Content uploaded by Gabriela Lavezzari
Author content
All content in this area was uploaded by Gabriela Lavezzari on Nov 09, 2015
Content may be subject to copyright.
Available via license: CC BY-NC-SA 4.0
Content may be subject to copyright.
Cellular/Molecular
Subunit-Specific Regulation of NMDA Receptor Endocytosis
Gabriela Lavezzari, Jennifer McCallum, Colleen M. Dewey, and Katherine W. Roche
National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, Maryland 20892
At excitatory synapses, both NMDA and AMPA receptors are localized to the postsynaptic density (PSD). However, unlike AMPA
receptors, synaptic NMDA receptors are stable components of the PSD. Even so, surface-expressed NMDA receptors undergo endocyto-
sis, which is more robust early in development and declines during synaptic development. We investigated the subunit-specific contri-
butions to NMDA receptor endocytosis, specifically defining the endocytic motifs and endocytic pathways preferred by the NR2A and
NR2B subunits. We find that NR2A and NR2B have distinct endocytic motifs encoded in their distal C termini and that these interact with
clathrin adaptor complexes with differing affinities. We also find that NR2A and NR2B sort into different intracellular pathways after
endocytosis, with NR2B preferentially trafficking through recycling endosomes. In mature cultures, we find that NR2B undergoes more
robust endocytosis than NR2A, consistent with previous studies showing thatNR2A is more highly expressed at stable synaptic sites. Our
findings demonstrate fundamental differences between NR2A and NR2B that help clarify developmental changes in NMDA receptor
trafficking and surface expression.
Key words: internalization; clathrin; endocytosis; glutamate receptors; trafficking; endocytic sorting
Introduction
Glutamate receptors mediate the majority of excitatory neuro-
transmission in the CNS and are essential for synaptic develop-
ment and plasticity. Although the synaptic localization of gluta-
mate receptors is tightly regulated, it is becoming clear that the
targeting and anchoring of the various subtypes of glutamate recep-
tors are differentially regulated. For example, synaptic AMPA recep-
tors undergo rapid and robust cycling at the postsynaptic membrane
(Luscher et al., 1999; Sheng, 2001; Malinow and Malenka, 2002),
whereas synaptic NMDA receptors are relatively stable (Luscher et
al., 1999; Wenthold et al., 2003). Although synaptic NMDA recep-
tors are tightly anchored to the postsynaptic density (PSD), it is clear
that NMDA receptors are quite mobile within neurons, via intracel-
lular pathways and lateral diffusion in the membrane (Rao and
Craig, 1997; Tovar and Westbrook, 1999; Standley et al., 2000; Scott
et al., 2001; Snyder et al., 2001; Xia et al., 2001; Washbourne et al.,
2002). In addition, surface-expressed NMDA receptors have been
shown to internalize both in primary neuronal cultures and when
expressed in heterologous cells (Roche et al., 2001). The regulation of
NMDA receptor endocytosis is therefore likely to be a critical deter-
minant of NMDA receptor surface expression, both at synaptic and
extrasynaptic sites.
NMDA receptors are tetramers comprising NR1, NR2
(NR2A–NR2D), and NR3 (NR3A–NR3B) subunits. In the fore-
brain, endogenous NMDA receptors are thought to be composed
of NR1 subunits combined with NR2B early in development and
heteromeric combinations of NR1, NR2B, and NR2A in mature
neurons (Kew et al., 1998; Li et al., 1998; Vicini et al., 1998;
Rumbaugh and Vicini, 1999; Tovar and Westbrook, 1999).
NMDA receptor endocytosis is developmentally regulated, with
robust endocytosis early in development that declines as neurons
mature and synapses form (Roche et al., 2001). Interestingly, the
decline in NMDA receptor endocytosis correlates well with the
known changes in NMDA receptor subunit composition during
development. Previous studies have defined an endocytic motif
encoded within the NR2B subunit. This motif, YEKL, is located
just upstream of the PSD-95–Discs large–zona occludens-1
(PDZ)-binding domain on the extreme C terminus of NR2B
(Roche et al., 2001). However, it is not known whether NR2A also
contains endocytic motifs within its C terminus.
In the current study, we have characterized the specific con-
tributions of the NR2B and NR2A subunits to NMDA receptor
endocytosis using a variety of approaches. We find that NR2A
and NR2B contain different sorting motifs within their distal C
termini that regulate trafficking to distinct intracellular pathways
after endocytosis. In neurons, we find that NR2B preferentially
sorts to recycling endosomes, consistent with the more robust
internalization of NR2B-containing NMDA receptors. NR2B ex-
pression drives NMDA receptor endocytosis in both immature
and mature neurons, whereas NR2A endocytosis is most notable
at early stages of development. These results are consistent with
the presence of stable NR2A-containing NMDA receptor com-
plexes at mature synapses. Our findings demonstrate a strong
subunit-specific contribution to NMDA receptor endocytosis
and stability on the plasma membrane.
Materials and Methods
Antibodies and DNA constructs. The primary antibodies Tac 7G7 (Amer-
ican Type Culture Collection, Manassas, VA) and FLAG M2 (Sigma, St.
Received Feb. 6, 2004; accepted May 28, 2004.
WethankDaynaHayesforcontributiontothepreparationoftheprimaryneuronalcultures,theNationalInstitute
of Neurological Disorders and Stroke (NINDS) Light Imaging Facility, in particular the facility manager Dr. Carolyn
Smith, for advice and expertise in the collection and analysis of the confocal images, and the NINDS Sequencing
Facility for automated DNA sequence analysis.
Correspondence should be addressed to K. W. Roche, National Institute of Neurological Disorders and Stroke,
National Institutes of Health, Building 36, Room 5B20, Bethesda, MD 20892. E-mail: rochek@ninds.nih.gov.
DOI:10.1523/JNEUROSCI.1890-04.2004
Copyright © 2004 Society for Neuroscience 0270-6474/04/246383-09$15.00/0
The Journal of Neuroscience, July 14, 2004 • 24(28):6383– 6391 • 6383
Louis, MO) were used for immunofluorescence. All secondary antibod-
ies were obtained from Molecular Probes (Eugene, OR), except for cya-
nine 5 (Cy5)-conjugated anti-rabbit, which was obtained from Jackson
ImmunoResearch (West Grove, PA). The following cDNA constructs
were obtained as gifts: FLAG–NR2B (F. A. Stephenson, School of Phar-
macy, University of London, London, UK), green fluorescent protein
(GFP)–Rab5, GFP–Rab7, GFP–Rab9,
1,
2,
3a, and
4 in pACTII,
and the trans-Golgi network (TGN)38 full C terminus in pGBT9 (Juan
Bonifacino, National Institute of Child Health and Human Develop-
ment, National Institutes of Health, Bethesda, MD); and GFP–Rab11
(James Goldenring, Vanderbilt University, Nashville, TN). The TGN38
C terminus was subcloned into the pBHA vector using EcoRI and PstI
restriction sites. Both the NR2A and NR2B C termini were amplified by
PCR. The amplified tails included amino acids 1315–1482 (NR2B) or
1304 –1464 (NR2A) and were subcloned using EcoRI restriction sites into
pBHA vector or with EcoRV and XbaI into Tac vector. The TacNR2A
truncations, TacNR2A⌬7 and TacNR2A⌬11, were obtained using PCR.
FLAG-NR2A was constructed in the laboratory using PCR with the fol-
lowing sequence: 5⬘-GAACTTCGAAATCTGGACTACAAGGACGA-
CGATGACAAGTGGGGCCCAGAGCAG-3⬘ and 3⬘-CTGCTCTGGGC-
CCCACTTGTCATCGTCGTCCTTGTAGTCCAGATTTCGAAGTTC-5⬘,
inserting the FLAG epitope between amino acids 54 and 55 of NR2A. The
bold sequence indicates the FLAG peptide. FLAG–NR2A⌬11 L1320A was
constructed in the laboratory using site-directed mutagenesis. All mutations
were confirmed using automated sequence analysis.
Yeast two-hybrid direct-interaction assays. Direct-interaction assays of
NR2B (amino acids 1315–1482), NR2A (1304 –1464), or the TGN38 C
terminus binding to adaptor medium chains were performed as de-
scribed previously (Kim et al., 1995). Briefly, NR2B (amino acids 1315–
1482), NR2A (1304–1464), or TGN38 in the LexA DNA binding domain
vector pBHA and medium chains in the GAL4 activation domain vector
pACTII were cotransformed into L40 yeast cells. For each condition,
one-half of the yeast cells were plated on dextrose plates containing his-
tidine but lacking leucine and tryptophan (His-containing), and the
other half were plated on dextrose plates lacking histidine, leucine, and
tryptophan (His-deficient). Nutritional selection was used as a measure
of a direct interaction. For nutritional selection, growth on His-deficient
plates was scored on a ⫺ to ⫹⫹⫹⫹ scale for conditions with equivalent
growth on His-containing plates.
Immunofluorescent internalization assay in HeLa cells. HeLa cells
(American Type Culture Collection) grown on coverslips were trans-
fected with the cDNAs indicated (4
g of DNA per well in a six well dish)
using the calcium phosphate coprecipitation method, and experiments
were performed 48 hr later. Transfected cells were washed in PBS, incu-
bated with anti-Tac antibody for 1 hr on ice, washed with PBS, and
returned to conditioned media at 37°C for 15 min to allow internaliza-
tion. The cells were washed in PBS, fixed in 4% paraformaldehyde in PBS
for 15 min, washed in PBS, and permeabilized in 0.25% Triton X-100 in
PBS for 5 min. The coverslips were washed in PBS, incubated with Alexa
568-conjugated anti-mouse secondary antibodies for 30 min at room
temperature, washed, and mounted with ProLong Antifade Kit (Molec-
ular Probes). When double labeling [early endosome autoantigen 1
(EEA1); PSD-95], the cells were incubated with the appropriate primary
antibody for 1 hr at room temperature after permeabilization. The cells
were washed in PBS, incubated with both anti-mouse and anti-rabbit
secondary antibodies (1:500) for 30 min at room temperature, washed in
PBS, and mounted. Images were collected with a 63⫻ objective on a Zeiss
(Oberkochen, Germany) LSM 510. Series of optical sections were col-
lected at intervals of 0.34
m. Figures show maximum projections. For
the statistical analysis, images from five different HeLa cells in three
independent experiments were collected at a 63⫻ objective with a Zeiss
Axioplan 2 microscope and analyzed with the OpenLab colocalization
module (Improvision, Lexington, MA). Values represent the mean ⫾
SEM of Pearson’s correlation.
Quantitative assay of internalization in HeLa cells. HeLa cells were
grown on 12 well dishes and transiently transfected with Tac–NMDA
receptor chimeras. The cells were incubated with anti-Tac antibody for 1
hr on ice to label surface-expressed protein. Cells were either left on ice or
washed and returned to conditioned media for 15 min at 37°C to allow
internalization. Cells were then fixed for 15 min in 4% paraformaldehyde
without detergent permeabilization. After fixation, the cells were washed
and incubated for 45 min with [
125
I]-labeled anti-mouse antibody (25
Ci) at room temperature. After extensive washing, the cells were incu-
bated with 1
M NaOH for 15 min at room temperature, and the lysate was
collected and analyzed. Each condition was assayed in triplicate. For each
experiment, background antibody binding was measured on cells trans-
fected with empty vector (also in triplicate), and the average background
value was subtracted from the average experimental counts. Quantitative
assessment of total surface labeling was calculated by measuring the
amount of anti-Tac antibody present on cells maintained on ice and not
subjected to the 15 min, 37°C incubation. Quantitative assessment of
internalization was calculated by subtracting the amount of anti-Tac
antibody remaining on cells after the 15 min, 37°C incubation from the
total surface counts. Results are expressed as a ratio of internalization to
total. The graph in Figure 2B represents the average of five independent
experiments.
Transfected hippocampal neurons. Preparation of primary hippocam-
pal cultures was performed as described previously (Roche and Huganir,
1995) with minor changes. Briefly, cells were dissected from embryonic
day 18 Sprague Dawley rats (Harlan, Indianapolis, IN), dissociated in
0.25% trypsin, and incubated with 0.01% DNase. Dispersed neurons
were plated onto polyornithine–fibronectin-coated glass coverslips at a
density of 100,000 cells per well of a 12 well dish. The neurons were grown
in serum-free media (Neurobasal; Invitrogen, Gaithersburg, MD) with
B27 supplement (Invitrogen). The cultures were maintained in a 5% CO
2
incubator until transfected and processed for immunofluorescence. Hip-
pocampal neurons [3 or 10 d in vitro (DIV)] were transfected with the
appropriate cDNA using the calcium phosphate coprecipitation method.
Six micrograms of total DNA were used per well of the 12 well dish.
Precipitation was allowed to form on cells for ⬃45 min, and the cells were
washed twice with serum-free media. The cells were returned to condi-
tioned media and maintained at 37°Cina5%CO
2
incubator for an
additional 48 hr. The use and care of animals used in this study followed
the guidelines of the National Institutes of Health Animal Research Ad-
visory Committee.
Internalization and immunocytochemistry of neurons. Transfected neu-
rons were washed in PBS and incubated with polyclonal FLAG antibody
for 45 min at room temperature to label surface-expressed protein. The
antibody was removed, and the coverslips were returned to conditioned
media for 15 or 30 min at 37°C. The cells were washed in PBS and fixed
with 4% paraformaldehyde– 4% sucrose in PBS for 15 min. The cells
were incubated for 30 min at room temperature with Alexa 568-
conjugated (red) anti-rabbit secondary antibody (Molecular Probes) for
labeling the surface population. The cells were washed and permeabilized
in 0.25% Triton X-100 in PBS for 5 min. After a 30 min incubation with
10% normal goat serum (Vector Laboratories, Burlingame, CA), the cells
were then incubated with Alexa 488-conjugated (green) anti-rabbit sec-
ondary antibody for 30 min to specifically label the internalized popula-
tion. The cells were washed in PBS and mounted with a ProLong Antifade
kit (Molecular Probes). When neurons were cotransfected with GFP
constructs, the surface population was labeled with Alexa 568-
conjugated (red) anti-rabbit secondary antibody, and after permeabili-
zation, the internalized population was specifically labeled with Cy5-
conjugated (blue) anti-rabbit secondary antibody from Jackson
ImmunoResearch. Images were collected with a 63⫻ objective on a Zeiss
LSM 510. Series of optical sections were collected at intervals of 0.34
m.
Figures show maximum projections. For quantitative analysis, images
from three dendrites per neuron (three neurons per experiment) were
collected, and colocalization analysis was measured based on the data
collected in three to five independent experiments with Volocity 2 soft-
ware (Improvision). Values represent mean ⫾ SEM; n ⫽ 5.
Results
The NR2A C terminus contains an internalization motif
Endogenous NMDA receptors undergo robust endocytosis that
decreases as synapses develop and neurons mature. We have
demonstrated previously that NR2B contains important regula-
tory motifs that influence NMDA receptor internalization and
6384 • J. Neurosci., July 14, 2004 • 24(28):6383– 6391 Lavezzari et al. •Regulation of NMDA Receptor Endocytosis
stability on the plasma membrane (Roche et al., 2001; Lavezzari et
al., 2003). Whereas NR2B is expressed early in development,
NR2A is only expressed in mature neurons. To determine
whether this developmental change in NR2 subunit expression
influences the surface stability of NMDA receptors, we have be-
gun to identify sequences within NR2A that might regulate its
surface expression. Like NR2B, NR2A contains a PDZ-binding
domain motif at the extreme C terminus (⫺ESDV) (Fig. 1 A,B).
In addition, NR2A contains several potential internalization mo-
tifs, including a putative tyrosine-based endocytic motif and sev-
eral dileucine motifs. Both of these are consensus endocytic mo-
tifs that can regulate clathrin-mediated endocytosis (Ohno et al.,
1995, 1996; Bonifacino and Traub, 2003). It is known that NR2B
contains a tyrosine-based endocytic motif (YEKL) that binds di-
rectly to the AP-2 adaptor complex (Lavezzari et al., 2003) and
regulates endocytosis. Although the tyrosine residue is conserved
in both NR2A and NR2B, the surrounding residues in each sub-
unit are unique (YKKM vs YEKL) (Fig. 1A,B).
To determine whether any of these putative endocytic motifs
within the NR2A C terminus regulate surface expression, we have
created a chimera of the distal portion of NR2A (amino acids
1304 –1464) and the stable surface integral membrane protein
Tac (TacNR2A) (Fig. 1B), and evaluated endocytosis of this chi-
mera compared with wild-type Tac and the TacNR2B chimera
(NR2B amino acids 1315–1482) (Fig. 1 B). We were unable to
compare the distal C terminus to the entire C terminus, because
Tac chimeras using the entire C termini of NR2A or NR2B are
retained by the endoplasmic reticulum (ER) (data not shown)
(Hawkins et al., 2004). Transfected HeLa cells were surface-
labeled on ice for 1 hr with Tac antibody and returned to condi-
tioned media for 15 min to allow internalization. Whereas Tac
alone was expressed almost exclusively on
the plasma membrane, TacNR2A was in-
ternalized into vesicular structures similar
to TacNR2B (Fig. 1C). These data show
that the C terminus of NR2A contains a
sequence that is sufficient to induce inter-
nalization of Tac. Within the NR2A C ter-
minus there is also the PDZ domain-
binding motif (⫺ESDV) that is completely
conserved with NR2B and binds to the
PSD-95 family of proteins (Kim et al.,
1995; Kornau et al., 1995; Sheng, 2001). To
examine the role of PSD-95 in regulating
surface expression, we coexpressed TacNR2A
with PSD-95 in HeLa cells and evaluated
internalization of TacNR2A. As we had
observed previously in our examination of
NR2B-mediated endocytosis (Roche et al.,
2001; Lavezzari et al., 2003), coexpression
of PSD-95 blocked the internalization of
TacNR2A and clustered TacNR2A into
patches on the cell surface (Fig. 1D). Co-
expression of PSD-95 with TacNR2A⌬7,
which does not contain the PDZ-binding
motif, did not affect internalization of the
chimera (data not shown), demonstrating
that the direct interaction with PSD-95 is
essential.
To determine whether the conserved ty-
rosine within the YXXA endocytic motif in
NR2A regulates endocytosis, we truncated
the last 11 amino acids of the TacNR2A C
terminus to remove this putative motif (TacNR2A⌬11) (Fig. 1 B).
However, we found that this mutation did not suppress internaliza-
tion (Fig. 2). This result is in sharp contrast to previous findings
demonstrating the importance of the analogous tyrosine (1472) on
the endocytosis of TacNR2B (Roche et al., 2001; Lavezzari et al.,
2003). This result led us to investigate whether the dileucine motifs
in the NR2A C terminus were critical for internalization. We used
site-directed mutagenesis and mutated the three dileucine motifs
from LL to LA (TacNR2A⌬11 L1320A; TacNR2A⌬11 L1337A;
TacNR2A⌬11 L1358A). Both the immunofluorescence assay for in-
ternalization (Fig. 2 A) and an independent quantitative measure
of endocytosis (Fig. 2B) demonstrated that endocytosis of
TacNR2A⌬11 L1320A was significantly less compared with the
other TacNR2A chimeras. This therefore indicates that leucine 1320
regulates endocytosis of TacNR2A.
Differential binding of NR2A and NR2B to adaptor
medium chains
The medium chain of AP-2,
2, binds directly to internalized
cargo proteins, thereby linking them to clathrin and regulating
endocytosis (Ohno et al., 1995, 1996; Bonifacino and Traub,
2003). Although we have shown previously that the C terminus of
NR2B interacts directly with
2 (Lavezzari et al., 2003), we
wanted to determine whether the conserved tyrosine in the NR2A
C terminus also binds to
2. Using the yeast two-hybrid assay, we
found that unlike NR2B, the NR2A C terminus did not interact
with
2 (Fig. 3). In addition to AP-2, there are other intracellular
adaptor complexes (AP-1, AP-3, and AP-4), each of which pos-
sesses a unique medium chain and each of which regulates dis-
tinct transport steps in the endocytic pathway. Therefore, we
examined the ability of NR2A and NR2B to interact with different
Figure 1. The Cterminus ofNR2A contains anendocytic motifthat is inhibited byPSD-95 binding. A, Theamino acid sequence
of the distal C terminus of NR2A. Potential endocytic motifs (LL and YXXA) and the PDZ ligand (ESDV) are indicated in bold. B,
Schematicdiagrams oftheTac–NMDA receptor chimeras.The distalCterminus ofNR2B (aminoacids1315–1482) orNR2A (amino
acids 1304 –1464) was appended to the plasma membrane reporter molecule Tac (TacNR2B; TacNR2A). TacNR2A⌬7 (NR2A
amino acids 1304 –1457) is a truncation removing the PDZ-binding domain. TacNR2A⌬11 (amino acids 1304 –1453) is a trun-
cation removing both the PDZ-binding domain and the putative tyrosine-based endocytic motif. The transmembrane domain is
depicted as a striped rectangle, and the putative tyrosine-based motif and the PDZ-binding domain are depicted in bold. C, The
NR2B and NR2A C termini are both sufficient to induce internalization of the surface protein Tac into endosomal structures. HeLa
cells weretransiently transfected withTac, TacNR2B,or TacNR2A. Cells were incubatedwith Tac monoclonal antibody onice for 1
hr and then returned to conditioned media and incubated at 37°C for 15 min. The cells were fixed and permeabilized, and the
immunoreactivitywas visualizedusing Alexa 568-conjugated(red)anti-mouse secondaryantibodies. Images were collectedwith
a63⫻ objective onaZeissLSM. Seriesof opticalsections werecollected atintervals of0.34
m.Maximum projectionsareshown.
D, PSD-95 inhibits internalization of TacNR2A andclusters the chimera onthe cell surface. HeLacells were transiently transfected
with PSD-95 and TacNR2A. Cells were incubated with Tac monoclonal antibody on ice for 1 hr and then returned to conditioned
media and incubated at 37°C for 15 min. The cells were fixed, permeabilized, and incubated with PSD-95 polyclonal antibodies.
Immunoreactivity was visualized using a combination of Alexa 568-conjugated (red) anti-mouse and Alexa 488-conjugated
(green) anti-rabbit secondary antibodies. Images were collected with a 63⫻ objective on a Zeiss LSM. Series of optical sections
were collected atintervals of 0.34
m. Maximum projections are shown.
Lavezzari et al. •Regulation of NMDA Receptor Endocytosis J. Neurosci., July 14, 2004 • 24(28):6383– 6391 • 6385
adaptor medium chains in the yeast two-hybrid assay. The well
characterized binding between the adaptor medium chains and
the TGN38 C terminus was used as a positive control (Aguilar et
al., 2001). Most notable were the interactions with
1 and
2.
Interestingly, we found that the NR2A and NR2B C termini bind
equally well to
1, the adaptor subunit that plays a role in clathrin
vesicle formation at the TGN. However, the plasma membrane
adaptor subunit
2 interacted robustly with NR2B but not
NR2A. This is consistent with the YKKM motif in NR2A not
playing a critical role in plasma membrane endocytosis but po-
tentially being important for other intracellular sorting steps.
This correlates quite well with results of our endocytosis assays
(Fig. 2), which also revealed that the YKKM motif does not play
an important role in endocytosis.
The NR2A and NR2B C termini regulate trafficking to distinct
intracellular pathways
After we determined that the C terminus of NR2A was sufficient
to induce endocytosis of the stable surface protein Tac, we began
to characterize the intracellular compartments that contained in-
ternalized TacNR2A. Previous data demonstrated that TacNR2B
is colocalized with internalized transferrin (Roche et al., 2001),
revealing its presence in early endosomes. To determine whether
TacNR2A follows the same intracellular trafficking pathway as
TacNR2B, we followed the same approach as before, but ob-
served that although TacNR2B and TacNR2A both traffic to early
endosomes initially, they subsequently traffic to distinct com-
partments over time. To characterize the intracellular sorting, we
analyzed TacNR2A and TacNR2B over time using immunofluo-
rescence microscopy to examine their subcellular localization.
We incubated the cells on ice for 1 hr with Tac antibody and then
returned the cells to 37°C for various times to allow internaliza-
tion. After 5 min, TacNR2A colocalized completely with the early
endosome marker GFP–Rab5 (Fig. 4 A). At 15 min, we saw that
the amount of colocalization with GFP–Rab5 decreased and
TacNR2A trafficked into a different compartment (data not
shown). To identify this compartment we coexpressed the late
endosome marker GFP–Rab9 in these cells. At 15 min, we were
able to see partial colocalization of TacNR2A with GFP–Rab9-
positive late endosomes, and by 30 min, we found almost com-
plete colocalization (Fig. 4B–D).
We then quantitated the colocalization of TacNR2A with the
early endosomal marker EEA1 and the late endosomal marker
GFP–Rab9 over time (5, 15, and 30 min) (Fig. 4E). At 5 min,
TacNR2A colocalized well with EEA1 but not well with GFP–
Rab9. At 15 min, there was moderate colocalization with both
endosomal markers, and at 30 min, TacNR2A strongly colocal-
ized with GFP–Rab9, demonstrating that the NR2A C terminus
regulates sorting from early to late endosomes.
We had originally observed strong colocalization of TacNR2B
with transferrin at both 5 and 15 min (data not shown) (Roche et
al., 2001). In contrast to TacNR2A, we found very little colocal-
ization of TacNR2B with GFP–Rab9 after 30 min, indicating that
the NR2B C terminus did not regulate sorting to late endosomes.
At this time point, most of the internalized TacNR2B was distrib-
uted throughout the cell, with some accumulation at the cell
edges. This expression pattern was very different from the strong
perinuclear distribution of GFP–Rab9 that colocalized with in-
ternalized TacNR2A after 30 min. To define the compartment to
which TacNR2B trafficked at later time points, we coexpressed
cells with GFP–Rab11, a marker for recycling endosomes. After
30 min of internalization, we observed that the majority of inter-
nalized TacNR2B colocalized with GFP–Rab11 (Fig. 5A). These
data are consistent with the idea that the NR2B C terminus reg-
Figure 3. The NR2A C terminus and NR2B C terminus differentially bind to the medium
chains of adaptor complexes. The NR2A C terminus (amino acids 1304 –1464), the NR2B C
terminus (amino acids 1315–1482), or the TGN38 C terminus in the LexA DNA binding domain
wascotransformedwith
1,
2,
3
␣
,or
4intheGAL4activation domain as follows: (1)
1
and NR2A, (2)
2 and NR2A, (3)
3
␣
and NR2A, (4)
4 and NR2A, (5)
1 and NR2B, (6)
2
and NR2B, (7)
3
␣
and NR2B, (8)
4 and NR2B, (9)
1 and TGN38, (10)
2 and TGN38, (11)
3
␣
and TGN38, and(12)
4 and TGN38.Growth onHis-deficient plates was scoredon ascale
of ⫺ to ⫹⫹⫹⫹ for conditions with equivalent growth on His-containing plates.
Figure 2. TacNR2A internalization is dependent on a dileucine motif. A, HeLa cells were
transiently transfected with TacNR2A⌬11 or TacNR2A⌬11 L1320A. Cells were incubated with
Tac monoclonalantibody on icefor 1 hrand thenreturned to conditionedmedia and incubated
at 37°C for 15 min. The cells were fixed and permeabilized, and the immunoreactivity was
visualized using Alexa 568-conjugated (red) anti-mouse secondary antibodies. Images were
collected with a 63⫻ objective on a Zeiss LSM. Series of optical sections were collected at
intervals of 0.34
m. Maximum projections are shown. B, HeLa cells were transiently trans-
fected with TacNR2A, TacNR2A⌬11, TacNR2A⌬11 L1320A, TacNR2A⌬11 L1337A, or
TacNR2A⌬11 L1358A. The amount of internalization was determined using an independent
quantitative internalization assay as described in Materials and Methods. Values are expressed
as internalization per total (the total represents initial surface labeling; mean ⫾ SEM; n ⫽ 5).
Statistical comparison was performed using ANOVA. *p ⬍ 0.01.
6386 • J. Neurosci., July 14, 2004 • 24(28):6383– 6391 Lavezzari et al. •Regulation of NMDA Receptor Endocytosis
ulates sorting from early endosomes into recycling endosomes.
We then quantitated the colocalization of TacNR2B with the
early endosomal marker EEA1 and the recycling endosomal
marker GFP–Rab11 over time (5, 15, and 30 min) (Fig. 5B),
demonstrating that TacNR2B strongly colocalized with EEA1 at 5
min and that colocalization with Rab11 increased over time. The
relatively high colocalization of TacNR2B with both EEA1 and
GFP–Rab11 at all time points is consistent with continual cycling
through this endocytic pathway.
Developmental regulation of NR2A and NR2B trafficking
in neurons
Having characterized the trafficking of NR2A and NR2B chime-
ras in HeLa cells, we set out to determine whether NMDA recep-
tors in neurons also displayed differential endocytosis dependent
on subunit composition. We expressed full-length NR2A or
NR2B proteins containing an extracellular FLAG epitope in hip-
pocampal neurons at different stages of development. The use of
the FLAG epitope tag allows us to specifically label surface-
expressed NR2A or NR2B proteins on living cells. We transfected
hippocampal neurons at 10 DIV with FLAG–NR2A or FLAG–
NR2B. At 12 DIV, we labeled the surface-expressed receptors
with anti-FLAG antibody and returned the cells to 37°C for 15
min to allow internalization. The cells were then fixed and stained
with Alexa 568 (red)-conjugated secondary antibody to label the
surface population. The stained cells were then permeabilized,
and Alexa 488 (green)-conjugated secondary antibody was added
to specifically label the internalized population. Both NR2A and
NR2B internalized in these neurons (Fig. 6A); however, the in-
Figure 4. The NR2A C terminus regulates trafficking from early endosomes to late endo-
somes. A, After endocytosis, TacNR2A is localized to early endosomes at early time points. HeLa cells
were transiently transfected with TacNR2A and GFP–Rab5. Cells were incubated with Tac polyclonal
antibody onice for1 hr andthen returnedto conditionedmedia for 5min at37°C. Thecells were fixed
and permeabilized, and the immunoreactivitywas visualized using Alexa 568-conjugated (red) anti-
mouse. B–D, Over time, TacNR2A traffics to late endosomes. HeLa cells were transiently transfected
with TacNR2A and GFP–Rab9. Cells were incubated with Tac polyclonal antibody on ice for 1 hr and
then returned to conditioned media for 5 min ( B), 15 min ( C), or 30 min ( D) at 37°C. The cells were
fixed and permeabilized, andthe immunoreactivity was visualized using Alexa568-conjugated (red)
anti-mouse. Images were collected with a 63⫻ objective on a Zeiss LSM. Series of optical sections
were collectedat intervalsof 0.34
m. Maximumprojections areshown. E,Analysis ofthe amountof
colocalization betweenTacNR2A andEEA1 orGFP–Rab9. HeLacells weretransiently transfected with
TacNR2A. Cells were incubated with Tac polyclonal antibody on ice for 1 hr and then returned to
conditionedmediafor5, 15,or30minat37°C.The cellswerefixed,permeabilized,andincubatedwith
antibody for early endosomal marker EEA1. Immunoreactivity was visualized using a combination of
Alexa568-conjugated(red) anti-mouseIgG2AandAlexa488-conjugated(green) anti-mouseIgG1.In
separate experiments, HeLa cells were transiently transfected with TacNR2A and GFP–Rab9. Cells
were incubated with Tac polyclonal antibody on ice for 1 hr and then returned to conditioned media
for 5, 15, or 30 min at 37°C. The cells were fixed and permeabilized, and the immunoreactivity was
visualizedusingAlexa 568-conjugated(red)anti-mousesecondary antibodies.Forthestatisticalanal-
ysis, imagesfrom five differentHeLa cells inthree independent experiments werecollected at a63⫻
objective with a Zeiss Axioplan 2 microscope and analyzed with the OpenLab colocalization module
(Improvision). Values represent the mean ⫾ SEM of Pearson’s correlation.
Figure 5. The NR2B C terminus regulates trafficking to recycling endosomes. A, HeLa cells
were transiently transfected with TacNR2B and GFP–Rab11. Cells were incubated with Tac
polyclonal antibody on ice for 1 hr and then returned to conditioned media for 30 min at 37°C.
The cellswere fixed and permeabilized,and the immunoreactivityof internalized TacNR2B was
visualizedusing Alexa568-conjugated(red) anti-mouse.GFP–Rab11is visualizedasgreen,and
the merged image shows the level of colocalization of internalized TacNR2B with Rab11. B,
Analysis of the amount of colocalization between TacNR2B and EEA1 or GFP–Rab11 was per-
formed as described in the legend to Figure 4 E. For the statistical analysis, images from five
different HeLa cellsin threeindependent experimentswere collectedat a63⫻ objective witha
Zeiss Axioplan 2 microscope and analyzed with the OpenLab colocalization module (Improvi-
sion). Values represent the mean ⫾ SEM of Pearson’s correlation.
Lavezzari et al. •Regulation of NMDA Receptor Endocytosis J. Neurosci., July 14, 2004 • 24(28):6383– 6391 • 6387
ternalization of FLAG–NR2B was significantly greater than that
of FLAG–NR2A (36 vs 20%) (Fig. 6B).
It is possible that the NR2B subunit internalizes more effi-
ciently than NR2A because of the extrasynaptic localization of
NR2B at sites separate from the scaffolding protein PSD-95.
NR2A is only expressed later in development and is known to be
synaptically localized, whereas NR2B is highly expressed at both
synaptic and extrasynaptic sites (Li et al., 2002). Therefore, we
evaluated the internalization of NR2A and NR2B at earlier devel-
opmental times, when the cells are less mature, when synapses are
still developing, and when PSD-95 is known to be less abundant.
We transfected hippocampal neurons at 3 DIV with FLAG–
NR2A or FLAG–NR2B and performed the immunofluorescence
2 d later as described above. Although FLAG–NR2A consistently
internalized less well than FLAG–NR2B, the difference was not
significant, and NR2A and NR2B displayed similar levels of en-
docytosis (26 vs 31%) (Fig. 6). We subsequently analyzed
whether the same mutations that inhibited the internalization of
TacNR2A in HeLa cells had the same effect in hippocampal neu-
rons. We transfected neurons at 3 DIV with FLAG–NR2A and
FLAG–NR2A⌬11 L1320A but found no significant difference in
the internalization rate between the two constructs (data not
shown), suggesting that heteromeric complexes of NR2A in neu-
rons may use this motif differently or less efficiently than the
monomeric chimera in HeLa cells. Unfortunately, with the high
expression of NR2B in primary neuronal cultures at all develop-
mental stages, it is impossible for us to specifically evaluate sub-
populations of heteromeric NR2A in neurons (for example,
NR1–NR2A vs NR1–NR2B–NR2A).
Differential sorting of NR2A and NR2B in
hippocampal neurons
Because both NR2A and NR2B internalize efficiently in hip-
pocampal neurons at 5 DIV, we decided to use this developmen-
tal time to analyze the endocytic sorting and intracellular distri-
bution of the internalized subunits. We coexpressed FLAG–
NR2A or FLAG–NR2B with various endosomal marker proteins
(GFP–Rab5, GFP–Rab9, or GFP–Rab11) and analyzed colocal-
ization at different time points. At 15 min, FLAG–NR2A colocal-
izes well with the early endosome marker GFP–Rab5 (Fig. 7A)
but to a lesser degree with the late endosome marker GFP–Rab9
(Fig. 7D) and the recycling endosome marker GFP–Rab11 (Fig.
7D). At 30 min, the colocalization of FLAG–NR2A with all three
markers was similar (Fig. 7D). In contrast, at 15 min, FLAG–
NR2B colocalizes with all three markers (Fig. 8A–C), but most
prominently with GFP–Rab5. At 30 min, the amount of colocal-
ization with the late endosome marker GFP–Rab9 decreased sub-
stantially (Fig. 8 B–D), whereas colocalization of FLAG–NR2B
with the recycling endosome marker GFP–Rab11 almost doubled
(Fig. 8C–D), indicating the preferential sorting of NR2B through
recycling endosomes. Quantitative analyses revealed that
whereas only 25% of FLAG–NR2A colocalized with GFP–Rab11
after 30 min of internalization, almost 50% of FLAG–NR2B co-
localized with the recycling endosome marker under these con-
ditions (Fig. 8 D), demonstrating that NMDA receptors contain-
ing NR2B rather than NR2A have a clear preference for the
recycling pathway. Together, these data extend our studies of
NMDA receptor trafficking in heterologous cells to primary neu-
ronal cultures and demonstrate distinct endocytic sorting motifs
encoded within NR2A and NR2B.
Discussion
Recent studies have demonstrated that NMDA receptors are
quite mobile, including intracellular pools of NMDA receptors
(Standley et al., 2000; Scott et al., 2001; Xia et al., 2001; Wash-
bourne et al., 2002), plasma membrane-expressed NMDA recep-
tors, and even synaptic NMDA receptors (Rao and Craig, 1997;
Figure 6. Differential internalization of NR2A and NR2B in hippocampal neurons during
development. A, Hippocampal neurons (3 or 10 DIV) were transfected with FLAG–NR2B or
FLAG–NR2A using thecalcium phosphatecoprecipitation method.After 2d (at5 or12 DIV),the
cells were incubated with FLAG polyclonal antibody for 45 min at room temperature. The anti-
bodywas removed,andthe cellswere returnedtoconditioned mediafor15 minat 37°Ctoallow
internalization. Neuronswere fixedand incubated withAlexa 568-conjugated (red) anti-rabbit
secondary antibody to visualize the surface pool. Cells were then washed, permeabilized, and
incubated with Alexa 488-conjugated (green) anti-rabbit secondary antibody to specifically
visualize theinternalized pool. Green fluorescenceindicated internalization compared withthe
total (red plus green). Images were collected with 63⫻ objectives on a Zeiss LSM. Series of
optical sections were collected at intervals of 0.34
m. Maximum projections are shown. B,
Histogram of the amount of internalization of FLAG–NR2A or FLAG–NR2B at 5 or12 DIV. Series
of optical sections were collected at intervals of 0.34
m. Colocalization was measured with
Volocity 2 software (Improvision). At 5 DIV, there is no significant difference between the
amount ofinternalization ofNR2A andNR2B, whereasat 12DIV, thereis astatistical difference
between the two constructs. Data represent means ⫾ SEM; n ⫽ 9; *p ⬍ 0.01; Student’s
unpaired t test.
6388 • J. Neurosci., July 14, 2004 • 24(28):6383– 6391 Lavezzari et al. •Regulation of NMDA Receptor Endocytosis
Rumbaugh and Vicini, 1999; Snyder et al., 2001; Grosshans et al.,
2002; Prybylowski et al., 2002; Tovar and Westbrook, 2002). Fur-
thermore, endocytosis of native NMDA receptors is develop-
mentally regulated such that endocytosis is robust early in devel-
opment but declines as neurons mature and synapses form
(Roche et al., 2001). Interestingly, this change in endocytosis par-
allels changes in NMDA receptor subunit composition. In the
forebrain early in development, NMDA receptors are thought to
be NR1–NR2B heteromers that switch to heteromeric combina-
tions of NR1, NR2A, and NR2B in the adult brain (Kew et al.,
1998; Li et al., 1998; Rumbaugh and Vicini, 1999; Tovar and
Westbrook, 1999). In the present study, we have probed the pos-
sibility that NR2A and NR2B differentially regulate plasma mem-
brane expression and endocytic sorting of NMDA receptors. We
found that NR2A and NR2B do indeed possess distinct endocytic
motifs and sorting information within their distal C termini. In
addition, we found that the NR2 subunit content of NMDA re-
Figure 7. Internalized NR2A traffics through early, late, and recycling endosomes in hip-
pocampal neurons.Hippocampal neurons(3 DIV)were transfectedwith FLAG–NR2Aand GFP–
Rab5 ( A), FLAG–NR2A and GFP–Rab9 ( B), or FLAG–NR2A and GFP–Rab11 ( C) using the cal-
cium phosphate coprecipitation method. At 5 DIV, the cells were incubated with FLAG
polyclonal antibody for 45min atroom temperature,antibody wasremoved, andthe cellswere
returned to conditioned media for 15 or 30 min at 37°C to allow internalization. Neurons were
then fixed and incubated with Alexa 568-conjugated (red) anti-rabbit secondary antibody to
visualize the surface pool. Cells were then washed, permeabilized, and incubated with Cy5-
conjugated (blue)anti-rabbit secondary antibody to specifically visualize theinternalized pool.
Blue fluorescence indicated internalization compared with the total (red plus blue). Images
were collected with 63⫻ objectives on a Zeiss LSM. Maximum projections are shown. D, Sta-
tistical analysis of the amount of colocalization between FLAG–NR2A and GFP–Rab5, GFP–
Rab9, or GFP–Rab11. Series of optical sections were collected at intervals of 0.34
m. Images
from three dendrites per neuron (3 neuronsper experiment) were collected,and colocalization
analysis was measured based on the data collected in three to five independent experiments
with Volocity 2 software (Improvision). Values represent mean ⫾ SEM; n ⫽ 5.
Figure 8. NR2B preferentially traffics through recycling endosomes in hippocampal neu-
rons. Hippocampal neurons (3 DIV) were transfected with FLAG–NR2B and GFP–Rab5 ( A),
FLAG–NR2B and GFP–Rab9 ( B), or FLAG–NR2B and GFP–Rab11 ( C) using the calcium phos-
phatecoprecipitationmethod.At5 DIV, the cells were incubated with FLAG polyclonal antibody
for 45 min at room temperature, antibody was removed, and the cells were returned to condi-
tioned media for 15 or 30 min at 37°C to allow internalization. Neurons were then fixed and
incubated with Alexa 568-conjugated (red) anti-rabbit secondary antibody to visualize the
surface pool. Cells were then washed, permeabilized, and incubated with Cy5-conjugated
(blue) anti-rabbit secondary antibody to specifically visualize the internalized pool. Blue fluo-
rescence indicated internalization compared with the total (red plus blue). Images were col-
lected with 63⫻ objectives on a Zeiss LSM. Series of optical sections werecollected at intervals
of 0.34
m. Maximum projections are shown. D, Statistical analysis of the amount of colocal-
ization between FLAG–NR2B and GFP–Rab5, GFP–Rab9, or GFP–Rab11. Series of optical sec-
tionswere collectedat intervalsof0.34
m.Images fromthree dendritesperneuron(3neurons
per experiment) were collected, and colocalization analysis was measured based on the data
collected in three to five independent experiments with Volocity 2 software (Improvision).
Values represent mean ⫾ SEM; n ⫽ 5.
Lavezzari et al. •Regulation of NMDA Receptor Endocytosis J. Neurosci., July 14, 2004 • 24(28):6383– 6391 • 6389
ceptors in hippocampal neurons regulates the amount of NMDA
receptor endocytosis and determines the intracellular sorting of
NMDA receptors after endocytosis. This demonstrates the im-
portant influence of subunit composition of NMDA receptors on
trafficking, plasma membrane expression, and endocytosis.
NMDA receptors, like all integral membrane proteins expressed
on the cell surface, must undergo regulated transport to the plasma
membrane, where they can be selectively stabilized, undergo endo-
cytosis–degradation, or undergo endocytosis followed by recycling
to the plasma membrane. Protein trafficking through distinct intra-
cellular pathways is tightly regulated primarily by specific protein–
protein interactions between cargo proteins and the trafficking ma-
chinery. One of our primary goals is to define motifs and molecular
binding sites on NMDA receptors critical for regulating trafficking,
surface expression, and endocytosis. Although in recent years there
have been numerous studies published on glutamate receptor traf-
ficking and endocytosis, it is interesting to note that AMPA recep-
tors, the best-characterized subtype of glutamate receptor thus far,
do not contain any of the strong consensus sequences known to play
a role in clathrin-mediated endocytosis. In sharp contrast, the cyto-
solic domains of NMDA receptors have numerous consensus endo-
cytic motifs. Thus it is likely that the mechanisms regulating the
surface expression and synaptic targeting of AMPA receptors and
NMDA receptors are distinct. In addition, NMDA receptors possess
a variety of well characterized protein-binding motifs that regulate
interactions with components of the PSD (Hung and Sheng, 2002).
These proteins are also likely to mediate synaptic expression of
NMDA receptors.
In this study, we have focused on elucidating differences be-
tween NR2A and NR2B that are important for NMDA receptor
trafficking and developmental changes in surface expression.
Both NR2 subunits need to associate with NR1 to get to the
surface (McIlhinney et al., 1998). Similarly, Tac chimeras includ-
ing the entire NR2A or NR2B C terminus are retained in the ER
(data not shown) (Hawkins et al., 2004). Therefore, we focused
on the distal portion of the NR2 C terminus for our studies and
found that the NR2A C terminus, like that of NR2B, contains a
dominant endocytic motif. Previous studies have characterized a
tyrosine-based endocytic motif on NR2B, YEKL, which binds
directly to the medium chain of AP-2 and can regulate endocy-
tosis in heterologous cells and in neurons (Roche et al., 2001;
Lavezzari et al., 2003). Although the tyrosine in NR2B (Tyr 1472)
is conserved in NR2A (Tyr 1454), the surrounding residues are
different, suggesting that the interaction of these proteins with
clathrin-associated adaptors might also differ. We found that this
tyrosine-based motif within the C terminus of NR2A did not
regulate endocytosis; in this case, a dileucine motif (Leu 1319,
Leu 1320) plays a critical role in NR2A-mediated internalization.
Interestingly, as described previously for NR2B, we still found
that coexpression with PSD-95 inhibited NR2A-mediated endo-
cytosis. Thus for both NR2A and NR2B, binding of PSD-95 in-
hibits clathrin-mediated endocytosis.
Our evidence that endocytic motifs in the distal C termini of
NMDA receptors were different in the individual NR2 subunits
prompted us to perform binding assays of the NR2A and NR2B C
termini with the medium chains of the various clathrin adaptor
complexes (
1–
4). Interestingly, we found that although both
NR2A and NR2B C termini could directly interact with adaptor
medium chains, the specificity varied. For example, the binding
to
1, the medium chain for AP-1 that regulates trafficking at the
TGN, was similar for NR2A and NR2B, whereas the binding to
2 was quite different. We found that NR2B binds very well to
2
(Fig. 3) (Lavezzari et al., 2003); however, the NR2A C terminus
displays almost no binding. This is consistent with the YEKL
motif within NR2B being important in plasma membrane endo-
cytosis, unlike the YKKM motif of NR2A. However, our findings
still clearly demonstrate that the C termini of NR2A and NR2B
both directly interact with medium chains of clathrin adaptor
proteins and are both likely to interact with the clathrin machin-
ery at various intracellular sorting steps. It is important to note
that the preference of NR2A for
1 versus
2 is somewhat rare. It
has even been proposed in the literature (Ohno et al., 1996; Boni-
facino and Traub, 2003) that
2 binding is almost a default in-
teraction for any sequence that interacts with clathrin adaptor
medium chains. Specificity for a particular medium chain is more
common for
1, for example. These data suggest that the lack of
2 binding to the NR2A C terminus is of particular importance.
In addition to the distal C terminus of NR2A and NR2B that
we have characterized in the present study, there is good evidence
that a region adjacent to transmembrane domain 4 (TM4) of the
NMDA receptor subunits is important for surface expression and
functional regulation of NMDA receptors (Vissel et al., 2001).
Interestingly, there is a conserved tyrosine in all NMDA receptor
subunits adjacent to TM4 that appears to be sensitive to phos-
phorylation, and mutation of this residue of NR2A influences
channel function and rundown in rat hippocampal pyramidal
neurons and heterologous cells (Vissel et al., 2001). It is possible
that these conserved tyrosines form a ring near the plasma mem-
brane of NMDA receptors and may regulate plasma membrane
expression or trafficking through intracellular pathways. One of
the challenges for the future will be to determine how these motifs
in different regions of NMDA receptors work together to mediate
trafficking, surface expression, and/or endocytosis of endoge-
nous NMDA receptors.
Our evidence indicates that NR2B undergoes more robust endo-
cytosis than NR2A. This could be attributable to several factors.
Many groups have shown that NR2A is highly synaptic, whereas
NR2B is expressed at both synaptic and extrasynaptic sites (Stocca
and Vicini, 1998; Tovar and Westbrook, 1999; Li et al., 2002). This is
consistent with NR2A being strictly colocalized with components of
the PSD that are likely to stabilize synaptic receptors, most notably
PSD-95. Additional support for this hypothesis is that NR2A expres-
sion and PSD-95 expression are coordinately increased during de-
velopment. Conversely, NR2B is expressed not only early in devel-
opment during synaptogenesis but also in mature neurons at
extrasynaptic sites. We also demonstrated that the NR2A and NR2B
subunits direct differential sorting into distinct intracellular path-
ways after endocytosis. The NR2A and NR2B C termini both initially
regulate internalization into early endosomes; however, over time,
these proteins diverge and NR2A traffics to late endosomes, whereas
NR2B preferentially sorts into recycling endosomes. These data fit
well with the described higher level of NMDA receptor endocytosis
early in development (Roche et al., 2001), with NR2B preferring the
recycling pathway (Figs. 5, 8), and with high expression of NR2B at
extrasynaptic sites in mature neurons. Our finding that NR2B is
more likely to recycle seems quite logical, because NR2B is highly
expressed early in development, when endocytosis is more robust.
Given the relatively long half-life of both surface and total pools of
glutamate receptors (Hall and Soderling, 1997; Mammen et al.,
1997; Huh and Wenthold, 1999) and the fact that NR2B endocytosis
occurs at a rapid rate (31–36% of NR2B-containing receptors in 15
min) (Fig. 6), if recycling of NR2B were not an ongoing process, the
neuron would rapidly deplete the surface pool of NMDA receptors.
Our findings support a model of NMDA receptor endocytosis
that is more robust early in development and at extrasynaptic sites,
conditions in which PSD-95 expression is limited and is therefore
6390 • J. Neurosci., July 14, 2004 • 24(28):6383– 6391 Lavezzari et al. •Regulation of NMDA Receptor Endocytosis
not available to stabilize plasma membrane-expressed NMDA.
When NMDA receptors are expressed at the plasma membrane at
nonsynaptic sites, their C termini are free to bind to the endocytic
machinery and the receptors undergo endocytosis. In support of our
model, other studies of NMDA receptor function at synaptic versus
extrasynaptic sites demonstrate a role for endocytosis regulated by
tyrosine phosphorylation of NMDA receptor and subsequent regu-
lation of rundown (Wang et al., 1996; Li et al., 2002). These data fit
well with our findings on a differential contribution of NR2A and
NR2B to NMDA receptor endocytosis and the differential sorting of
distinct NMDA receptor complexes. Our evidence of the direct in-
teraction of NR2A and NR2B with clathrin adaptor medium chains
is also supported by recent findings that NMDA receptors coimmu-
noprecipitate with adaptor complexes (Nong et al., 2003), demon-
strating the importance of clathrin-mediated endocytosis and
NMDA receptor regulation.
Our studies elucidated critical differences between the NR2
subunits that are expressed in the hippocampus and cortex. Us-
ing a variety of approaches, we demonstrate that the NR2A and
NR2B subunits have dominant endocytic motifs that regulate
sorting into distinct intracellular pathways. These differences are
also seen when NR2A and NR2B are expressed and their endocy-
tosis is monitored in primary neuronal culture. These findings
provide a framework for understanding developmental changes
in NMDA receptor subunit expression and how it regulates
NMDA receptor trafficking and synaptic expression.
References
Aguilar RC, Boehm M, Gorshkova I, Crouch RJ, Tomita K, Saito T, Ohno H,
Bonifacino JS (2001) Signal-binding specificity of the
4 subunit of the
adaptor protein complex AP-4. J Biol Chem 276:13145–13152.
Bonifacino JS, Traub LM (2003) Signals for sorting of transmembrane pro-
teins to endosomes and lysosomes. Annu Rev Biochem 72:395–447.
Grosshans DR, Clayton DA, Coultrap SJ, Browning MD (2002) LTP leads to
rapid surface expression of NMDA but not AMPA receptors in adult rat
CA1. Nat Neurosci 5:27–33.
Hall RA, Soderling TR (1997) Quantitation of AMPA receptor surface ex-
pression in cultured hippocampal neurons. Neuroscience 78:361–371.
Hawkins LM, Prybylowski K, Chang K, Moussan C, Stephenson FA,
Wenthold RJ (2004) Export from the endoplasmic reticulum of assem-
bled NMDA receptors is controlled by a motif in the C terminus of the
NR2 subunit. J Biol Chem, in press.
Huh KH, Wenthold RJ (1999) Turnover analysis of glutamate receptors iden-
tifies a rapidly degraded pool of the N-methyl-D-aspartate receptor subunit,
NR1, in cultured cerebellar granule cells. J Biol Chem 274:151–157.
Hung AY, Sheng M (2002) PDZ domains: structural modules for protein
complex assembly. J Biol Chem 277:5699–5702.
Kew JN, Richards JG, Mutel V, Kemp JA (1998) Developmental changes in
NMDA receptor glycine affinity and ifenprodil sensitivity reveal three
distinct populations of NMDA receptors in individual rat cortical neu-
rons. J Neurosci 18:1935–1943.
Kim E, Niethammer M, Rothschild A, Jan YN, Sheng M (1995) Clustering
of Shaker-type K⫹ channels by interaction with a family of membrane-
associated guanylate kinases. Nature 378:85–88.
Kornau HC, Schenker LT, Kennedy MB, Seeburg PH (1995) Domain inter-
action between NMDA receptor subunits and the postsynaptic density
protein PSD-95. Science 269:1737–1740.
Lavezzari G, McCallum J, Lee R, Roche KW (2003) Differential binding of
the AP-2 adaptor complex and PSD-95 to the C-terminus of the NMDA
receptor subunit NR2B regulates surface expression. Neuropharmacol-
ogy 45:729 –737.
Li B, Chen N, Luo T, Otsu Y, Murphy TH, Raymond LA (2002) Differential
regulation of synaptic and extra-synaptic NMDA receptors. Nat Neurosci
5:833– 834.
Li JH, Wang YH, Wolfe BB, Krueger KE, Corsi L, Stocca G, Vicini S (1998)
Developmental changes in localization of NMDA receptor subunits in
primary cultures of cortical neurons. Eur J Neurosci 10:1704–1715.
Luscher C, Xia H, Beattie EC, Carroll RC, von Zastrow M, Malenka RC, Nicoll
RA (1999) Role of AMPA receptor cycling in synaptic transmission and
plasticity. Neuron 24:649 – 658.
Malinow R, Malenka RC (2002) AMPA receptor trafficking and synaptic
plasticity. Annu Rev Neurosci 25:103–126.
Mammen AL, Huganir RL, O’Brien RJ (1997) Redistribution and stabiliza-
tion of cell surface glutamate receptors during synapse formation. J Neu-
rosci 17:7351–7358.
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.
Nong Y, Huang YQ, Ju W, Kalia LV, Ahmadian G, Wang YT, Salter MW
(2003) Glycine binding primes NMDA receptor internalization. Nature
422:302–307.
Ohno H, Stewart J, Fournier MC, Bosshart H, Rhee I, Miyatake S, Saito T, Gal-
lusser A, Kirchhausen T, Bonifacino JS (1995) Interaction of tyrosine-based
sorting signals with clathrin-associated proteins. Science 269:1872–1875.
Ohno H, Fournier MC, Poy G, Bonifacino JS (1996) Structural determi-
nants of interaction of tyrosine-based sorting signals with the adaptor
medium chains. J Biol Chem 271:29009–29015.
Prybylowski K, Fu Z, Losi G, Hawkins LM, Luo J, Chang K, Wenthold RJ,
Vicini S (2002) Relationship between availability of NMDA receptor
subunits and their expression at the synapse. J Neurosci 22:8902–8910.
Rao A, Craig AM (1997) Activity regulates the synaptic localization of the
NMDA receptor in hippocampal neurons. Neuron 19:801–812.
Roche KW, Huganir RL (1995) Synaptic expression of the high-affinity kai-
nate receptor subunit KA2 in hippocampal cultures. Neuroscience
69:383–393.
Roche KW, Standley S, McCallum J, Dune Ly C, Ehlers MD, Wenthold RJ
(2001) Molecular determinants of NMDA receptor internalization. Nat
Neurosci 4:794 – 802.
Rumbaugh G, Vicini S (1999) Distinct synaptic and extrasynaptic NMDA
receptors in developing cerebellar granule neurons. J Neurosci
19:10603–10610.
Scott DB, Blanpied TA, Swanson GT, Zhang C, Ehlers MD (2001) An
NMDA receptor ER retention signal regulated by phosphorylation and
alternative splicing. J Neurosci 21:3063–3072.
Sheng M (2001) Molecular organization of the postsynaptic specialization.
Proc Natl Acad Sci USA 98:7058–7061.
Snyder EM, Philpot BD, Huber KM, Dong X, Fallon JR, Bear MF (2001)
Internalization of ionotropic glutamate receptors in response to mGluR
activation. Nat Neurosci 4:1079–1085.
Standley S, Roche KW, McCallum J, Sans N, Wenthold RJ (2000) PDZ do-
main suppression of an ER retention signal in NMDA receptor NR1 splice
variants. Neuron 28:887– 898.
Stocca G, Vicini S (1998) Increased contribution of NR2A subunit to syn-
aptic NMDA receptors in developing rat cortical neurons. J Physiol
(Lond) 507:13–24.
Tovar KR, Westbrook GL (1999) The incorporation of NMDA receptors
with a distinct subunit composition at nascent hippocampal synapses in
vitro. J Neurosci 19:4180– 4188.
Tovar KR, Westbrook GL (2002) Mobile NMDA receptors at hippocampal
synapses. Neuron 34:255–264.
Vicini S, Wang JF, Li JH, Zhu WJ, Wang YH, Luo JH, Wolfe BB, Grayson DR
(1998) Functional and pharmacological differences between recombi-
nant N-methyl-
D-aspartate receptors. J Neurophysiol 79:555–566.
Vissel B, Krupp JJ, Heinemann SF, Westbrook GL (2001) A use-dependent
tyrosine dephosphorylation of NMDA receptors is independent of ion
flux. Nat Neurosci 4:587–596.
Wang YT, Yu XM, Salter MW (1996) Ca
2⫹
-independent reduction of
N-methyl-
D-aspartate channel activity by protein tyrosine phosphatase.
Proc Natl Acad Sci USA 93:1721–1725.
Washbourne P, Bennett JE, McAllister AK (2002) Rapid recruitment of
NMDA receptor transport packets to nascent synapses. Nat Neurosci
5:751–759.
Wenthold RJ, Prybylowski K, Standley S, Sans N, Petralia RS (2003) Traf-
ficking of NMDA receptors. Annu Rev Pharmacol Toxicol 43:335–358.
Xia H, Hornby ZD, Malenka RC (2001) An ER retention signal explains
differences in surface expression of NMDA and AMPA receptor subunits.
Neuropharmacology 41:714 –723.
Lavezzari et al. •Regulation of NMDA Receptor Endocytosis J. Neurosci., July 14, 2004 • 24(28):6383– 6391 • 6391
