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INTRODUCTION |
Fast excitatory synaptic transmission in the mammalian central
nervous system is primarily mediated by multimeric ligand-gated ion
channels, which are activated by the neurotransmitter glutamate. Based
on pharmacological properties and sequence similarities, these
receptors fall into three main classes: the glutamate receptor (GluR)1 subunits 1-4 form
the family of
-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid
(AMPA) receptors, subunits GluR5-7 and KA1 and KA2 are the family of
kainate receptors, and subunits NR1 and NR2A-D are components of
N-methyl-D-aspartate receptors (1, 2).
Functional receptors of each type are composed of multiple subunits.
Recent experiments suggest that each receptor complex contains four
subunits (3-6), whereas previously a pentameric structure has been
favored (7-9). Recombinant expression of an individual type of subunit
in nonneuronal cells in many cases leads to the formation of functional
homomeric ion channels. Coexpression of two or more subunits of the
same class, however, mainly directs the formation of heteromeric
channels (10-13). Their electrophysiological characteristics are
different from those of their homomeric counterparts and often are
quite similar to channels present in neurons. In vivo, GluRs
are thought to be predominantly or perhaps exclusively composed of
heteromeric channels, based on their functional similarity with
recombinant channels as well as co-immunoprecipitation experiments (7,
14-16).
The subunit composition of GluRs determines the electrophysiological
characteristics of these ligand-gated ion channels. For example, AMPA
receptors lacking the GluR2 subunit are permeable for calcium, whereas
channels containing at least one GluR2 subunit do not allow calcium
entry into the cells (10, 17). A number of studies have demonstrated
that many neurons express several subunits from different subfamilies
of GluRs (15, 18-20). This diversity is further enhanced by
alternative splicing of most subunits (21, 22).
The rules guiding the assembly of GluRs from individual subunits are
not known. One possibility is that association occurs randomly and the
composition of the GluRs in each cell is simply determined by the
availability of individual subunits. An alternative possibility is that
subunits preferentially interact with one or a small group of other
subunits and thereby favor the formation of GluRs with a distinct
subunit composition. A classical example for such a stereotypic
receptor assembly pathway is the muscle nicotinic acetylcholine
receptor.N-terminal domains of individual subunits of this
neurotransmitter receptor initiate association of specific subunits in
an ordered process (23-26).
Regions on GluR subunits mediating assembly of homomeric or heteromeric
receptors have not been identified so far. All GluR subunits are
thought to share a common transmembrane topology and domain structure
(27-30). The glutamate-binding region is formed by two extracellular
domains, which are separated in the protein sequence by two
transmembrane regions (31-33). A loop between these hydrophobic
domains is part of the ion channel. A third transmembrane region
defines the border of a C-terminal intracellular domain of variable
size, which in many subunits is a target for binding of several
PDZ-domain-containing proteins (34-37). No function has been ascribed
to date to the proximal N-terminal region. It forms a separate domain
homologous to the bacterial periplasmic leucine-isoleucine-valine-binding protein (38).
Here, we addressed the question of which domains of AMPA receptor
subunits mediate their assembly into receptor complexes. We expressed
different pairs of subunits in COS7 cells and analyzed subunit
association by immunoprecipitation. All AMPA receptor subunits but not
kainate receptor subunits associated with the GluR1 subunit with high
efficiency. Using chimeric receptors and truncation fragments of
subunits, we show that this subfamily specificity of assembly is
determined by N-terminal regions of subunits.
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EXPERIMENTAL PROCEDURES |
Antibodies
Biotinylated sheep anti-rabbit F(ab')2 fragment was
purchased from Roche Molecular Biochemicals; horseradish
peroxidase-conjugated secondary antibodies were from Amersham Pharmacia
Biotech; streptavidin-agarose was obtained from Sigma; and immobilized
monomeric avidin (ImmunoPure) was from Pierce. Anti-myc (9E10) mAb was
affinity-purified from hybridoma supernatants. Cy3-conjugated
anti-mouse antibody was obtained from Jackson Immunoresearch.
cDNA Clones
The cDNA clones GluR1 (flop), GluR2 (flip) GluR4 (flip), and
GluR7 were kind gifts from R. Sprengel and P. Seeburg (Heidelberg); the
clones of the AMPA receptor subunit GluR3 (flip) and the kainate receptor subunit GluR6 as well as the GluR1- and GluR6 derivatives (R1-PCS and R6-PCS) used for the construction of the GluR1-GluR6 chimeras were kind gifts from M. Hollmann (Göttingen).
Vector Constructs
HV Vector with N-terminal Hexa-myc-tag (HV-N-myc)--
The
hexa-myc-tag was amplified from the pCS2+MT vector (39) by PCR
(primers: TCC CAT CGA TCT GCA GCT ATG GAG and GAG AGG CCT TGC ATG CAA
GTC CTC TTC) and subcloned into the HV vector (40) between the
PstI and SphI sites.
pCMV2 vector with C-terminal Hexa-myc-tag (pCMV2-C-myc)--
The
polylinker of the pCMV2-expression vector (41) was expanded by
introducing a NotI site; the plasmid Sac-KiSS-
(42) was
digested with XbaI, and the resulting
fragment
containing the NotI site was inserted into the
XbaI site of pCMV2. By digestion with NotI and
re-ligation, the
insert was eliminated resulting in a pCMV2 with a
NotI site between two XbaI sites (pCMV2-Not). The
PCR-amplified hexa-myc-tag (primers: CAT CGA TTT AAA GCG GCC GCT ATG
GAG CAA and TAG TTC TAG AGT CTA GAG AGG CCT TGA) was introduced between
the NotI and the second XbaI site, the TAG of
XbaI forming the stop codon.
myc-tagged Constructs
Constructs that were to be tagged at their N terminus were first
amplified by PCR (the 5'-primer contained an SphI site, the 3'-primer either an XbaI site or a NotI site),
inserted into HV-N-myc between SphI and XbaI and
then subcloned into pCMV2 using ClaI and XbaI
except for GluR7N, where KpnI and XbaI was used.
Constructs with a C-terminal myc-tag were PCR-amplified, inserted into
the HV vector without myc-tag between SphI and
NotI, and then subcloned into pCMV2-C-myc using
ClaI and NotI.
Since ClaI and XbaI cut in the coding region of
the AMPA subunit GluR4, the polylinker of HV-N-myc was modified by
introducing a MluI site between SalI and
ClaI. The PCR-amplified GluR4 sequence was inserted into the
modified HV-N-myc vector with SphI and NotI and
subcloned into the pCMV2-Not vector using MluI and
NotI.
MuSK-cDNA (43) was subcloned into pCMV2-C-myc between the
ClaI and NotI site.
All full-length constructs start at position 1 behind the signal
sequence (13). The position of the first and last amino acid of the
deletion fragments are numbered according to Keinänen et
al. (13): C1 (1-365), C2 (1-524), C3 (1-546), C4 (1-618), C5
(1-812), N1 (378-862), C1-TMD A (1-381 and 525-544), C1-TMD C
(1-381 and 792-862), C2-TMD C (1-524 and 792-862), TMD A-B
(525-618), and TMD C (792-862).
Chimera with the Pore Loop Region of GluR6 (R1-PL6-R1)
GluR1-PCS and GluR6-PCS (44) were digested with NruI
and EcoRI, and the fragment containing the pore loop region
of GluR6 was inserted at the corresponding position of GluR1-PCS. The
pore loop cassette was then excised using BglII and
introduced into the GluR1-expression vector (pCDM8, Ref. 13).
Untagged GluR6-GluR1 Chimeras (R6-PL1-R1 and R6-PL6-R1)
R1-PCS and R6-PCS were digested either by NotI and
NruI (for R6-PL1-R1) or NotI and EcoRI
(for R6-PL6-R1). The R6 fragment consisting of the N terminus with or
without the pore loop was inserted into R1-PCS. Then these chimeras
were digested with XhoI, the ends treated with the Klenow
fragment of DNA polymerase I, and digested with NarI. The
NarI/(XhoI) fragment was inserted between
NarI and SmaI of untagged GluR6 (in pCMV2), thus
yielding the final chimeric constructs in the pCMV2 expression vector.
GluR1-GluR6 Chimeras with an N-terminal myc-tag (R1N-PL6-R6 and
R1N-PL1-R6)
R1-PCS and R6-PCS were digested either by NotI and
NruI (for R1N-PL6-R6) or NotI and
EcoRI (for R1N-PL1-R6). The R1 fragment consisting of the N
terminus with or without the pore loop was inserted into R6-PCS. These
chimeras were digested with XmaCI and inserted into the
N-terminally myc-tagged GluR1 (in the pCMV2) that had been digested
with the same enzyme.
Deletion Fragments Fused to Transmembrane Domains (C1TMD C,
C2-TMD C, and C1-TMD A)
Fragments C1 and C2 as well as the C-terminal part containing
the last putative transmembrane domain of GluR2 (TMD C) were amplified by PCR. The PCR products C1 and C2 were digested by SphI and NotI and inserted into HV-N-myc (see above).
PCR product TMD C was inserted into pCMV2 by exchanging the myc-tag of
pCMV2-C-myc for TMD C using NotI and XbaI.
The N-terminally myc-tagged sequences for C1 and C2 were then subcloned
from HV into pCMV2 (with TMD C) using ClaI and
NotI yielding C1-TMD C and C2-TMD C.
C1-TMD A was constructed using two sense and two antisense
oligonucleotides representing the first putative transmembrane domain
of GluR2 (TMD A) containing a NotI site at the 5'-end and an
XbaI site at the 3'-end as well as sticky ends corresponding to the mid-region of TMD A. Equal amounts of each oligonucleotide were
used for ligation into pCMV2-Not between NotI and
XbaI. C1 was then subcloned from HV-N-myc into pCMV2
containing the first transmembrane domain using ClaI and
NotI.
The integrity of the constructs was verified by specific restriction
analysis, expression in COS7 cells, and partial sequencing across
regions connecting original sequences with the myc-tag or the
transmembrane domains.
Antiserum Directed against the GluR1 Subunit
A cDNA fragment corresponding to the C terminus of GluR1
(amino acids 811-889, Ref. 13) was amplified by PCR (primers: CCT TAA
TCG GAT CCT GCT ACA AAT and GGC ACT GCA GGG CTT GG) and inserted into
the pQE30 vector (Qiagen) between BamHI and PstI.
The fusion protein containing an N-terminal hexahistidine tag was
overexpressed in Escherichia coli and purified as described
(43). Rabbits were immunized with the fusion protein in Freund's
adjuvant. After the fourth boost, the serum was purified over an
affinity column with the fusion protein coupled to a mixture of
Affi-Gel 10 and 15 (Bio-Rad).
Cell Culture and Transient Transfections
One day prior to transfection, COS7 cells were seeded at a
density of 700,000 cells/10-cm culture dish in COS medium (10% (v/v)
fetal calf serum in Dulbecco's modified Eagle's medium). COS7 cells
were transfected transiently as described previously (45) with some
minor variations that were found to improve transfection efficiency;
the transfection mix contained 40 µg of DNA/10-cm culture dish, and
the incubation time was reduced to 6 h, thereby increasing cell
survival. Usually, cells were harvested on the third day after
transfection in phosphate-buffered saline containing 5 mM
EDTA, 5 mM EGTA, 1 mM phenylmethylsulfonyl
fluoride, and 1 mM benzamidine.
Cell Lysis
COS7 cells were lysed with 500 µl of ice-cold solubilization
buffer (SB: 1% (w/v) Triton X-100, 500 mM NaCl, 5 mM EGTA, 5 mM EDTA in phosphate-buffered
saline, pH 7.1) containing freshly added protease inhibitors (1 mM phenylmethylsulfonyl fluoride, 1 mM
benzamidine, 1 µg/ml leupeptin, 1 µg/ml pepstatin, 1 µg/ml aprotinin) for 2 h at 4 °C in a head-over-head shaker. Then,
the lysate was centrifuged at 100,000 × g for 1 h
at 4 °C and the protein concentration of the supernatant was
determined using bicinchoninic acid (BCA, Pierce) with bovine serum
albumin as standard. In the solubilization experiments shown in Table
I, proteins from 200 µl of the detergent extract were precipitated and together with a corresponding aliquot of the insoluble fraction analyzed by SDS-PAGE and Western blotting.
Co-immunoprecipitation Assay with Streptavidin-Agarose
25 µl of streptavidin-agarose were incubated with 1 µg of
biotinylated anti-rabbit F(ab')2 fragment in SB for 1 h at 4 °C in a head-over-head shaker. After three washes with SB,
the beads were incubated with anti-GluR1 pAb in SB (1 h, 4 °C). The
beads were washed three times in SB before adding 500-600 µl of
cleared lysate containing 0.5 mg/ml protein. After incubation overnight at 4 °C followed by three washes with SB, the beads were incubated with anti-myc mAb in SB for 1 h, followed by another three washes with SB. Finally, peroxidase-linked anti-mouse Ig was added in SB and
incubated for 1 h at 4 °C. The beads were then washed three times with SB and once with peroxidase reaction buffer (100 mM sodium acetate, 50 mM
NaH2PO4, pH 6). 300 µl of peroxidase reaction buffer containing 2 mM
2,2'-azinobis(3-ethylbenzthiazolinesulfonic acid) and 0.016%
H2O2 were added and incubated for approximately 10 min at room temperature. The beads were pelleted by centrifugation, and the absorbance of the supernatant measured at 405 nm.
Normalization of Co-immunoprecipitation Data
The amount of co-immunoprecipitated myc-tagged protein was
normalized with respect to the amount of the same protein present in
the cell lysate. The data were then corrected for the amount of either
GluR1 or the chimeras R1-PL6-R1, R6-PL1-R1, or R6-PL6-R1 by using the
calibration curve given in Fig. 2. For better comparison between single
results, the association efficiency was expressed as percentage of the
association obtained for the combination of GluR1 and myc-tagged GluR2
("R1 + R2N").
Determination of the Extent of Co-immunoprecipitation of GluR2
with GluR1
25 µl of monomeric avidin-beads were incubated with 2 mM D-biotin in solubilization buffer at 4 °C
(three times, 20 min each) to block biotin-binding sites of residual
oligomeric avidin. The biotin was then removed from monomeric avidin by
washing the beads three times (5 min each) with elution buffer (100 mM glycine, pH 2.5, 1% (w/v) Triton X-100). The beads were
incubated first with biotinylated anti-rabbit F(ab')2
fragment in SB, then with anti-GluR1 pAb (see above) before 400 µl of
detergent extract (protein concentration 0.5 mg/ml) were added and
incubated overnight. The beads were washed three times with SB, and
then the precipitated proteins were eluted by incubating the beads
twice with 100 µl of elution buffer for 15 min at room temperature.
The two eluates were combined, and the amount of co-precipitated
myc-tagged protein as well as its amount present in the solubilized
fraction was determined by dot blotting (see below).
Western Blotting
Proteins were precipitated from detergent extracts (46) and
separated by SDS-PAGE (47). After transfer to nitrocellulose (48) using
the buffer system described by Eckerskorn et al. (49),
incubation with the primary antibodies as indicated in the figure
legends and the corresponding secondary antibodies, immunoreactive
bands were visualized by chemiluminescence using SuperSignal (Pierce)
as a substrate.
Dot-Blot Analysis
Dot-blot analysis was performed as described (50) with slight
modifications: 25-30 µl of cleared lysate (protein concentration: 1 mg/ml) were immobilized on a nitrocellulose membrane (diameter: 15 mm).
The air-dried membrane was incubated with either the anti-myc mAb or
the anti-GluR1 pAb and secondary antibodies conjugated to horseradish.
After the last washing step, membrane dots were punched out and
incubated with 250 µl of reaction buffer (100 mM
NaH2PO4, pH 4.5, containing 0.4 mg/ml
o-phenylene diamine and 0.01% H2O2)
for 5-20 min. The reaction was stopped by adding 105 µl of 30%
H2SO4 and the absorbance measured at 492 nm.
Surface Staining of myc-tagged Proteins
COS7 cells were transfected with cDNAs coding for various
myc-tagged proteins. On the third day after transfection, anti-myc mAb
was added to the culture medium and incubated for 2 h at room temperature. After three washes with phosphate-buffered saline containing 1 mM Ca2+ and 0.5 mM
Mg2+ (PBS Ca/Mg), the cells were incubated for 30 min with
blocking buffer (5% goat serum and 1.5% bovine serum albumin in PBS
Ca/Mg). The cells were then exposed for 1 h to Cy3-conjugated
secondary antibody in blocking buffer and washed extensively with PBS
Ca/Mg.
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RESULTS |
Expression of Recombinant GluR Subunits and Generation of a
Subunit-specific Antiserum--
We wanted to monitor the assembly of
AMPA and other receptor subunits into heteromeric receptors by
co-expressing epitope-tagged and untagged subunits in COS7 cells and
immunoprecipitating heteromeric receptors with a subunit-specific
antibody. To this end, we cloned the following cDNAs into
expression vectors appropriate for transient transfection in COS7
cells: the four AMPA receptor subunits (GluR1-4), two kainate receptor
subunits (GluR6 and 7), and a nonrelated membrane protein of comparable
size, the muscle-specific kinase (MuSK). These subunits were either
expressed untagged or contained six repeats of a peptide derived from
the Myc protein. This epitope tag was fused either to the C-terminal
end of the proteins or to the N terminus behind a signal sequence and
allowed us to detect all expressed receptor proteins with a single
antibody. Thus, we avoided quantification problems originating from the
use of multiple antibodies with different affinities. Transfection of all constructs into COS7 cells yielded proteins of the expected size, as seen on Western blots probed with the myc-tag-specific antibody (Fig. 1A).

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Fig. 1.
Specificity of anti-GluR1 antibody and
expression of myc-tagged proteins. COS7 cells were transfected
with various full-length constructs of either AMPA receptor subunits
(R1-R4), the kainate receptor subunits GluR6 and 7 (R6, R7), or the control protein MuSK. Cells were
solubilized, and the same amounts of specific protein were subjected to
SDS-PAGE. After transfer to a nitrocellulose membrane, the blots were
probed with either the anti-myc (9E10)-mAb (A) or the
anti-GluR1-pAb (B). All constructs but one (R1,
first lane) contained a myc-tag at either the N- or C
terminus.
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For specific immunoprecipitation of homomeric and heteromeric GluRs, we
generated an antiserum recognizing selectively the AMPA receptor
subunit GluR1. The serum was directed against a fusion protein that
consisted of an N-terminal hexahistidine tag followed by the C-terminal
sequence of GluR1. This part of the subunit is localized
intracellularly and represents the most divergent region between
different GluR subunits. Antibodies affinity-purified from this serum
recognized a band of the expected size in COS7 cells that had been
transfected with expression constructs encoding both untagged and
myc-tagged GluR1 subunits. No cross-reactivity with any of the three
other AMPA receptor subunits: the kainate receptor subunits GluR6 and 7 or the control protein MuSK, was detected (Fig. 1B). The
serum was able to immunoprecipitate more than 90% of GluR1 subunit
from detergent extracts of transfected COS7 cells, as determined by
Western blotting and dot blotting (data not shown).
Heteromeric Assembly of AMPA Receptors--
In order to assess the
influence of subunit concentration on heteromeric receptor assembly, we
expressed a constant amount of epitope-tagged GluR2 subunit with
variable amounts of untagged GluR1 subunit. Receptors containing at
least one GluR1 subunit were immunoprecipitated, and the fraction of
GluR2 subunits present in heteromeric complexes was measured by a
quantitative immunoassay. In the presence of a considerable excess of
GluR1 subunits, about 40% of total GluR2 subunits were
immunoprecipitated by the GluR1-specific antiserum and therefore had
been assembled into heteromeric complexes (Fig.
2). This fraction only slightly decreased
over a wide range of expression levels of the GluR1 subunit. Only below
a threshold when GluR1 presumably was present in limiting amounts, the
fraction of GluR2 subunit in heteromeric receptors strongly decreased
with lower GluR1 concentrations. In all of the following
co-transfection experiments, the GluR1 concentration was above the
critical concentration of 50% (Fig. 2).

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Fig. 2.
Association of GluR2 with GluR1 at different
concentrations of GluR1. COS7 cells were transfected with constant
amounts of GluR2-cDNA and increasing amounts of GluR1-cDNA. The
cells were then solubilized, immunoprecipitated, and the extent of
co-precipitation determined as described under "Experimental
Procedures." The figure represents accumulated data from five
different experiments.
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To estimate the size of receptor complexes present in COS cells, we
analyzed detergent extracts containing either GluR2N alone or GluR2N
and GluR1 by sucrose gradient centrifugation. In both cases, two peaks
sedimentating at about 5.2 and 9.8 S were found, the first probably
representing monomeric subunits, the second partially or fully
assembled complexes.2
Apparently, about half of GluR2N did not assemble into homomeric or
heteromeric complexes.
Next, we addressed the question whether individual GluR subunits differ
in their ability to co-assemble with the GluR1 subunit. The AMPA
receptor subunits GluR2, 3, and 4, the kainate receptor subunits GluR6
and 7, as well as the MuSK as control protein were each transfected
into COS7 cells together with the GluR1 subunit. The extent of
heteromeric assembly was analyzed by co-immunoprecipitation.
Each of the AMPA receptor subunits co-assembled with the GluR1 subunit
with comparable efficiency (Fig. 3). We
found no evidence for a preference of the GluR1 subunit for assembly
with one of the other AMPA receptor subunits. In contrast, the kainate
receptor subunits GluR6 and 7 reached only about 40% of this level of
association. Nevertheless, both subunits had a higher tendency to
assemble with AMPA receptor subunits as compared with the control
protein MuSK. This non-GluR-related protein displayed approximately
20% of the association seen with GluR2, which as our standard
represented 100% assembly in all of the following experiments.

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Fig. 3.
Co-immunoprecipitation of myc-tagged proteins
with GluR1. COS7 cells were co-transfected with cDNAs coding
for GluR1 and myc-tagged GluR subunits or the control protein MuSK.
After solubilization of the cells, the GluR1 subunit was
immunoprecipitated and the amount of co-precipitated myc-tagged protein
was determined. All values were corrected for expression levels of both
GluR1 and the myc-tagged protein, using the dot-blot procedure
described under "Experimental Procedures" and the curve shown in
Fig. 2. Relative association of myc-tagged proteins with GluR1 is shown
as percentage of the association obtained for the combination of GluR1
with GluR2 (R1 + R2N, 100%). R2N
indicates the association obtained from cells transfected with GluR2N
alone. R1 mixed with MuSK, R6N, or R2N
describes the association obtained from cells transfected separately
with cDNAs coding for GluR1 or the myc-tagged protein; before
precipitation with the anti-GluR1 pAb, the solubilized fractions of the
singly transfected COS7 cells were mixed at a ratio of 1:1. The data
represent the mean ± S.E. of three different transfection
experiments except R1+R7N and the mixing experiments, which were
duplicates.
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The great majority of protein association took place within cells; only
a little occurred during the immunoprecipitation procedure. When
extracts of cells expressing GluR1 or another GluR subunit or MuSK
separately were mixed after solubilization, at most 10% of the level
observed upon co-expression of AMPA receptor subunits was
co-precipitated. Furthermore, no GluR2 immunoreactivity was detected in
precipitates from cells expressing GluR2 but not GluR1. Control
experiments were performed in parallel to all our
co-immunoprecipitation experiments with other constructs with similar
results (data not shown).
To identify any effects of epitope tags on the association of subunits
in our assay, we expressed GluR2 with a tag added either to the N
terminus (R2N) or to the C terminus (R2C). Both proteins were
co-precipitated with similar efficiency, suggesting that the tags did
not interfere with receptor assembly (Fig. 3). Addition of a different
N-terminal tag to GluR1 has no effect on functional properties and
synaptic targeting of this subunit (51).
Assembly of Chimeric AMPA Receptor/Kainate Receptor
Subunits--
In our co-immunoprecipitation experiments, AMPA receptor
subunits preferentially assembled with each other into heteromeric receptors. To determine which regions of the GluRs were responsible for
selective oligomerization, we constructed a number of chimeric receptor
subunits consisting of parts of the AMPA receptor subunit GluR1 fused
to complementary regions of the kainate receptor subunit GluR6.
Chimeras containing the epitope for the GluR1-specific antibody were
co-expressed with tagged GluR2 or GluR6 subunits, and their ability to
assemble with one of these subunits was determined by
co-immunoprecipitation (Fig. 4). The
chimeric subunit R1-PL6-R1, which contains only the channel-lining
segment of GluR6, assembled with GluR2 with an efficiency similar to
that for the GluR1 subunit (Fig. 4C). Replacing the
N-terminal extracellular domain and the first transmembrane region of
GluR1 with the homologous part of GluR6 strongly reduced the
association with GluR2. Conversely, chimeric receptors containing the
N-terminal region of GluR6 efficiently co-precipitated GluR6 subunits
(Fig. 4C), whereas chimeras including the N-terminal half of
GluR1 did not associate efficiently with this subunit.

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Fig. 4.
The N-terminal domain mediates specific
subunit association of either AMPA receptor subunits or the kainate
receptor subunit GluR6. A, schematic representation of
chimeras consisting of an N-terminal part of GluR6 (black)
and a C-terminal part of GluR1 (white); transmembrane
domains are shown as vertical bars:
R6-PL1-R1, R1 fused directly to the end of TMD A of GluR6;
R6-PL6-R1, part of GluR6 containing the pore loop (see Fig.
6) fused to the C terminus of GluR1 including TMD B; R1-PL6-R1, pore
loop of GluR1 exchanged for the same region of GluR6. B,
COS7 cells were transfected with cDNA coding for GluR1 or the
chimeric constructs, lysed, and the cell extract subjected to SDS-PAGE.
After transfer to a nitrocellulose membrane, the blot was probed with
the anti-GluR1 pAb. C, COS7 cells were co-transfected with
various combinations of GluR2 or GluR6 and the chimeras. The extent of
association was determined as described previously. The result for the
association of GluR6 with GluR1 was taken from Fig. 3. The
bars represent the mean ± S.E. of three different
transfection experiments, except R6-PL6-R1+R6N, which was done in
duplicate. GluR2 was co-precipitated efficiently with the chimera
containing the N-terminal half of GluR1, whereas GluR6 was
co-precipitated with the chimera containing the N-terminal half of
GluR6. The pore loop region did not contribute to subfamily-specific
association.
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The complementary constructs containing C-terminal GluR6 sequence could
not be assessed by the same type of co-immunoprecipitation assay, since
they do not contain the epitope for the antibody used for
precipitation. To study assembly of these constructs, we cloned the
myc-epitope tag between the signal sequence and the N terminus of the
chimeric receptors (Fig. 5A).
After Western blot analysis (Fig. 5B), these constructs were
co-transfected with unlabeled GluR1 into COS7 cells and the expressed
proteins were immunoprecipitated using the GluR1 antiserum. Chimeras
containing N-terminal GluR1 sequence either with or without the pore
loop and C-terminal GluR6 sequence co-immunoprecipitated with the GluR1 subunit with an efficiency indistinguishable from that of GluR2 (Fig.
5C).

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Fig. 5.
The first extracellular domain together
with the first transmembrane domain of GluR1 mediates association.
A, schematic representation of chimeras consisting of an
N-terminal part of GluR1 (white) and a C-terminal part of
GluR6 (black); transmembrane domains are shown as
vertical bars: R1N-PL6-R6, R6 fused
directly to the end of TMD A of GluR1 (with N-terminal hexa-myc-tag,
black ball); R1N-PL1-R6, N-terminal
part of GluR1 containing the pore loop fused to the C terminus of GluR6
including TMD C. B, COS7 cells were transfected with
cDNA coding for myc-tagged GluR1 or the myc-tagged chimeric
constructs, lysed, and the cell extract subjected to SDS-PAGE. After
transfer to a nitrocellulose membrane, the blot was probed with the
anti-myc mAb. C, COS7 cells were co-transfected with various
combinations of GluR1 and the chimeras or GluR2. The extent of
association was determined as described above. The bars
represent the mean ± S.E. of three different transfection
experiments.
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Taken together, these data show that the regions responsible for the
subclass-specific assembly of GluRs largely reside in the N-terminal
half of their subunits.
Recombinant Expression and Co-immunoprecipitation of Fragments of
the GluR2 Subunit--
In order to further characterize regions in
AMPA receptor subunits mediating receptor assembly, we expressed
different epitope-tagged fragments of the GluR2 subunit together with
the GluR1 subunit and measured their degree of association. We
constructed a series of truncation fragments of increasing length,
starting from the C terminus (Fig.
6A). All deletion fragments
could be detected on Western blots as bands migrating at positions
expected from their calculated molecular weights (Fig. 6B).
In some lanes, an additional band at much higher apparent molecular
weight was present, which most likely represented nonreduced multimeric
forms.

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Fig. 6.
Association of GluR2 deletion fragments with
GluR1. A, domain structure of the AMPA receptor subunit
GluR2 and its C- and N-terminal deletion fragments. Individual domains
are represented by the following symbols: X, black
box, leucine/isoleucine/valine-binding protein-like domain
(LIVBP); ECD, extracellular domain; S1 and
S2, hatched boxes,
lysine/arginine/ornithine-binding protein-like domain (LAOBP),
glutamate binding domains; PL, open box: pore
loop; vertical black bars: transmembrane domains (TMD) A, B,
and C; punctuated box: C-terminal domain located
intracellularly; black ball: myc-tag. B,
expression of C- and N-terminal deletion fragments. Detergent extracts
of COS7 cells transfected with cDNAs coding for C1-C5 or N1 were
subjected to SDS-PAGE, transferred to nitrocellulose membranes and
probed with the anti-myc mAb. C, association of deletion
fragments with GluR1. COS7 cells were co-transfected with cDNAs
coding for GluR1 and one of the deletion fragments. The extent of
association was determined as described above. The bars
represent the mean ± S.E. of three different transfection
experiments.
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Deletion of the very C-terminal cytoplasmic region resulted in fragment
C5, whose association with the GluR1 subunit was virtually indistinguishable from the association of the unaltered GluR2 subunit
(Fig. 6C). Deletion of the extracellular region between transmembrane domains 2 and 3 as in construct C4 and deletion of the
small loop thought to be part of the ion channel pore as in fragment C3
only slightly reduced the association with GluR1. These results
demonstrate that in agreement with the data obtained with the chimeric
receptors the C-terminal half of the receptor is not required for
specific assembly. In contrast, deletion of the most N-terminal domain
X as in fragment N1 reduced association with GluR to a value slightly
above the level observed for kainate receptor subunits.
Removal of the first transmembrane domain resulted in soluble fragment
C2, which did not specifically associate with GluR1. In fragment C1,
which also co-precipitated at a low level, additionally the adjacent
extracellular region S1 involved in the formation of the glutamate
binding region was deleted.
These data implied an important role for the first putative
transmembrane region in subunit assembly of AMPA receptors since its
deletion strongly reduced the assembly of GluR2 fragments with GluR1.
This domain might contain sequence stretches mediating specific
association with other transmembrane domains. Alternatively, the
presence of a transmembrane region might be necessary to keep the
N-terminal fragments close to the membrane in an orientation favorable
for assembly. In this case, the sequence of this domain would not be
expected to be of importance for assembly. To distinguish between these
possibilities, we replaced the transmembrane domain 1 of fragment C3 by
transmembrane domain 3 including the C-terminal intracellular domain
and assessed the capacity of the resulting transmembrane protein
(C2-TMD C) to assemble with GluR1 (Fig. 7). Fragment C2-TMD C
co-immunoprecipitated with GluR1 with nearly the same efficiency as
fragment C3 or the unaltered GluR2 subunit indicating that the second
alternative is correct, i.e. the presence of a transmembrane
region irrespective of its sequence appears to be required for
efficient subunit assembly. This was corroborated by control
experiments, which showed that transmembrane domain C expressed in the
absence of fused sequence did not co-precipitate above the level of the
control protein MuSK. A similar low level of association was found for
a very hydrophobic fragment comprising the transmembrane domains A and
B and the pore loop (Fig. 7).

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Fig. 7.
Association of C-terminal GluR2 deletion
fragments fused to transmembrane domains with GluR1. A,
schematic representation of the C-terminal deletion fragments fused to
TMD A or C (compare with legend to Fig. 6A). B,
Western blot analysis of expression of the deletion fragments.
Detergent extracts of COS7 cells transfected with cDNAs coding for
C2-TMD C, C1-TMD C, or C1-TMD A were subjected to SDS-PAGE, transferred
to nitrocellulose membranes, and probed with the anti-myc mAb.
C, association of the deletion fragments with GluR1. COS7
cells were co-transfected with cDNAs of GluR1 and one of the
deletion fragments. The extent of association was determined as
described above. The bars represent the mean ± S.E. of
three different transfection experiments.
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These results prompted us to reassess the assembly of the most
N-terminal domain C1 in the presence of a transmembrane domain. We
fused either transmembrane domain A or C to this fragment and transfected the fusion fragments (C1-TMD A and C1-TMD C) together with
GluR1 into COS7 cells. Both fragments had the expected size when
analyzed by SDS-PAGE. They co-precipitated with the GluR1 subunit with
only slightly reduced efficiency (about 80%) as compared with the
GluR2 subunit (Fig. 7). These results indicate that the most important
domain directing subunit assembly is localized in the more N-terminal
part of the extracellular region, which is not part of the glutamate
binding region. The still lower capability of fragments C1-TMD A and C
in comparison with the complete subunit could suggest that other parts
of the N-terminal domain also participate in subunit assembly.
Alternatively, the slightly reduced assembly could be due to distorted
conformations of these constructs caused by our splicing of normally
separated domains.
Structural instability of proteins often causes their nonspecific
aggregation and retention in the endoplasmic reticulum by the quality
control system located there (52). To obtain an indication of how much
our constructs were affected by these processes, we investigated which
portion of each fragment could be solubilized in a Triton
X-100-containing buffer and which constructs were expressed on the cell
surface (Table I). Upon detergent
extraction, more than 40% of the full-length subunits and the chimeric
proteins were found in the soluble fraction. Many of our deletion
fragments could be solubilized to a similar or even higher extent.
However, fragments C1 and C2, which do not contain transmembrane
domains, as well as these fragments artificially fused to transmembrane domains were found to a higher degree in the insoluble fraction. The
apparent structural instability of these fragments could be caused by
the presence of only one half of the glutamate binding region, which is
normally formed by an association of domains S1 and S2 (31-33). Even
these fragments, however, could be solubilized to at least one third of
the level of the entire subunit. Only association of the solubilized
fraction was measured in our co-immunoprecipitation assay.
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Table I
Detergent solubility and surface localization of full-length subunits,
chimeras, and deletion fragments of GluR2
COS7 cells were transfected with cDNA coding for the full-length
GluR subunits, deletion fragments, or the chimeric proteins R1N-PL6-R6
and R1N-PL1-R6. For the determination of detergent solubility, cells
were treated for 1 h with a Triton X-100-containing extraction
buffer. Proteins from the supernatant and pellet were analyzed by
SDS-PAGE and Western blotting. Indicated is the mean percentage of
total immunoreactivity recovered in the supernatant from two
experiments. For the assessment of surface localization,
nonpermeabilized cells were stained with the anti-myc mAb. ND, not
determined; surface localization of fragment C5 could not be analyzed
since its epitope tag is located in an intracellular region. A fraction
of the fragments C1 and C2, which do not contain a transmembrane
region, most likely precipitated upon secretion or unspecifically
attached to the cell surface and was therefore detectable by our
staining procedure. Both fragments were also detected in the medium.
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While the nature of the transmembrane domain did not influence assembly
with GluR1, it modified surface expression of fusion proteins. In
nonpermeabilized COS7 cells, fragments C1-TMD C and C2-TMD C were
accessible to antibodies added into the medium and therefore were
expressed on the cell surface (Table I). In contrast, fragment C1-TMD A
could not be detected in the plasma membrane and most likely was
retained within the cell. Similarly, the deletion fragment C4 also was
not detected on the surface, although its association with GluR1 was
comparable to that of construct C3.
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DISCUSSION |
Transient transfection of subunits into eukaryotic cell lines of
nonneuronal origin has been used in many studies to identify different
regions of neurotransmitter receptors directing assembly into
functional receptors (10, 12, 53, 54). In this study, we applied this
approach to identify regions that mediate specific association of AMPA
receptor subunits.
Subfamily Specificity of AMPA Receptor Assembly--
Upon
heterologous expression in COS7 cells, antibodies against the GluR1
subunit co-precipitated each of the co-expressed AMPA receptor subunits
GluR2, 3, or 4 with indistinguishable efficiency. In contrast, a
kainate receptor subunit was co-precipitated to a much lower extent,
which, however, was still above the level of our control protein. Thus
the subfamily specificity of GluR assembly was conserved in COS7 cells,
an important feature known from functional analysis of heterologously
expressed GluRs. Immunoprecipitation experiments from detergent extract
of neurons show a similar segregation of subunits between subfamilies
(14-16, 55). Whether a small amount of receptor complexes containing
both AMPA and kainate receptor subunits is present in neurons is still
a matter of debate (15, 55). The number of mixed complexes in neurons
might be reduced not only by preferential subunit assembly, but also by differential targeting and stabilization of receptor complexes.
In order to quantitatively compare assembly of subunits by
co-immunoprecipitation, we first assessed its dependence on the expression level of transfected receptors. Above a critical
concentration, co-precipitation only slightly increased with higher
subunit levels. By investigating subunit assembly under conditions when
expression was above this concentration and carefully monitoring and
correcting for individual expression levels of subunits, it was
possible to determine subunit association with considerable accuracy.
Despite the high sensitivity of our method, we were unable to detect
differences in assembly between the subunit GluR1 and GluR2, 3, or 4.
Electrophysiological studies have shown that upon heterologous
expression of combinations of two or more AMPA receptor subunits glutamate-gated channels are formed, which differ in their
characteristics from homomeric channels (10, 11). It has been difficult
to directly compare the efficiency of subunit assembly in these
functional studies because the expression levels of individual subunits
are not known. In addition, the abundance of ion channels with low opening probabilities and ion conductivities tend to be underestimated in whole cell recordings. Similar difficulties were avoided by our
experimental approach, in which we controlled expression levels and did
not rely on functional properties for detection of receptor complexes.
Our study indicated that AMPA receptor subunits do not carry intrinsic
signals directing a preferential assembly of distinct subunit
combinations. A similar random association of closely related subunits
was shown for different
-subunits of the glycine receptor, which can
freely substitute for each other (56).
N-terminal Domains of AMPA Receptor Subunits as Well as a Membrane
Anchor Are Important for Subfamily-specific Receptor
Assembly--
Using chimeric receptors and truncation fragments of
GluR subunits, we identified domains of AMPA receptors directing
specific assembly. Both approaches indicated that regions important for association are localized in the N-terminal half of these subunits. Chimeric receptors, in which the N-terminal part of an AMPA receptor was combined with a C-terminal half derived from a kainate receptor subunit, efficiently co-precipitated with the GluR1 subunit. Similarly, truncation fragments of an AMPA receptor subunit retaining the N-terminal X domain as well as at least one transmembrane region associated efficiently with GluR1.
The specificity of co-precipitation of the chimeric receptors is
highlighted by the fact that the replacement of the N-terminal half of
subunit GluR1 with the corresponding region of GluR6 had opposite
effects on the assembly with AMPA and kainate receptors; this exchange
increased the co-precipitation with GluR6, whereas it decreased the
co-precipitation with GluR2 to the level normally observed with kainate
receptor subunits. Such an association pattern could not easily be
explained by abnormal folding of the chimeric protein. Rather, the
reduced association with GluR2 and increased association with GluR6 had
to be ascribed to the removal or addition of regions mediating
subfamily-specific subunit binding. Furthermore, these observations
imply that, for the assembly of kainate receptors, N-terminal domains
of the corresponding subunits are instrumental as well, although this
aspect was not further analyzed.
The specificity of association is more difficult to assess for deletion
fragments. Co-immunoprecipitation of fragments with GluR1 might be lost
either because specific assembly domains were deleted or because of
misfolding of fragments induced by truncations. Differences in
detergent solubility and surface expression between our fragments
indeed suggest that the structural integrity of some constructs was
affected. Therefore, we based our conclusions mainly on fragments
retaining the capability of association with GluR1 at a level
comparable to that of the parental subunit. Structural alterations of
fragments might give rise to unspecific hydrophobic interactions. To
exclude this possibility, we measured the co-precipitation of fragments
in the absence of GluR1, which was very low for all fragments presented here.
Upon heterologous expression in mammalian cells, about 30-40% of
homomeric or heteromeric AMPA receptors are found on the cell surface
(57). Many of our chimeras and subunit fragments also were targeted to
the plasma membrane, but cell surface expression clearly was no
prerequisite for association: For example, fragments C3 and C4
co-immunoprecipitated with similar efficiencies with GluR1, although
only C3 was expressed on the cell surface. Subunits of many receptors
associate to larger complexes immediately after synthesis in the
endoplasmic reticulum (58). Our observation that fragments that were
retained within the cell did not lose their ability to interact with
other subunits suggests that folding of the assembly region is to some
extent independent from other regions.
Deletion of the C-terminal half of GluR2 up to the first transmembrane
domain did not strongly affect its association with GluR1. These data
were in agreement with our observations obtained from chimeric
receptors. They indicate that this half of the protein does not play an
important role in subunit assembly. Deletion of the first transmembrane
region strongly reduced association, as has been shown for the assembly
of nicotinic acetylcholine receptors and potassium channels (26, 59,
60). The association of two fusion proteins in which the first
transmembrane domain was replaced by the third showed that the sequence
of this domain was not important for assembly.
Membrane domains have been shown to direct the multimerization of a
number of proteins, for example the T cell receptor, the major
histocompatibility complex class II complex, and glycophorin (61-63).
Apparently, these domains do not play a comparable role for GluR
subunits. Our data suggest that a transmembrane domain is simply
required for efficient assembly because it provides a membrane anchor
leading to an enrichment of the attached domains in close vicinity to
the membrane where assembly occurs. In addition, such a domain may
support subunit association by orienting subunits in a way that favors
their assembly into larger complexes.
Considerable evidence identifies the proximal N-terminal X domain that
is homologous to the bacterial leucine/isoleucine/valine-binding protein (38) as the major determinant of subfamily-specific subunit
association. This region fused to a transmembrane domain directed an
association with GluR1, almost reaching the level of the entire GluR2
subunit. Additional chimeric proteins and fragments should in future
experiments allow a more detailed analysis of this assembly domain. The
key role of N-terminal domains for subunit assembly found here mirrors
a similar importance of these regions for assembly of other ion
channels, for example nicotinic acetylcholine, GABAA, and
glycine receptors, as well as potassium channels (26, 56, 59, 60, 64,
65). The advantages of such an arrangement are presently unclear.
It remains to be seen whether assembly of GluRs in neurons follows a
similar pattern as the one described here for COS7 cells or if subunit
association is modified by neuron-specific factors. A number of
proteins have recently been identified that can associate with the
cytoplasmic domain of individual GluR subunits (34, 35, 66, 67).
Binding of these components could shift the assembly pattern in favor
of specific subunit combinations while preventing others. In addition,
neurons might be able to selectively direct distinct GluR subtypes to
particular synapses, either by local synthesis (68) and assembly of
AMPA receptor subunits in dendrites or by selective targeting of AMPA
receptors to synaptic regions based on their subunit composition. One
of these mechanisms or their combination could be responsible for an
unequal distribution of certain types of AMPA receptors at different
synapses (16, 69, 70).