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INTRODUCTION |
Glutamate is the major excitatory neurotransmitter in the
mammalian central nervous system and three distinct subtypes of ionotropic glutamate receptors mediate fast excitatory transmission (1). AMPA1 receptors and NMDA
receptors are the principle ionotropic receptors, while kainate
receptors are widely distributed but less abundant (2, 3). AMPA
receptors are composed of four homologous subunits, GluR1, -2, -3, and
-4, which assemble into a receptor complex, probably containing five
subunits (2, 4). While homomeric AMPA receptors are functional and
appear to exist in neurons, most native AMPA receptors consist of two
or more different subunits (5-7). For NMDA receptors, two classes of
subunits, NR1 and NR2A-D, have been cloned (2, 8). In addition, various
splice variants have been identified for the NR1 subunits (9-11). The
NR1 subunit may be functional by itself, but it is generally thought
that native NMDA receptors are heteromeric complexes with NR1 serving as an obligatory subunit (12, 13). Unlike AMPA receptors, all NMDA
receptors are calcium-permeable, and gating is
voltage-dependent.
NMDA and AMPA receptors are expressed in the same neuron and often are
co-localized at postsynaptic membranes, where they are anchored through
their C termini to PDZ (PSD-95/discs large protein/zona occludens protein 1) domain-containing
proteins in the postsynaptic density (14-19). The number and type of
receptors at the postsynaptic membrane will determine the nature as
well as the magnitude of the response to release of the
neurotransmitter, glutamate. Two general mechanisms appear to control
the number and composition of glutamate receptors at the postsynaptic
membrane. The first is the control of the expression of a receptor at
the levels of transcription and translation. Expression of mRNA for all glutamate receptors is developmentally and regionally regulated (20-23). Selective changes in expression of receptor subunits also occur under certain pathophysiological conditions and may have a
critical impact on neurons. For example, it has been proposed that a
selective decrease in GluR2 mRNA and protein following ischemia
generates calcium-permeable AMPA receptors, which eventually lead to
neuronal degeneration due to excess calcium influx through their
channels (24). Receptor expression is also regulated at the level of
the individual synapse such that different synaptic populations of a
neuron may contain different glutamate receptors (25-27). The synaptic
receptor composition may vary under different physiological conditions;
for example, it has been proposed that long term potentiation is
generated by the conversion of synapses that previously did not contain
AMPA receptors or contained electrophysiologically silent AMPA
receptors to synapses that contain functional AMPA receptors through
the activation of NMDA receptors (28, 29). A similar up-regulation of
AMPA receptors may occur during synapse development (30, 31).
A critical parameter in a protein's response to changes in its rate of
synthesis or degradation is the turnover rate of the protein. Turnover
rates, usually expressed in half-lives, vary widely for proteins with
half-lives ranging from less than 1 min to many days (32). A protein
with a short half-life is much more effectively regulated by changing
synthesis or degradation rates than a protein with a long half-life.
Therefore, if glutamate receptors have short half-lives, changes in
synthesis, resulting from either changes in transcription or
translation, as well as changes in degradation could be effective in
rapid regulation of levels of synaptic receptors. In the present study,
we investigated the turnover characteristics of two key ionotropic
receptors, AMPA and NMDA receptors, in cultured cerebellar granule
cells. These cultures have the advantage of representing a nearly
homogenous population of neurons that express several glutamate
receptors, including functional AMPA and NMDA receptors. We demonstrate
that subunits of both receptors have similar and relatively long
half-lives, with values of about 20 h, as measured both by
pulse-chase and surface biotinylation. However, a pool of the NR1
subunit, which is not assembled with NR2 and represents about half of
the total NR1, is rapidly degraded with a half-life of about 2 h.
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EXPERIMENTAL PROCEDURES |
Materials--
Tissue culture media, sera, and supplies were
purchased from Life Technologies, Inc.; cytosine arabinoside from
Sigma; methionine and cysteine-free medium from NIH Media Service
branch; [35S]methionine and Enlightening from NEN Life
Science Products; protein A-agarose beads, Ultralink Plus immobilized
streptavidin beads, Sulfo-NHS-LC-biotin, and NHS-SS-biotin from Pierce;
Kodak X-Omat AR films from Eastman Kodak Co.; autoradiographic
14C-labeled microscales and horseradish
peroxidase-conjugated secondary antibodies from Amersham Pharmacia
Biotech; SDS electrophoresis gels from Novex; and horseradish
peroxidase-conjugated streptavidin from Southern Biotechnology Associates.
Cerebellar Granule Cell Culture--
Cultures of cerebellar
granule neurons were prepared as described by Gallo et al.
(33). Briefly, cerebella were obtained from 7-8-day-old Sprague-Dawley
rat pups. They were chopped by using a tissue slicer and treated with
0.025% trypsin for 15 min at 37 °C. Following trypsinization,
tissue was dissociated by passage through fire-polished pipettes, and
tissue debris was separated from dissociated cells by sedimentation.
Cells were centrifuged and suspended in the plating medium, which
consisted of basal medium Eagle, 25 mM KCl, 10 µg/ml
gentamycin, 2 mM glutamine, 10% fetal calf serum, and were
plated on 3.5-cm culture plates coated with poly-L-lysine
at a density of 2.5 × 106 cells/dish. After 19-20 h,
proliferation of glial cells was inhibited by treatment with 10 µM cytosine arabinoside. All experiments were performed
on 8-9-day in vitro cells.
Pulse-chase Labeling of Cultured Cells--
Cells were washed
twice with prewarmed Hanks' balanced salt solution (HBSS) containing
25 mM KCl, incubated for 30 min at 37 °C in depletion
media, which consisted of methionine- and cysteine-free basal medium
Eagle, 25 mM KCl, 2 mM glutamine, 10 µg/ml
gentamycin, 5% dialyzed fetal calf serum, and then pulse-labeled with
250 µCi of [35S]methionine (1175 Ci/mmol) in depletion
medium for 20 min at 37 °C. Some experiments were performed in
medium lacking glutamine. After 20 min, cells were washed once with
basal medium Eagle containing 2 mM methionine and then were
incubated with conditioned medium containing 2 mM
methionine and 2.5 mM HEPES for variable times. KCl (25 mM) was included in all media throughout the experiment. To
block glycosylation, tunicamycin was added to a final concentration of
1 µg/ml to the medium 12 h before pulse labeling and was
included during pulse labeling. To harvest, cells were washed twice
with cold Dulbecco's phosphate-buffered saline, scraped into
phosphate-buffered saline containing protease inhibitor mixture (1 mM EDTA, 1 mM 4-(2-aminoethyl)benzenesulfonyl
fluoride, 20 µM leupeptin, 2 µg/ml pepstatin, 2 µg/ml
aprotinin), and centrifuged at 2000 × g for 15 min.
Pellets were stored frozen at
70 °C until use.
Antibodies--
Monoclonal antibodies (54.2) to NR1 subunits and
NR2B subunits were purchased from Pharmingen (San Diego, CA) and
Transduction Laboratories (Lexington, KY), respectively. Antibodies
specific to NR2A, NR2B, and NR2C subunits were generated against
polyhistidine fusion proteins containing the C-terminal region of each
subunit encompassing 934-1203 for NR2A, 935-1856 for NR2B (34), and 1110-1242 for NR2C subunits. The NR2C antibody was specific to NR2C
subunits as tested by Western blot analysis using human embryonic kidney (HEK293) cells transfected with different NR2 subunits. Other
antibodies to splice variants of NR1, NR2A/B, GluR1, GluR2/3, and GluR4
subunits were generated against synthetic peptides which correspond to
the sequences at the C termini, and characterization and demonstration
of the specificity of the antibodies was made in previous studies (5,
35, 36).
Immunoprecipitation and Fluorography--
Cell pellets were
suspended in buffer A (50 mM Tris, pH 7.5, 150 mM NaCl, 0.02% NaN3, 1 mM EDTA, 20 µM leupeptin, 1 mM
4-(2-aminoethyl)benzenesulfonyl fluoride, 2 µg/ml pepstatin, 2 µg/ml aprotinin) by trituration and solubilized with 2% SDS in
buffer A by incubating for 3 min at 90 °C. The soluble extract was
obtained by centrifugation at 100,000 × g for 30 min
at 15 °C, and 100 µl of the soluble extract was diluted 6-fold
with 2% Triton X-100 in buffer A. To retain NMDA receptor subunit
associations, cells were solubilized with 1% deoxycholate at pH 9 in
buffer A (36) (200 µM phenylmethylsulfonyl fluoride was
used instead of 1 mM ABESF). After centrifugation, the
soluble extract was diluted 10-fold with 0.1% Triton X-100. SDS or
deoxycholate soluble fractions were incubated overnight at 4 °C with
50 µl of protein A-agarose beads to which 10 µg of polyclonal
antibodies or 2.5 µg of monoclonal antibodies were preattached.
Immunoprecipitated pellets were washed three times with buffer A
containing 2% Triton X-100, 10% glycerol, and the above protease
mixture, followed by two washes with buffer A containing 0.1% Triton
X-100 and protease inhibitor mixture. Immunoprecipitated proteins were
eluted with SDS-sample buffer (60 µl) by incubating at 90 °C for 3 min and subjected to SDS-PAGE on 4-20% gradient gels. For
fluorography, electrophoresed gels were fixed with the solution
containing 30% methanol and 10% glacial acetic acid for 30 min and
treated with fluorophore (Enlightening) for 30 min. Gels were dried
onto Whatman filter papers and exposed to films (Kodak X-Omat AR) at
70 °C. Fluorograms were scanned with a Molecular Dynamics
densitometer, and densities were normalized using standard curves
generated with autoradiographic 14C-labeled microscales.
Deglycosylation of the Subunits--
Membrane homogenates from
cultured cells or cerebellum were suspended in 10 mM
NaH2PO4 buffer containing 10 mM
EDTA, 0.2 mM leupeptin, and 10 µg/ml pepstatin and
solubilized with 1% SDS containing 5%
-mercaptoethanol by
incubating at 90 °C for 2-3 min. The soluble fraction was diluted
with 1%
-octyl glucopyranoside in 10 mM
NaH2PO4 containing the above protease mixture
to a final concentration of 0.1% SDS and incubated with
endoglycosidase H (15 milliunits) or N-glycosidase F (3 units) overnight at 37 °C. An equal volume of 2× SDS-sample buffer
was added for SDS-PAGE analysis.
Gel Electrophoresis and Western Blot Analysis--
Membrane
homogenates or soluble extracts of granule cells or adult rat cerebella
were subjected to SDS-PAGE using 4-20% gradient gels. Proteins were
transferred to nitrocellulose membranes, and the membranes were blocked
with Tris-buffered saline containing 0.1% Tween (TBST) and 5% nonfat
dry milk overnight at 4 °C, incubated with primary antibodies in
TBST for 1.5 h, and washed three times for 15 min each. Membranes
were incubated with horseradish peroxidase-conjugated secondary
antibodies for 1 h and washed three times, and bound antibodies
were visualized by the chemiluminescence detection method.
Biotinylation of Cell Surface Proteins--
Cultured granule
cells were washed four times with HBSS, and surface proteins were
biotinylated with Sulfo-NHS-LC-biotin or NHS-SS-biotin (1 mg/ml) in
HBSS for 30 min at 4 °C. Cells were washed with HBSS four times and
incubated in conditioned medium for 15 min at 37 °C in a humidified
incubator before harvest. For determination of the cytoplasmic pool of
the receptors, cells were harvested immediately after washing. KCl (25 mM) was included in all HBSS solutions for washing and
biotinylation. Cells were harvested at various times as described
above, by using phosphate-buffered saline containing 0.1 M
glycine and protease mixture. Cells were stored at
70 °C. Pellets
were solubilized with 2% SDS, diluted with 2% Triton X-100,
immunoprecipitated using appropriate antibodies as described above, and
subjected to SDS-PAGE. After transferring the gel to nitrocellulose
membranes, membranes were blocked with 5% nonfat dry milk in TBST,
incubated for 1.5 h with horseradish peroxidase-conjugated
streptavidin (1:10,000) in TBST containing 0.5% milk, and washed three
times for 15 min each. Biotinylated proteins were visualized by
chemiluminescence. When NHS-SS-biotin was used, Ultralink-immobilized
streptavidin beads were used to precipitate biotinylated proteins, and
subunit proteins were detected by Western blot analysis.
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RESULTS |
Expression of Glutamate Receptors in Cerebellar Granule
Cells--
Cultured cerebellar granule cells, 8-9 days in
vitro, were chosen for this study because it has been shown
previously that these cells express functional AMPA and NMDA receptors
(37-41). Using antibodies selective for AMPA and NMDA receptor
subunits and NR1 splice variants, Western blot analysis shows that
multiple subunits and splice variants are expressed in these cultures
(Fig. 1). It has been previously shown
that NR2A and NR2C are the predominant NR2 subunits in granule cells
in vivo in the adult animal, while NR2B is the major subunit
in granule cells at early developmental stages (20, 23). This
developmental change of the subunit expression also occurs in cultured
granule cells (42). The pattern of NR2 subunit expression in the
present study is consistent with previous findings of mRNA
analysis, where NR2A was the major subunit expressed and NR2B and NR2C
were expressed in a lower abundance at 7-8 days in vitro
(37, 41). Since all four alternatively spliced cassettes of NR1 are
found in cultured granule cells, it is possible that they are
differentially assembled and that multiple functionally distinct NMDA
receptors are expressed. NR1 C2-containing receptors appear to be more
abundant than those containing the other C-terminal cassette, C2', as
determined by using different antibodies selective for the two
cassettes. This was confirmed in a separate experiment where NR1 PAN
antibodies were used to probe receptors immunoprecipitated with NR1 C2
and C2' antibodies (data not shown). At least three AMPA receptor subunits were expressed in granule cells, GluR1, GluR2/3, and GluR4
(the antibody to GluR2/3 recognizes both GluR2 and GluR3 (5); mRNA
for GluR2 and GluR3 subunits have been shown to be expressed (42)).
GluR1 is not expressed in granule cells in vivo in adult
rats but is expressed in cultured cells, as previously reported by Hack
et al. (39), who showed a greater than 3-fold increase in
GluR1 between 2 and 9 days in culture, while GluR2/3 and GluR4 remained
relatively constant. Because of these rapidly changing levels of GluR1,
this subunit was not included in the turnover analyses.

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Fig. 1.
Expression of glutamate receptor subunits in
cultured cerebellar granule cells. Membrane homogenates were
prepared from adult cerebellum (cb) and cerebellar granule
cells (gr) of 8-9 days in vitro and analyzed by
SDS-PAGE, followed by Western blot analysis using subunit-specific
antibodies. NR1 PAN recognized all NR1 subunits. NR1 C2, C2', C1, and N
are splice variants of NR1 subunits containing C2, C2', C1, and N
cassettes, respectively. Subunits were visualized by the
chemiluminescence detection method. For a given sample, the same amount
of protein was loaded for each lane for Western blot analysis.
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Pulse Labeling and Turnover of NMDA and AMPA Receptor
Subunits--
To measure the degradation rates of receptor subunits,
cells were pulse-labeled with [35S]methionine and chased
in conditioned medium containing unlabeled methionine. Sufficient
radioactivity was incorporated under these conditions to permit
quantitation of receptor subunits immunoprecipitated with antibodies to
NR1, NR2A, GluR2/3, and GluR4. Incubation with 2% SDS at 90 °C was
used to ensure optimal solubilization of subunits; NMDA receptor
subunits, in particular, have been reported to be only partially
solubilized from the brain with nonionic or weaker ionic detergents
(36, 43). Under the conditions used in the present study, about 90% of
NR1 and 80% of NR2A immunoreactivities were present in the soluble
fraction (data not shown).
After a 20-min incubation with [35S]methionine, the
predominant species of NMDA and AMPA receptor subunits appeared to be
mature, fully glycosylated forms (Fig.
2), since molecular weights of pulse-labeled subunits are similar to those of subunits that are expressed in cultured cells and the brain as determined by Western blotting (data not shown). Treatment of the culture with tunicamycin before and during pulse labeling resulted in the complete loss of the
mature subunit and the appearance of lower molecular weight bands that
migrate at a position consistent with that of the deglycosylated forms
of subunits (44-47). The absence of significant lower molecular weight
unglycosylated forms indicates a rapid glycosylation of the subunits,
and this appears to occur for nicotinic acetylcholine receptor subunits
as well (48, 49). To characterize the glycosylation properties of NMDA
and AMPA receptor subunits, membrane homogenates of cultured cells and
the cerebellum were analyzed by Western blotting after treatment with
endoglycosidase H, which recognizes N-linked high mannose
carbohydrates or N-glycosidase F (Fig.
3). Treatment with endoglycosidase H
generated low molecular weight species of NR1 subunits, which comigrate
with those that were treated with N-glycosidase F,
indicating glycosylation of NR1 subunits is of the high mannose type.
NR2A subunits showed significant sensitivity but were not completely
sensitive to endoglycosidase H. GluR2/3 and GluR4 subunits appeared to
be resistant; however, there was a slight shift in molecular weights,
indicating that some glycosylation moieties have high mannose
structures (Fig. 3).

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Fig. 2.
Treatment with tunicamycin demonstrates that
pulse-labeled subunits are obtained as mature glycosylated
subunits. Cerebellar granule cells were pulse-labeled with 250 µCi of [35S]methionine and solubilized with 2% SDS.
After diluting SDS with 2% Triton X-100, glutamate receptor subunits
were immunoprecipitated using subunit-specific antibodies and subjected
to SDS-PAGE analysis. Gels were dried and analyzed by fluorography as
described under "Experimental Procedures." For deglycosylation,
cells were incubated with tunicamycin (final concentration of 1 µg/ml) for 12 h before and during pulse labeling. Glycosylated
and deglycosylated subunits are indicated by
arrowheads.
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Fig. 3.
Glycosylation properties of glutamate
receptor subunits. Membrane homogenates from cultured cells
(gr) and cerebellum (cb) were solubilized with
1% SDS in the presence of 5% -mercaptoethanol. Soluble extracts,
after dilution with 1% -octyl glucoside, were incubated with
endoglycosidase H or N-glycosidase F and analyzed by
SDS-PAGE and Western blotting. Migration of glycosidase-treated samples
is compared with that of the control samples, which were treated
identically but without enzymes.
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Quantitation of radioactivity associated with the receptor subunits
from 0-48 h after the pulse labeling gave half-lives of 18 ± 5 h and 23 ± 8 h for the AMPA receptor subunits GluR2/3
and GluR4, respectively, and 16 ± 5 h for NR2A (Fig.
4, A and B). For
the NR1 subunit, at least two different half-lives were detected, with
the majority of the NR1 subunit being degraded rapidly with a half-life
of 2 h. Although it was difficult to accurately measure the
half-life of the slowly degrading pool due to low radioactivity remaining, we estimate a half-life of about 34 h for the slow pool. After 14 h, only about 2% of the total radioactivity
remains; since most of the rapidly degraded pool is gone at this time, we conservatively estimate that less than 10% of the total
pulse-labeled NR1 is associated with the slowly degraded pool. The
biphasic decay of NR1 could reflect different degradation rates for NR1 splice variants, since the relative abundance of C2 and C2' would be
consistent with a larger, rapidly degraded pool of C2 and a minor,
slowly degraded pool of C2'. The differential assembly of NR1 splice
variants with NR2 subunits has been proposed by Sheng et al.
(50). To determine if the C2 and C2' cassette-containing variants of
NR1 have similar or different degradation properties, immunoprecipitation was done using antibodies selective for these cassettes. As shown in Fig. 5, similar
degradation patterns were obtained for the two variants.

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Fig. 4.
Turnover rates of NMDA and AMPA receptor
subunits determined by pulse-chase labeling. After pulse labeling
with [35S]methionine, cells were harvested at the
indicated times. Harvested cells were solubilized and treated for
fluorography as described in the legend to Fig. 2. Fluorograms were
scanned for densitometric analysis using 14C-labeled
microscales as standards to quantify radioactivity. Values were expressed as percentages of the control
(radioactivity at time 0), and half-lives are mean ± S.E. from
3-7 separate experiments. A, half-lives of NR1 subunits
were estimated to be 1.7 ± 0.3 h and 34 h (three
separate determinations for each time point), and those of NR2A
subunits were 16 ± 5 h (for 5, 14, 18, 20, 30, and 48 h; 5, 4, 2, 5, 2, and 1 separate determination(s), respectively).
B, degradation of GluR2/3 and GluR4 subunits displays
half-lives of 18 ± 5 and 23 ± 8 h, respectively (for
5, 14, and 20 h, two separate determinations; for 18, 30, and
48 h, three separate determinations, respectively).
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Fig. 5.
Turnover rates of NR1 splice variants.
Antibodies made against the two different C-terminal cassettes of NR1,
C2 and C2', were used to immunoprecipitate corresponding subunits from
SDS-solubilized extracts of pulse-labeled cells at the indicated times.
Following SDS-PAGE and fluorography, radioactive bands were analyzed by
densitometric analysis as described in Fig. 4. Half-lives for the rapid
degradation phase of C2 and C2' cassette-containing NR1 subunits were
estimated as 3.0 ± 0.4 h (n = 3) and
2.3 ± 0.3 h (n = 2), respectively.
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Degradation of Glutamate Receptors on the Cell Surface--
The
rapidly degraded pool of NR1 could represent unassembled subunits that
are not expressed on the cell surface or, less likely, homomeric NR1
receptors expressed on the cell membranes. We measured the degradation
of surface receptors by determining the amount of biotinylated NMDA and
AMPA receptor subunits remaining 0-14 h after biotinylation of surface
proteins. These results (Fig.
6A) show similar degradation
properties for NR1 and NR2A as well as for the AMPA receptor subunits
and suggest that functional NMDA receptors have long half-lives similar
to those of AMPA receptors and, also, support an intracellular location
for the rapidly degraded pool of NR1. Biotinylation of surface
proteins, but not cytoplasmic proteins, was verified by showing that
SNAP25, which is present in the cytoplasm, is not biotinylated (Fig.
6B).

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Fig. 6.
Turnover of biotinylated surface glutamate
receptors. A, surface proteins of cerebellar granule
cells were biotinylated using Sulfo-NHS-LC-biotin or
Sulfo-NHS-SS-biotin, and, after solubilization with 2% SDS, they were
treated as described under "Experimental Procedures." Biotinylated
proteins were detected by chemiluminescence. Half-lives of the surface
glutamate receptors were estimated as 20 ± 4 and 21 ± 7 h for NR1 and NR2A/B, and 17 ± 4 and 21 ± 5 h
for GluR2/3 and GluR4 subunits, respectively. These values are from two
or three separate experiments. Data shown were obtained using
Sulfo-NHS-LC-biotin. B, as a control for biotinylation of
intracellular proteins, SNAP25 was immunoprecipitated from soluble
extracts of biotinylated cell membranes, and immunoprecipitates were
analyzed as described for A.
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NR1 Subunits in the Rapidly Degraded Pool Are Not Associated with
NR2--
Using pulse labeling and co-immunoprecipitation, we were not
able to clearly demonstrate that degradation of NR1 is dependent on
association with NR2 subunits due to low amounts of radioactivity in
the co-immunoprecipitating bands. To determine if the rapidly degraded
and slowly degraded pools of NR1 are differentially associated with
NR2, the co-immunoprecipitation of the subunits was investigated by
Western analysis after treatment of cultures with cycloheximide. Rapidly degraded proteins are lost more quickly than slowly degraded proteins in the presence of a protein synthesis inhibitor such as
cycloheximide, and since degradation continues in the absence of
synthesis, this approach has been used to estimate degradation rates
(51, 52). Receptors were solubilized with deoxycholate using conditions
that were previously shown to preserve complexes of NR1 and NR2
subunits (36). The solubilized receptor complex was immunoprecipitated
with an antibody to NR2, and the amount of co-immunoprecipitating NR1
was determined. As shown in Fig. 7, NR1
is found in both the bound and unbound fractions in untreated cultures.
Its presence in the unbound fraction was not due to insufficient first
round immunoprecipitation, since no NR2 immunoreactivity is detected in
the unbound fraction. However, after treatment with cycloheximide, the
amount of unbound NR1 is greatly diminished (a decrease of 76%,
n = 2), while the amount in the bound fraction is not
changed. These data show that the unassembled fraction of NR1 is
selectively affected by the block of protein synthesis and indicate
that the rapidly degraded pool of NR1 is not assembled with NR2.

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Fig. 7.
Effect of cycloheximide on unassembled and
assembled pools of NMDA receptors. After treatment of cultured
cells with cycloheximide (CHX) (50 µg/ml) or
Me2SO vehicle (0.02%) for 5 h, NMDA receptors were
solubilized with deoxycholate as described under "Experimental
Procedures." Soluble extracts were used for immunoprecipitation with
antibodies specific to NR2A/B subunits. The unbound fraction was
reimmunoprecipitated with antibodies to NR1 C2 subunits.
Immunoprecipitates were probed with NR1 PAN or NR2A antibody. Changes
in immunoreactivity were quantitated by the chemiluminescence detection
method using a standard curve generated from serial dilutions of
unbound NR1, which was not treated with cycloheximide.
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Estimation of the Size of the Rapidly Degraded Pool of
NR1--
While our pulse labeling data indicate that a relatively
small amount of the newly synthesized NR1 is associated with the slowly
degraded pool, the total amount of the slowly degraded pool is expected
to be much larger because of its relatively slow rate of degradation.
Two approaches were taken to estimate the amount of NR1 associated with
the two pools. First, we estimated the relative amounts of NR1
associated with the surface and intracellular pools, based on our
findings that surface NR1 subunits are not rapidly degraded. Intact
cells were biotinylated, and the biotinylated surface receptors were
removed from SDS-solubilized whole cell extract using streptavidin
beads. By quantifying the unbound, nonbiotinylated receptors, our
results show that 60% of the NR1 is intracellular, while only 10% of
the NR2A is intracellular (Table I).
Under the conditions which were used, we detected no biotinylated
proteins in the unbound fraction after incubation with streptavidin
beads (data not shown). Based on these findings, the rapidly degraded
pool of NR1 would account for 60% or less of the total pool of NR1.
Unless surface expression itself, rather than assembly with NR2, is the
determining factor in the degradation rate of NR1, we would expect that
some slowly degraded NR1 is also intracellular. Since a rather small
amount of NR2 is intracellular, we can conclude that the intracellular
pool of NR1 that is assembled with NR2 is also relatively small.
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Table I
Estimation of the cytoplasmic pool of the glutamate receptor subunits
Cell surface proteins of cerebellar granule cells were biotinylated as
described under "Experimental Procedures." After solubilization
with 2% SDS, biotinylated proteins were separated from nonbiotinylated
proteins using streptavidin beads. The unbound fraction was used for
Western blot analysis to estimate the cytoplasmic pool of the
receptors. Quantification was made by the chemiluminescence detection
method using a standard curve generated from serial dilutions of the
original soluble extracts. Data were expressed as mean ± S.E. for
three or four determinations.
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A second approach was to block protein synthesis with cycloheximide and
measure the remaining NR1 at a time such that most of the rapidly
degraded NR1 is lost. After treatment of granule cells with
cycloheximide, changes of NMDA and AMPA receptor subunits were
determined by Western blot analysis. The major effect was on NR1 with
40% reduction after 5 h, while NR2 did not change (Table
II). The AMPA receptor subunits were only
slightly affected under these conditions. These results suggest that
about 40% of NR1 subunits are associated with the rapidly degraded
pool. Therefore, based on these two results, most of intracellular NR1
subunits are in a pool with short half-lives.
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Table II
The effect of cyclohexamide on the expression and degradation of the
glutamate receptor subunits
Granule cells were treated with cyclohexamide (50 µg/ml in 0.02%
Me2SO vehicle) for 5 h. Cells were pelleted and subjected
to SDS-PAGE followed by Western blot analysis. Subunits were detected
by chemiluminescence. Data were expressed as percentage of the control
(from Me2SO vehicle-treated cells) and are mean ± S.E.
from four or five determinations.
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DISCUSSION |
In the present study, we characterized the turnover properties of
AMPA and NMDA receptor subunits in cultured cerebellar granule neurons.
Our studies show that, with the exception of a pool of NR1, the AMPA
and NMDA receptor subunits are degraded relatively slowly with
half-lives of about 20 h. These values, which are similar to that
recently obtained for GluR1 in cultured spinal cord neurons (31 h for
11 day cultures) (53), suggest that regulation of synthesis is not an
efficient mechanism for rapid modulation of the levels of AMPA and NMDA
receptors in neurons. This serves as indirect evidence supporting local
mechanisms, such as insertion of receptors from an intracellular pool,
in cases where rapid changes in the number of synaptic receptors have
been proposed to occur, such as long term potentiation (28, 29, 54).
Our studies using pulse labeling and treatment with cycloheximide identified a large pool of NR1 that is rapidly degraded with a half-life of about 2 h. This pool of NR1 is not assembled with NR2
based on co-immunoprecipitation analysis. Biotinylation of surface
proteins indicates that the rapidly degraded pool of NR1 is not
expressed on the cell surface and therefore would not form functional
homomeric receptors. This is consistent with results on transfected
cells showing that only NR1 and NR2 subunits that are assembled are
expressed on the cell surface (55). These findings support the
interpretation that NR1 becomes metabolically stable when it assembles
with NR2. Since we find that only a small percentage of NR2 is
intracellular, assembled receptors may become quickly associated with
the plasma membrane.
The function of the rapidly degraded pool of NR1 subunits is unclear.
The pool may simply represent misfolded or unfolded subunits that are
retained in the endoplasmic reticulum and degraded (56). A number of
other ion channel proteins, which are formed from multiple subunits,
have been shown to have subunits that are rapidly degraded and/or do
not assemble with other subunits. The
-subunit of the muscle
nicotinic acetylcholine receptor is synthesized in excess of the other
subunits, and the unassembled
-subunits undergo rapid degradation
(57). Similarly, unassembled Kv1.2 subunits of potassium channels as
well as the
-subunit of the voltage-sensitive sodium channel are
rapidly degraded (58, 59), although the
-subunits of sodium channels
have a metabolically stable pool of free subunits that has a half-life
similar to those of the assembled subunits (58).
Since all our studies have been done using cultured cells, a key
question is whether or not a similar pool of NR1 is found in neurons
in vivo. Although turnover analyses have not been done on
intact brain, there is considerable evidence supporting the presence of
a relatively large pool of unassembled NR1 in brain. Analysis of
detergent solubility of rat brain membranes showed that more NR1
subunits than NR2 can be solubilized with 1% Triton X-100 in the
presence of high salt (43, 60). Subfractionation shows that this
Triton-soluble pool is associated with the microsomal fraction, while
the synaptic membrane fraction contains little Triton-soluble NR1 (36).
Immunocytochemical studies also showed a large intracellular pool of
NR1 (61). The NR1 subunit, unassembled with NR2, could exist either as
an assembled homomeric complex or unassembled as a single subunit that
is ready to combine with NR2 subunits. The fact that NR1 mRNA alone
can produce a functional NMDA receptor in oocytes indicates that under
some conditions NR1 can form a homomeric receptor complex (8, 11).
However, a functional homomeric receptor cannot be formed in
transfected cell lines, although it appears that the NR1 subunit can
form a receptor complex that has the binding sites for the co-agonist, glycine (62, 63). Size fractionation of the Triton-soluble fraction of
NR1 from rat brain indicated a peak at 125,000 daltons, which fits the
size of a single unassembled NR1 subunit (60). However, those studies
cannot rule out the possibility that the complex was disrupted into
individual subunits with detergent treatment.
The most likely functional role of a large, rapidly degraded pool of
receptor subunits in neurons would be to serve as a reserve for
assembly into receptor complexes when the synthesis of its co-assembling subunit is increased. Since unassembled NR1 appears not
to be expressed on the surface of a neuron, the presence of an excess
pool of NR1 implies that the expression of NR2 subunits controls the
number of functional NMDA receptors. NR2 subunits define the functional
properties of the NMDA receptor, and NR2 subunits are more finely
regulated than NR1 subunits with respect to regional and developmental
expression (20, 23, 35, 64). A recent study indicates that NR2
expression during development may be regulated by synapse formation
(65). Therefore, a large pool of unassembled NR1 could be a reserve
awaiting changes in expression of NR2 subunits arising, for example,
during synapse development or other changes in synaptic strength. None
of the studies addresses the subcellular location of the NR1 pool other than indicating that it is not expressed on the cell surface. An
interesting possibility is that in neurons, unassembled NR1 subunits
are present in dendrites and assembly with NR2 occurs immediately prior
to insertion into the postsynaptic membrane. Such a mechanism would
allow assembly and surface expression to be controlled locally, by
synaptic activity, for example. The presence of an endoplasmic
reticular structure, as well as the translational machinery associated
with the endoplasmic reticulum in dendrites lends support to this idea
(66, 67). The fact that we do not see a similar pool of AMPA receptors
with rapid turnover rates may suggest that these two subtypes are
differently regulated, perhaps reflecting the different functional
roles of these receptors. A key point may be the fact that AMPA
receptors can form functional homomeric receptors, which have been
identified in neurons, while NMDA receptors appear to require both NR1
and NR2 subunits (2, 6).
The relatively long half-lives of NMDA and AMPA receptors indicate that
the number and composition of postsynaptic receptors is likely to be
finely controlled at the level of the individual synapse. While
receptors are believed to be anchored at the synapse by specific
proteins (68, 69), little is known about the mechanisms involved in
adding and removing receptors that are critical factors for determining
turnover rates. Our results together with earlier findings on the
surface biotinylation of receptors (54), indicate that surface
receptors are stable and have turnover rates that are similar to those
of the entire pool of receptors, except NR1. Therefore, half-lives of
glutamate receptors appear to reflect the half-lives of the surface
pool of the receptors, suggesting that degradation may be closely
linked to the removal of the receptor from the postsynaptic membrane.
The C terminus of some glutamate receptor subunits can be cleaved by
the calcium-sensitive enzyme, calpain (70), and since AMPA and NMDA
receptors are believed to be anchored to the postsynaptic density
through their C termini, this cleavage would irreversibly free the
receptor from its anchor and may begin the process of internalization
and degradation. In support of such a mechanism, we have observed a
rapid loss of AMPA and NMDA receptor C termini in cultured granule
cells after treatment with agonist or removal of conditioned medium (71).
Additional information will be required to determine whether or not
this is related to the normal mechanism of receptor removal from the
postsynaptic membrane.