strong surface expression in
both COS7 cells (Fig. 5A) and hippocampal neurons (Fig. 5B).
Flow cytometric quantification of bound surface antibody showed
Figure 1. The C1 domain of NR1 regulates surface expression of chimeric
receptors in both heterologous cells and hippocampal neurons. A, Sche-
matic representation of NR1 splice variants possessing distinct intracellular
domains is shown. The N terminal (N) and C terminal ( C) ends of the
protein are indicated. B, Surface and total expression of Tac–NR1 recep-
tors in COS cells is shown. Live cells were labeled with a monoclonal
anti-Tac antibody (Surface, left), permeabilized, and labeled with polyclonal
anti-Tac (Tac, Tac–C0), anti-C1 (Tac–C1, Tac–NR1a), anti-C2 (Tac–C2,
Tac–NR1c), or anti-C2⬘ (Tac–C2⬘) antibodies (Total, right). The C1-
containing receptors Tac–C1 and Tac–NR1a are not expressed on the cell
surface. C, Quantification of Tac–NR1 chimeric receptor surface expres-
sion by flow-assisted cytometry is shown. Data represent means ⫾ SEM of
fluorescence intensity of transfected HEK293 cells stained live for the
presence of Tac on the cell surface (*p ⬍ 0.05 relative to Tac minus primary
antibody control, one-way ANOVA). D, C1-containing receptors do not
reach the plasma membrane of cultured hippocampal neurons. Surface
(left) and total (right) receptors were localized by immunofluorescence as
described in A. Scale bars: B,10
m; D,25
m.
Figure 2. Introduction of the C1 domain into the C terminal of GluR1
attenuates functional expression. A, Currents evoked by the application of
glutamate (10 mM for 100 msec) were recorded from HEK293 cells
expressing either wild-type homomeric GluR1 AMPA receptors
[GluR1(o); top trace] or GluR1 with the NR1 C1 domain inserted within
the cytoplasmic C terminus [GluR1(o)–C1; bottom trace]. Representative
traces for both receptor types are shown. The gray horizontal bar indicates
the time of glutamate application. Cells were voltage clamped at ⫺70 mV.
B, The mean peak current amplitude was significantly larger for GluR1(o)
receptors compared with GluR1(o)–C1 receptors (767 ⫾ 287 vs 17 ⫾ 10
pA; p ⬍ 0.05 in an unpaired t test; n ⫽ 14 and 10, respectively).
3066 J. Neurosci., May 1, 2001, 21(9):3063–3072 Scott et al. • ER Retention of NMDA Receptors
that Tac–NR1a SS896–7EE receptors were expressed at ⬃2.5-fold
higher levels at the plasma membrane compared with the expres-
sion of Tac–NR1a (Fig. 5C), whereas none of the other phosphor-
ylation site mutants (S890A, S890E, and SS896–7AA) showed
greater surface expression than that of Tac–NR1a (Fig. 5C).
To determine whether PKC phosphorylation dynamically reg-
ulates ER retention and release of Tac–NR1 receptors, we trans-
fected COS7 cells with either Tac–NR1a or Tac–NR1a SS896–
7AA and treated them with the cell-permeable PKC activator
PMA (100 n
M). Activation of PKC resulted in the appearance of
Tac–NR1a on the cell surface (Fig. 5D). Interestingly, very little
surface expression was detected 1 hr after incubating with PMA
(data not shown). However, by 2–3 hr after a 30 min incubation
with PMA, Tac–NR1a surface expression was readily detectable
(Fig. 5D). This delayed appearance of Tac–NR1a is consistent
with the previously reported transport kinetics of membrane
proteins through the secretory pathway (Hirschberg et al., 1998).
Furthermore, mutation of serines 896 and 897 to alanine com-
Figure 3. The C1 domain contains an
ER retention motif. Tac–C1 and Tac–
NR1a localize to the ER. A, B, COS7
cells transfected with either Tac–C1 or
Tac–NR1a were permeabilized and
stained with antibodies against Tac
( green) and the ER resident protein
Trap
␣
(red)(A) or with antibodies against
the C1 domain (red) and the ER marker
BiP ( green)(B). Image overlays showed
extensive colocalization ( yellow). C,
Tac–C1 and Tac–NR1a do not localize to
Golgi compartments. Rat-1 fibroblast cells
were transfected with either Tac–C1 or
Tac–NR1a and stained for Tac (red) and
mannosidase II ( green) to label the me-
dial Golgi compartment. D, Tac–C1 and
Tac–NR1a are sensitive to deglycosyla-
tion by endo H, indicating that these re-
ceptors are retained in the ER. Tac–NR1
receptors without C1 are resistant to endo
H but remain sensitive to PNGase F, in-
dicating that these receptors are able to
leave the ER and enter the Golgi. Arrows
designate mature Tac species. Arrowheads
designate immature forms of Tac. Hori-
zontal bars on the right indicate the loca-
tion of the 49.5 kDa molecular mass
marker. Scale bars: A, B,5
m; C,10
m.
Scott et al. • ER Retention of NMDA Receptors J. Neurosci., May 1, 2001, 21(9):3063–3072 3067
pletely abolished phorbol ester-induced surface expression (Fig.
5D, bottom panels), indicating that phosphorylation at these res-
idues is required for PKC-mediated ER release. Quantification of
these results revealed an approximately sixfold increase in the
number of Tac–NR1a-expressing cells showing surface expres-
sion after PMA treatment compared with that of untreated cells
(Fig. 5E). Mutation of serines 896 and 897 to alanine completely
abolished this effect (Fig. 5E). Together, these findings establish
a role for PKC phosphorylation of NR1 in the ER retention and
plasma membrane delivery of NMDA receptors and suggest that
surface levels of NMDA receptors may reflect, in part, the pre-
vious history of kinase activation at a given synapse.
Suppression of RXR-mediated ER retention by an
adjacent consensus PDZ-binding domain
In addition to inserting or removing the RXR-containing C1
domain, alternative mRNA splicing of the NR1 C terminal do-
main introduces a consensus type I PDZ-binding sequence
(VSTVV) (Songyang et al., 1997) adjacent to C1 by replacing the
C2 domain with the C2⬘ domain (Fig. 1A). To determine the
effect of the C2⬘ domain on plasma membrane trafficking of NR1,
we compared the surface expression of Tac–NR1a and Tac–C0–
C1–C2⬘ (Tac–NR1e). Insertion of the C2⬘ domain distal to C1
(Tac–NR1e) eliminated ER retention and promoted robust sur-
face expression of Tac–NR1 in both COS7 cells (Fig. 6A) and
hippocampal neurons (Fig. 6B). Tac–NR1e surface expression
was approximately sevenfold greater than that of Tac–NR1a and
did not differ significantly from that of Tac–NR1c or Tac–C0–
C2⬘ (Tac–NR1g) (Fig. 6C). The suppression of ER retention was
caused by the consensus PDZ-binding domain of C2⬘, because
deletion of the terminal five amino acids (⌬VSTVV) abolished
delivery of Tac–NR1e to the plasma membrane (Fig. 6A–C).
Consistent with these immunocytochemical results, glycosylation
state analysis revealed that Tac–NR1e was resistant to endo H
and thus had been processed through the Golgi, whereas Tac–
NR1e ⌬VSTVV remained sensitive to endo H and was thus
trapped in the ER (Fig. 6D). These data indicate that the alter-
natively spliced consensus PDZ-binding domain of C2⬘ sup-
presses ER retention mediated by the RXR motif of C1 and
promotes ER export and trafficking to Golgi compartments.
DISCUSSION
A variety of quality control mechanisms operate in the ER to
ensure that only properly folded and assembled proteins are
transported to their target organelles and compartments (Brodsky
and McCracken, 1999; Ellgaard et al., 1999). ER exit serves as an
important checkpoint both in coordinating the sequential assem-
bly of multisubunit protein complexes within the ER and in
defining the number of receptors expressed at the plasma mem-
brane (Blount et al., 1990; Green and Claudio, 1993; Brodsky and
McCracken, 1999; Ellgaard et al., 1999; Zerangue et al., 1999;
Bichet et al., 2000; Margeta-Mitrovic et al., 2000). Here we have
provided a first description of ER quality control mechanisms
that regulate the plasma membrane delivery of NMDA receptors.
In particular, our findings reveal the presence of an ER retention
signal in the alternatively spliced C terminal domain of the NR1
subunit and show that release of NR1 from the ER is regulated by
PKC phosphorylation and an alternatively spliced consensus
PDZ-binding domain. By altering the degree or efficiency of
NMDA receptor surface expression, such quality control mech-
anisms could modify the magnitude of NMDA receptor-
mediated signals at the synapse, thus influencing synaptic
strength, plasticity, and vulnerability to excitotoxicity.
Previous studies of ours and others have shown that the C
Figure 4. Identification of an RXR-type ER retention/retrieval motif in
NR1 and key residues required for ER retention. A, Sequence alignment
between C-terminal domains of rat K
ATP
channel subunits, the rat
GABA
B
receptor GB1 subunit, and the C1 domain of the rat NR1
subunit. The location of the RXR ER retention/retrieval motif is desig-
nated by asterisk s. Note that the C1 domain of NR1 shares homology
around this motif. B, Surface expression of Tac–NR1a point mutants in
COS7 cells and hippocampal neurons. Tac–NR1 receptors containing
mutations in the putative RXR ER retention/retrieval motif (Tac–NR1a
KRRR892–895AAAA) are able to exit the ER and are expressed at the
plasma membrane. C, Flow cytometric quantification of Tac–NR1a RXR
mutant surface expression in HEK293 cells. Data represent means ⫾
SEM of fluorescence intensities from 4,000 to 10,000 transfected cells
determined as described in Figure 1C (*p ⬍ 0.05 relative to Tac minus
primary antibody controls, one-way ANOVA). Scale bars, COS7,10
m;
Neuron,25
m.
3068 J. Neurosci., May 1, 2001, 21(9):3063–3072 Scott et al. • ER Retention of NMDA Receptors
terminus of NR1 influences intracellular distribution (Ehlers et
al., 1995) and surface expression (Okabe et al., 1999; Standley et
al., 2000) of NR1/NR2 heteromers. To identify the relevant
trafficking signals in NR1 and to minimize effects of NR2 sub-
units and other NR1 domains, we chose to isolate the contribu-
tion of NR1 C-terminal domains using Tac–NR1 chimeras. Such
an approach has been used extensively to identify signals involved
in secretory and endocytic membrane trafficking (Milgram et al.,
1996; Ghosh et al., 1998; Jansen et al., 1998; Tan et al., 1998; El
Meskini et al., 2001). The experimental advantages of this ap-
Figure 5. PKC phosphorylation regulates ER
retention of Tac–NR1a. A, Surface and intracel-
lular localization of Tac–NR1a receptors mu-
tated at phosphorylation sites in COS7 cells is
shown. Mutations that block (S3 A) or mimic
(S3 E) phosphorylation were introduced at
serines 890, 896, and 897. Note that unlike wild-
type Tac–NR1a, Tac–NR1a receptors contain-
ing mutations that mimic phosphorylation at
serines 896 and 897 (SS896–7EE) trafficked to
the cell surface. B, Surface and intracellular lo-
calization of Tac–NR1a phosphorylation site
mutants in hippocampal neurons is shown. C,
Flow cytometric quantification of Tac–NR1a
phosphorylation mutants in HEK293 cells is pre-
sented. Data represent means ⫾ SEM of surface
fluorescence intensities from 5,000 to 10,000
transfected cells determined as described in Fig-
ure 1C (*p ⬍ 0.05 compared with Tac minus
primary antibody control, one-way ANOVA). D,
Activation of PKC releases Tac–NR1a receptors
from the ER. COS7 cells transfected with either
Tac–NR1a or Tac–NR1a SS896–7AA were
treated with 100 nM PMA for 30 min and then
assayed for surface and intracellular expression
2–3 hr later. E, Quantification of PMA-induced
ER release of Tac–NR1a receptors is shown.
Data represent means ⫾ SEM of the percentage
of Tac–NR1a- or Tac–NR1a SS897–7AA-trans-
fected cells showing surface expression before or
2–3 hr after PKC activation. PKC activation was
accomplished as described in C (*p ⬍ 0.05 com-
pared with Tac–NR1a untreated, one-way
ANOVA; **p ⬍ 0.05 compared with Tac–NR1a
plus PMA, one-way ANOVA). Scale bars: A, D,
10
m; B,25
m.
Scott et al. • ER Retention of NMDA Receptors J. Neurosci., May 1, 2001, 21(9):3063–3072 3069
proach include the well characterized nature of the Tac protein
and its trafficking, the existence of highly specific extracellular
epitope antibodies, and the monomeric nature of the Tac chime-
ras. Moreover, by isolating specific domains of NR1 via Tac
fusions, we were able to identify RXR trafficking signals that
might normally be masked during subunit assembly (Zerangue et
al., 1999).
The RXR motif: a growing family of ER
retention/retrieval signals
Recent work on K
ATP
potassium channels and GABA
B
recep-
tors has revealed a novel class of RXR ER retention/retrieval
motifs (Zerangue et al., 1999; Margeta-Mitrovic et al., 2000).
These cytoplasmic RXR motifs differ from the classic KKXX
cytoplasmic ER retention motifs in that location of the RXR is
not limited to the most C-terminal domain of the molecule
(Teasdale and Jackson, 1996; Zerangue et al., 1999; Margeta-
Mitrovic et al., 2000). Here we report the identification of a
similar RXR motif in an intracellular domain of NR1. This
motif, also recently reported by Wenthold and colleagues
(Standley et al., 2000), extends the reported RXR family to
include ligand-gated ion channels, as well as potassium chan-
nels, ATP-binding cassette proteins, and G-protein-coupled
receptors (Zerangue et al., 1999; Bichet et al., 2000; Margeta-
Mitrovic et al., 2000), and suggests that the RXR ER reten-
tion/retrieval motif is a general quality control mechanism
governing the ER exit of multisubunit membrane proteins.
In other RXR-containing proteins, RXR motifs are masked by
assembly with additional subunits and thus coordinate the assem-
bly and stoichiometry of mature receptor complexes (Zerangue et
al., 1999; Margeta-Mitrovic et al., 2000). As with K
ATP
channels
and GABA
B
receptors, NMDA receptors are heteromeric pro-
tein complexes comprised of multiple polypeptide subunits (NR1
and NR2) that must coassemble to form a functional receptor
channel. Although this study has focused on the NR subunit, it is
likely that efficient assembly and trafficking of NMDA receptors
through the secretory pathway will depend on additional as yet
unidentified domains in both NR1 and NR2. Indeed, C-terminal
domains of NR2 are required for efficient synaptic targeting of
NMDA receptors (Mori et al., 1998; Sprengel et al., 1998; Steiger-
wald et al., 2000), and N-terminal extracellular regions are re-
quired for efficient assembly and ER exit of AMPA receptor
subunits (Leuschner and Hoch, 1999).
It is tempting to speculate that the obligate heteromeric nature
of NMDA receptors arises, in part, because of NR2-dependent
masking of NR1 ER retention domains. Alternatively, NR2
subunits may facilitate surface expression by providing an ER
export signal (Ma et al., 2001) that competes with RXR-mediated
ER retention. The recently appreciated complexity of ER reten-
tion and ER export signals present in K
⫹
channels (Zerangue et
al., 1999; Schwappach et al., 2000; Ma et al., 2001) raises the
possibility that multiple mechanisms and signals regulate NMDA
receptor trafficking through the ER and Golgi. In particular, NR2
forward-trafficking signals, NR2-dependent masking of ER re-
tention motifs, and phosphorylation-dependent masking of ER
retention motifs could each contribute at different stages in the
secretory pathway.
It will be important for future studies to determine how ER
retention motifs in NR1 interact with NR2 to orchestrate NMDA
receptor assembly, as well as identify the relationship between
NR1 RXR domains and other trafficking signals within mature
NMDA receptor complexes. Furthermore, additional intracellu-
Figure 6. The PDZ-binding domain located in the C2⬘ domain of NR1
suppresses RXR-mediated ER retention. A, Surface and intracellular
expression of Tac–NR1 receptors in COS7 cells is shown. Receptors
with the C2⬘ domain (Tac–NR1e, Tac–NR1g) reach the plasma mem-
brane, but deletion of the terminal PDZ-binding domain (Tac–NR1e
⌬VSTVV) prevents surface expression. B, Surface and intracellular
localization of Tac–NR1e, Tac–NR1e ⌬VSTVV, and Tac–NR1g in
hippocampal neurons is shown. C, Quantification of Tac–NR1 chime-
ras in HEK293 cells is presented. The fluorescence intensity of 5,000–
10,000 transfected cells was determined as described in Figure 1C
(*p ⬍ 0.05 compared with Tac minus primary antibody control, one-
way ANOVA). D, Tac–NR1e ⌬VSTVV is sensitive to deglycosylation
by endo H, indicating that it is retained in the ER. Tac–NR1e is
resistant to endo H but remains sensitive to PNGase F, indicating that
it is able to leave the ER and enter the Golgi. Arrows designate mature
forms of Tac. Arrowheads designate immature Tac proteins. Horizontal
bars on the right indicate the location of the 49.5 kDa molecular mass
marker. Scale bars: A,10
m; B,25
m.
3070 J. Neurosci., May 1, 2001, 21(9):3063–3072 Scott et al. • ER Retention of NMDA Receptors
lar signaling mechanisms may control the plasma membrane
expression of RXR-containing protein complexes independent of
multisubunit assembly. For example, our work demonstrates that
protein phosphorylation and the introduction of a consensus
PDZ-binding domain near the RXR motif can block RXR-
mediated ER retention/retrieval.
PDZ interactions and intracellular trafficking of
NMDA receptors
PDZ proteins play a prominent role in efficiently trafficking,
localizing, and/or anchoring membrane proteins and signaling
molecules to discrete cellular subdomains (Fanning and Ander-
son, 1999; Garner et al., 2000). In the case of NMDA receptors,
NR2 C-terminal domains interact with PDZ domains of the
synaptic scaffolding protein postsynaptic density-95 and related
family members to target and anchor NMDA receptor complexes
at synapses (Garner et al., 2000; Sheng and Lee, 2000). Unlike the
PDZ-binding motifs of NR2 subunits (IESDV), the binding part-
ners and cellular function of the consensus C2⬘ PDZ-binding
motif (VSTVV) of NR1 remain unknown [Bassand et al. (1999),
but see Kornau et al. (1995)].
Our work demonstrates that the consensus C2⬘ PDZ-binding
motif of NR1 antagonizes the RXR ER retention/retrieval motif
found in C1. These results are in agreement with a recent study
identifying suppression of NR1 ER retention via the C2⬘ consen-
sus PDZ-binding domain (Standley et al., 2000). This ER-
releasing activity is the first ascribed function for this potential
PDZ-binding domain and raises the possibility that PDZ-
mediated interactions play a more “active” role in the early events
of NMDA receptor trafficking. Indeed, efficient anterograde traf-
ficking and plasma membrane delivery of pro-TGF-
␣
is depen-
dent on the PDZ-mediated interaction between a very similar
C-terminal TVV sequence in pro-TGF-
␣
and syntenin/TACIP18
(Fernandez-Larrea et al., 1999). In addition, PDZ domain-
mediated active transport has been implicated recently in the
trafficking of NMDA receptor-containing vesicles along microtu-
bules in neuronal dendrites by linking NR2 subunits to the motor
protein KIF17 via a Lin7/Lin2/Lin10 PDZ protein complex
(Setou et al., 2000). Importantly, because C2⬘ domain-binding
partners are currently unknown, we cannot exclude the possibility
that the VSTVV motif mediates forward trafficking of NMDA
receptors via non-PDZ interactions. It will be important for
future studies to identify C2⬘ domain-binding partners and de-
termine their functional role in ER–Golgi transport of NMDA
receptors.
PKC phosphorylation and plasma membrane insertion
of NMDA receptors
Accumulation of glutamate receptors in the postsynaptic mem-
brane is regulated by synaptic activity and is a critical feature of
synapse formation (Luscher et al., 2000; Malinow et al., 2000).
The plasma membrane insertion of new NMDA receptors occurs
within 1–2 hr of initial contact by an active presynaptic terminal
(Friedman et al., 2000), and at more mature synapses, synaptic
insertion is proposed to occur within hours of experience-
dependent activation (Quinlan et al., 1999). In addition, chronic
increases or decreases in the level of synaptic activity cause
reciprocal changes in the synaptic accumulation of NMDA re-
ceptors (Rao and Craig, 1997; Liao et al., 1999; Watt et al., 2000).
Such dynamic control of NMDA receptor membrane insertion
may occur by regulated release of NMDA channels from the ER.
Our results indicate that potential PDZ interactions and phos-
phorylation dynamically regulate NMDA receptor ER retention.
Interestingly, previous studies have shown that PKC activation
potentiates NMDA receptor activity in neurons (Chen and
Huang, 1992; MacDonald et al., 1998), perhaps by promoting the
surface delivery of receptors. However, PKC potentiation of
NMDA receptor activity is not dependent on phosphorylation of
the C1 domain (Sigel et al., 1994; Zheng et al., 1999) and occurs
within minutes of phorbol ester treatment (Chen and Huang,
1992; Sigel et al., 1994). In contrast, we find that PKC phosphor-
ylation relieves NMDA receptor ER retention, leading to robust
surface expression of receptors after 2–3 hr. This latency in
surface expression suggests that the number of NMDA receptors
at the plasma membrane may reflect kinase signaling events that
occurred hours earlier. In addition, the delay we observe in the
insertion of NMDA receptors at the plasma membrane is consis-
tent with the time course of synaptogenesis- and experience-
dependent insertion of NMDA receptors (Quinlan et al., 1999;
Friedman et al., 2000). Although the initial appearance of
NMDA receptors at the plasma membrane after PKC activation
may occur earlier than the peak surface expression at 2–3 hr,
these findings suggest that phorbol ester potentiation of NMDA
receptor activity and PKC-mediated suppression of NMDA re-
ceptor ER retention are mechanistically distinct processes.
Note added in proof. After submission of this manuscript, two addi-
tional studies have reported ER retention/retrieval motifs in GABA
B
receptors (Calver et al., 2001; Pagano et al., 2001).
REFERENCES
Bassand P, Bernard A, Rafiki A, Gayet D, Khrestchatisky M (1999)
Differential interaction of the tSXV motifs of the NR1 and NR2A
NMDA receptor subunits with PSD-95 and SAP97. Eur J Neurosci
11:2031–2043.
Bichet D, Cornet V, Geib S, Carlier E, Volsen S, Hoshi T, Mori Y, De
Waard M (2000) The I-II loop of the Ca
2⫹
channel alpha1 subunit
contains an endoplasmic reticulum retention signal antagonized by the
beta subunit. Neuron 25:177–190.
Blount P, Smith MM, Merlie JP (1990) Assembly intermediates of the
mouse muscle nicotinic acetylcholine receptor in stably transfected
fibroblasts. J Cell Biol 111:2601–2611.
Brodsky JL, McCracken AA (1999) ER protein quality control and
proteasome-mediated protein degradation. Semin Cell Dev Biol
10:507–513.
Burke B, Griffiths G, Reggio H, Louvard D, Warren G (1982) A mono-
clonal antibody against a 135-K Golgi membrane protein. EMBO J
1:1621–1628.
Calver AR, Robbins MJ, Cosio C, Rice SQ, Babbs AJ, Hirst WD,
Bayfield I, Wood MD, Russell RB, Price GW, Couve A, Moss SJ,
Pangalos MN (2001) The C-terminal domains of the GABA
B
recep-
tor subunits mediate intracellular trafficking but are not required for
receptor signaling. J Neurosci 21:1203–1210.
Chen L, Huang LY (1992) Protein kinase C reduces Mg
2⫹
block of
NMDA-receptor channels as a mechanism of modulation. Nature
356:521–523.
Craven SE, Bredt DS (2000) Synaptic targeting of the postsynaptic den-
sity protein PSD-95 mediated by a tyrosine-based trafficking signal.
J Biol Chem 275:20045–20051.
Ehlers MD, Tingley WG, Huganir RL (1995) Regulated subcellular dis-
tribution of the NR1 subunit of the NMDA receptor. Science
269:1734–1737.
Ellgaard L, Molinari M, Helenius A (1999) Setting the standards: quality
control in the secretory pathway. Science 286:1882–1888.
El Meskini R, Galano GJ, Marx R, Mains RE, Eipper BA (2001) Tar-
geting of membrane proteins to the regulated secretory pathway in
anterior pituitary endocrine cells. J Biol Chem 276:3384–3393.
Fanning AS, Anderson JM (1999) PDZ domains: fundamental building
blocks in the organization of protein complexes at the plasma mem-
brane. J Clin Invest 103:767–772.
Fernandez-Larrea J, Merlos-Suarez A, Urena JM, Baselga J, Arribas J
(1999) A role for a PDZ protein in the early secretory pathway for the
targeting of proTGF-alpha to the cell surface. Mol Cell 3:423–433.
Friedman HV, Bresler T, Garner CC, Ziv NE (2000) Assembly of new
individual excitatory synapses: time course and temporal order of
synaptic molecule recruitment. Neuron 27:57–69.
Garner CC, Nash J, Huganir RL (2000) PDZ domains in synapse assem-
bly and signalling. Trends Cell Biol 10:274–280.
Scott et al. • ER Retention of NMDA Receptors J. Neurosci., May 1, 2001, 21(9):3063–3072 3071
Ghosh RN, Mallet WG, Soe TT, McGraw TE, Maxfield FR (1998) An
endocytosed TGN38 chimeric protein is delivered to the TGN after
trafficking through the endocytic recycling compartment in CHO cells.
J Cell Biol 142:923–936.
Green WN, Claudio T (1993) Acetylcholine receptor assembly: subunit
folding and oligomerization occur sequentially. Cell 74:57–69.
Hall RA, Soderling TR (1997) Differential surface expression and phos-
phorylation of the N-methyl-D-aspartate receptor subunits NR1 and
NR2 in cultured hippocampal neurons. J Biol Chem 272:4135–4140.
Hartmann E, Prehn S (1994) The N-terminal region of the alpha-subunit
of the TRAP complex has a conserved cluster of negative charges.
FEBS Lett 349:324–326.
Hirschberg K, Miller CM, Ellenberg J, Presley JF, Siggia ED, Phair RD,
Lippincott-Schwartz J (1998) Kinetic analysis of secretory protein
traffic and characterization of Golgi to plasma membrane transport
intermediates in living cells. J Cell Biol 143:1485–1503.
Huh KH, Wenthold RJ (1999) Turnover analysis of glutamate receptors
identifies a rapidly degraded pool of the N-methyl-D-aspartate receptor
subunit, NR1, in cultured cerebellar granule cells. J Biol Chem
274:151–157.
Jansen EJ, Holthuis JC, McGrouther C, Burbach JP, Martens GJ (1998)
Intracellular trafficking of the vacuolar H⫹-ATPase accessory subunit
Ac45. J Cell Sci 111:2999–3006.
Kornau HC, Schenker LT, Kennedy MB, Seeburg PH (1995) Domain
interaction between NMDA receptor subunits and the postsynaptic
density protein PSD-95. Science 269:1737–1740.
Leonard WJ, Depper JM, Robb RJ, Waldmann TA, Greene WC (1983)
Characterization of the human receptor for T-cell growth factor. Proc
Natl Acad Sci USA 80:6957–6961.
Leuschner WD, Hoch W (1999) Subtype-specific assembly of alpha-
amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptor subunits
is mediated by their N-terminal domains. J Biol Chem
274:16907–16916.
Liao D, Zhang X, O’Brien R, Ehlers MD, Huganir RL (1999) Regula-
tion of morphological postsynaptic silent synapses in developing hip-
pocampal neurons. Nat Neurosci 2:37–43.
Luscher C, Nicoll RA, Malenka RC, Muller D (2000) Synaptic plasticity
and dynamic modulation of the postsynaptic membrane. Nat Neurosci
3:545–550.
Ma D, Zerangue N, Lin Y, Collins A, Yu M, Jan Y, Jan L (2001) Role
of ER export signals in controlling surface potassium channel numbers.
Science 291:316–319.
MacDonald JF, Xiong XG, Lu WY, Raouf R, Orser BA (1998) Modu-
lation of NMDA receptors. Prog Brain Res 116:191–208.
Malenka RC, Nicoll RA (1999) Long-term potentiation—a decade of
progress? Science 285:1870–1874.
Malinow R, Mainen ZF, Hayashi Y (2000) LTP mechanisms: from si-
lence to four-lane traffic. Curr Opin Neurobiol 10:352–357.
Margeta-Mitrovic M, Jan YN, Jan LY (2000) A trafficking checkpoint
controls GABA(B) receptor heterodimerization. Neuron 27:97–106.
McIlhinney RA, Molnar E, Atack JR, Whiting PJ (1996) Cell surface
expression of the human N-methyl-D-aspartate receptor subunit 1a
requires the co-expression of the NR2A subunit in transfected cells.
Neuroscience 70:989–997.
McIlhinney RA, Le Bourdelles B, Molnar E, Tricaud N, Streit P, Whiting
PJ (1998) Assembly intracellular targeting and cell surface expression
of the human N-methyl-D-aspartate receptor subunits NR1a and NR2A
in transfected cells. Neuropharmacology 37:1355–1367.
Milgram SL, Mains RE, Eipper BA (1996) Identification of routing
determinants in the cytosolic domain of a secretory granule-associated
integral membrane protein. J Biol Chem 271:17526–17535.
Monyer H, Sprengel R, Schoepfer R, Herb A, Higuchi M, Lomeli H,
Burnashev N, Sakmann B, Seeburg PH (1992) Heteromeric NMDA
receptors: molecular and functional distinction of subtypes. Science
256:1217–1221.
Mori H, Mishina M (1995) Structure and function of the NMDA recep-
tor channel. Neuropharmacology 34:1219–1237.
Mori H, Manabe T, Watanabe M, Satoh Y, Suzuki N, Toki S, Nakamura
K, Yagi T, Kushiya E, Takahashi T, Inoue Y, Sakimura K, Mishina M
(1998) Role of the carboxy-terminal region of the GluR epsilon2 sub-
unit in synaptic localization of the NMDA receptor channel. Neuron
21:571–580.
O’Brien RJ, Lau LF, Huganir RL (1998) Molecular mechanisms of
glutamate receptor clustering at excitatory synapses. Curr Opin Neu-
robiol 8:364–369.
Okabe S, Miwa A, Okado H (1999) Alternative splicing of the
C-terminal domain regulates cell surface expression of the NMDA
receptor NR1 subunit. J Neurosci 19:7781–7792.
Ozawa S, Kamiya H, Tsuzuki K (1998) Glutamate receptors in the
mammalian central nervous system. Prog Neurobiol 54:581–618.
Pagano A, Rovelli G, Mosbacher J, Lohmann T, Duthey B, Stauffer D,
Ristig D, Schuler V, Meigel I, Lampert C, Stein T, Prezeau L, Blahos
J, Pin JP, Froestl W, Kuhn R, Heid J, Kaupmann K, Behler B (2001)
C-terminal interaction is essential for surface trafficking but not for
heteromeric assembly of GABA
B
receptors. J Neurosci 21:1189–1202.
Quinlan EM, Philpot BD, Huganir RL, Bear MF (1999) Rapid,
experience-dependent expression of synaptic NMDA receptors in vi-
sual cortex in vivo. Nat Neurosci 2:352–357.
Rao A, Craig AM (1997) Activity regulates the synaptic localization of
the NMDA receptor in hippocampal neurons. Neuron 19:801–812.
Sanders SL, Schekman R (1992) Polypeptide translocation across the
endoplasmic reticulum membrane. J Biol Chem 267:13791–13794.
Schwappach B, Zerangue N, Jan YN, Jan LY (2000) Molecular basis for
K(ATP) assembly: transmembrane interactions mediate association of
aK⫹ channel with an ABC transporter. Neuron 26:155–167.
Setou M, Nakagawa T, Seog DH, Hirokawa N (2000) Kinesin superfam-
ily motor protein KIF17 and mLin-10 in NMDA receptor-containing
vesicle transport. Science 288:1796–1802.
Sheng M, Lee SH (2000) Growth of the NMDA receptor industrial
complex. Nat Neurosci 3:633–635.
Sigel E, Baur R, Malherbe P (1994) Protein kinase C transiently acti-
vated heteromeric N-methyl-D-aspartate receptor channels indepen-
dent of the phosphorylatable C-terminal splice domain and of consen-
sus phosphorylation sites. J Biol Chem 269:8204–8208.
Songyang Z, Fanning AS, Fu C, Xu J, Marfatia SM, Chishti AH, Cromp-
ton A, Chan AC, Anderson JM, Cantley LC (1997) Recognition of
unique carboxyl-terminal motifs by distinct PDZ domains. Science
275:73–77.
Sprengel R, Suchanek B, Amico C, Brusa R, Burnashev N, Rozov A,
Hvalby O, Jensen V, Paulsen O, Andersen P, Kim JJ, Thompson RF,
Sun W, Webster LC, Grant SG, Eilers J, Konnerth A, Li J, McNamara
JO, Seeburg PH (1998) Importance of the intracellular domain of
NR2 subunits for NMDA receptor function in vivo. Cell 92:279–289.
Standley S, Roche KW, McCallum J, Sans N, Wenthold RJ (2000) PDZ
domain suppression of an ER retention signal in NMDA receptor NR1
splice variants. Neuron 28:887–898.
Steigerwald F, Schulz TW, Schenker LT, Kennedy MB, Seeburg PH,
Kohr G (2000) C-terminal truncation of NR2A subunits impairs syn-
aptic but not extrasynaptic localization of NMDA receptors. J Neurosci
20:4573–4581.
Tan PK, Waites C, Liu Y, Krantz DE, Edwards RH (1998) A leucine-
based motif mediates the endocytosis of vesicular monoamine and
acetylcholine transporters. J Biol Chem 273:17351–17360.
Tarentino AL, Gomez CM, Plummer Jr TH (1985) Deglycosylation of
asparagine-linked glycans by peptide:N-glycosidase F. Biochemistry
24:4665–4671.
Teasdale RD, Jackson MR (1996) Signal-mediated sorting of membrane
proteins between the endoplasmic reticulum and the Golgi apparatus.
Annu Rev Cell Dev Biol 12:27–54.
Tingley WG, Roche KW, Thompson AK, Huganir RL (1993) Regula-
tion of NMDA receptor phosphorylation by alternative splicing of the
C-terminal domain. Nature 364:70–73.
Tingley WG, Ehlers MD, Kameyama K, Doherty C, Ptak JB, Riley CT,
Huganir RL (1997) Characterization of protein kinase A and protein
kinase C phosphorylation of the N-methyl-D-aspartate receptor NR1
subunit using phosphorylation site-specific antibodies. J Biol Chem
272:5157–5166.
Trimble RB, Maley F (1984) Optimizing hydrolysis of N-linked high-
mannose oligosaccharides by endo-beta-N-acetylglucosaminidase H.
Anal Biochem 141:515–522.
Watt AJ, van Rossum MC, MacLeod KM, Nelson SB, Turrigiano GG
(2000) Activity coregulates quantal AMPA and NMDA currents at
neocortical synapses. Neuron 26:659–670.
Zerangue N, Schwappach B, Jan YN, Jan LY (1999) A new ER traffick-
ing signal regulates the subunit stoichiometry of plasma membrane
K(ATP) channels. Neuron 22:537–548.
Zheng X, Zhang L, Wang AP, Bennett MV, Zukin RS (1999) Protein
kinase C potentiation of N-methyl-D-aspartate receptor activity is not
mediated by phosphorylation of N-methyl-D-aspartate receptor sub-
units. Proc Natl Acad Sci USA 96:15262–15267.
3072 J. Neurosci., May 1, 2001, 21(9):3063–3072 Scott et al. • ER Retention of NMDA Receptors
