From the ¶ Neuroscience Center of Excellence and the
Department of Neurology, Louisiana State University Health Sciences
Center, New Orleans, Louisiana 70112 and the Department
of Pathology and Laboratory Medicine, Texas A&M University System
Health Science Center, College Station, Texas 77843
Received for publication, July 26, 2000, and in revised form, November 10, 2000
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ABSTRACT |
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High resolution structural studies of models of
glutamate receptors (GluRs) have been limited to monomeric models of
the ligand-binding site. To obtain oligomeric models of glutamate
receptors that can reveal more complete structural information, we
examined the assembly and ligand binding properties of two truncated
versions of the GluR1 subunit. The first version, GluR1-WS, consisted
of only the N-terminal extracellular segment
(Ala1-Glu520) bridged by a synthetic
linker to the second extracellular domain (Asn615-Gly790). The second version, GluR1-M1,
consisted of the first N-terminal extracellular domain
(Ala1-Glu520) bridged by a synthetic linker to
a second segment containing the second extracellular domain, the third
transmembrane domain, and the intracellular C-terminal domain
(Asn615-Leu889). When expressed in
Xenopus oocytes, GluR-WS was secreted and water-soluble;
GluR1-M1 was displayed on the surface of oocytes. GluR1-WS exhibited a
velocity sedimentation profile that was consistent with assembly of
homooligomers and bound the glutamate receptor agonist
Ionotropic glutamate receptors
(GluRs)1 mediate fast
synaptic excitatory neurotransmission in the nervous system (1). Their biochemical, pharmacological, and physiological properties have been
extensively studied (2-5) because they are important therapeutic targets for the treatment of many neurological disorders (6). The
rational design of drugs that selectively target different subtypes of
glutamate receptors critically depends on high resolution structural
studies. As membrane proteins, however, they belong to a general class
of proteins for which there is a paucity of high resolution x-ray
crystallographic information. Structural studies of membrane proteins,
in contrast to those of soluble proteins, have been hampered by
numerous hurdles including those associated with obtaining large
quantities of recombinant membrane proteins through expression in
heterologous expression systems and those associated with the
deleterious effects of detergents on the formation of crystals suitable
for X-crystallography.
To circumvent some of these problems, water-soluble extracellular
domains of many membrane proteins have been processed for crystallographic studies (7-15). Neurotransmitter-gated ion channels, however, represent a greater challenge for this strategy than monomeric
single transmembrane domain membrane proteins because of the
contribution of residues from multiple subunits to their pharmacological properties. Thus, establishing which domains of their
subunits are required for assembly and assessing whether they are
capable of forming water-soluble species with ligand binding properties
that mimic those of native receptors are essential intermediate goals
toward establishing suitable models of neurotransmitter-gated ion
channels for long term structural studies.
In recent years it has become clear that for GluRs, two discontinuous
extracellular domains of GluR subunits contribute to the formation of
their ligand-binding site (16). In elegant work done since then, it has
been shown that a fusion protein consisting of two short segments
termed S1 and S2, one from each of these two discontinuous
extracellular domains when bridged by an artificial linker, forms a
monomer that is capable of binding GluR ligands (17, 18). The crystal
structure of a S1-S2 monomer derived from the GluR2 subunit has also
been recently obtained (19). However, full-length glutamate receptors
are thought to exist as either tetramers (20-22) or pentamers (23-25)
in which neighboring subunits influence their ligand binding properties (26, 27). Thus, a more complete understanding of the contributions of
neighboring subunits and other regions of GluR subunits to their
overall structure and pharmacological properties is desirable. Recently, the feasibility of obtaining extracellular domains of ligand-gated ion channels that assemble into an oligomer whose size and
pharmacological properties are consistent with the formation of
water-soluble receptors for the AChR To achieve a similar goal for GluR subunits, we have examined the
assembly and ligand binding properties of two truncated GluR1 subunits
GluR1-WS and GluR-M1. Based on its velocity sedimentation profile on
sucrose gradient, high binding affinity for
Design of GluR1-142, GluR1-236, GluR1-M1, and GluR1-WS
Constructs--
All site-directed mutagenesis was performed using the
Altered Sites II in vitro mutagenesis system (Promega,
Madison, WI). The amino acid numbering system used refers to that of
the mature GluR1 polypeptide (29). To tag the GluR1 subunit at its C
terminus, an XbaI restriction enzyme site was introduced
into the GluR1 subunit cDNA by site-directed mutagenesis after the
C-terminal residue 889 and then double-stranded synthetic DNA cassettes
encoding epitope tags were ligated into the engineered XbaI
sites. Two different epitopes and mAbs were used in this study and both
correspond to well characterized epitopes derived from the sequence of
the Torpedo AChR GluR Protein Expression in Xenopus Oocytes--
cRNA from
linearized cDNA templates was synthesized in vitro using
SP6 RNA polymerase in conjunction with reagents from the mMessage
mMachine Kit (Ambion, Austin, TX). Oocytes were prepared for injection
as previously described (31). Oocytes were injected with 50-100 ng of
RNA/oocyte and incubated at 18 °C in ND-96 solution (96 mM NaCl, 2 mM KCl, 1 mM
MgCl2, 1.8 mM CaCl2, 5 mM HEPES, pH 7.6) for 2-3 days prior to harvesting them.
Solubilization of GluR Proteins--
Oocytes were homogenized
using a microcentrifuge tube plastic pestle in buffer A (50 mM
Na2HPO4-NaH2PO4, pH
7.5, 50 mM NaCl, 5 mM EDTA, 5 mM
EGTA, 5 mM benzamidine, 15 mM iodoacetamide, 2 mM phenylmethylsulfonyl fluoride). The homogenized
membranes were collected by centrifugation in a microcentrifuge at
15,000 rpm for 25 min. Receptors were solubilized by gentle agitation
of oocyte membranes in buffer A containing 2% Triton X-100 at 4 °C for 1 h. After removing cellular debris by centrifugation at
15,000 rpm for 25 min, the cleared extracts were used in all experiments.
Immunopurifications--
Triton X-100-solubilized oocyte
membrane extracts (100-200 µl) were incubated with ~10 µl of
mAb-coupled Actigel ALD bead (0.5 mg/ml) at 4 °C using gentle
agitation overnight. The beads were then washed five times with 1 ml of
solubilization buffer and eluted with sample buffer (lacking
Immunoblotting--
Where indicated, direct detection of the
protein was performed by incubating the membranes with 20 nM 125I-mAb 142 or 125I-mAb 236 in
phosphate-buffered saline solution containing 0.1% Tween and 5%
nonfat milk powder. In some cases, membranes were incubated with 20 nM primary mAbs in phosphate-buffered saline solution
containing 0.1% Tween and 5% nonfat milk powder, and the binding of
the primary mAbs was detected using goat anti-rat secondary Abs
conjugated to horseradish peroxidase in conjunction with a
chemiluminescence detection kit (SuperSignal; Pierce) after washing off
the unbound primary mAb.
Surface Binding Assays--
Oocytes (10 oocytes/200 µl) were
incubated with 20 nM 125I-mAb 142 (specific
activity, ~1018 cpm/mol) in ND-96 containing 10% heat
inactivated horse serum, washed five times with 1 ml of ND-96, and
counted in a Sucrose Gradient Sedimentation--
200 µl of Triton
X-100-solubilized membrane proteins from 10-20 oocytes expressing the
various GluR1 proteins were layered onto 5-ml sucrose gradients
(5-20% (w/v)) in 0.5% Triton solution containing 100 mM
NaCl, 10 mM sodium phosphate, 5 mM EGTA, 5 mM EDTA, and 1 mM NaN3, at pH 7.5. The gradients were centrifuged 50 min at 40,000 rpm at 4 °C in a
Beckman NVT90 rotor. Aliquots of 11 drops (~130 µl) from the
gradients were collected from the bottom of the tubes into Immulon 4 plastic microwells coated with the appropriate mAb. The entire gradient
was collected in 40 fractions. After gentle agitation of the microwells
for 24 h at 4 °C, the microwells were washed and incubated with
either 2 nM 125I-mAb 142 or 2 nM
125I-mAb 236 for 24 h, and following washing, the
presence of immobilized protein was detected by Ligand Binding Assays--
2% Triton X-100-solubilized membrane
proteins from oocytes expressing the GluR proteins were incubated (100 µl/well) overnight on Immulon 4 (Dynatech) wells coated with mAb 142 or mAb 236 as previously described (30). The wells were washed three
times with phosphate-buffered saline containing 0.05% Triton X-100 and incubated with various concentrations of [3H]AMPA (49.3 Ci/mmol; Amersham Pharmacia Biotech) for 1 h at 4 °C in the
presence of 100 mM NaSCN. The wells were then washed with
ice-cold phosphate-buffered saline, 0.05% Triton solution, and the
bound radioactivity was measured by scintillation counting. Nonspecific
binding was measured with Triton X-100 membrane extracts of uninjected
oocytes. The equilibrium dissociation constant Kd for the [3H]AMPA binding was determined by least squares,
nonlinear fitting to a Hill-type equation C = C0(1/(1+(Kd/L)n)),
where C is the measured signal (in cpm),
C0 is the maximal signal, L is the
concentration of [3H]AMPA, and n is the Hill coefficient.
Expression of GluR1 Proteins in Xenopus Oocytes--
Schematic
representations of the GluR1 subunit constructs used in this study are
shown in Fig. 1. GluR1-142 represents
the full-length GluR1 subunit tagged with an epitope for mAb 142, and
GluR1-236 represents the full-length GluR1 subunit tagged with an
epitope for mAb 236. Both mAbs are against well defined epitopes
derived from the Torpedo AChR
These constructs were created to evaluate the necessity of different
regions of the GluR1 subunit for subunit assembly and ligand binding.
GluR1-WS would allow us to test whether the extracellular domains of
the GluR1 subunit alone is sufficient for subunit assembly. The
hyphenated "WS" of GluR1-WS refers to the expectation that this
construct would be expected to be water-soluble. GluR1-M1 would allow
us to evaluate whether the presence of a single TMD was sufficient for
oligomerization of GluR1 subunits. The expression levels of each of
these subunits was checked by immunoblot analysis of Triton X-100
extracts from oocytes expressing the tagged subunit using either
125I-mAb 142 or 125I-mAb 236 as appropriate.
The expression of the truncated subunits GluR1-WS and GluR1-M1 were
expressed at approximately the same level (Fig.
2), indicating that deletions of portions
of the subunit did not grossly affect their stability in oocytes.
GluR1-WS Is a Secreted Water-soluble Protein, and GluR1-M1 Is
Membrane-bound--
The topology of GluR subunits has been extensively
investigated (34-36). They are proposed to have an extracellular
N-terminal domain, followed by a TMD, a membrane reentrant loop, a
second TMD, a second extracellular domain, a third TMD, and a
C-terminal domain. Based on this topology, GluR1-WS was expected to be
devoid of TMDs, and GluR1-M1 was expected to contain one TMD. Thus, we tested whether GluR1-WS would form a water-soluble secreted protein and
whether GluR1-M1 as predicted would be expressed as a membrane-bound protein on the surface membrane of oocytes. The expression of GluR1-WS
in the aqueous (secreted) and Triton X-100-solubilized fractions of
oocyte proteins was monitored by immunoblot analysis. To eliminate the
possibility that proteins in the aqueous media originated from cellular
debris from unhealthy microinjected oocytes, care was taken to collect
aqueous media only from oocytes that were deemed healthy by microscopic
examination following an incubation period of 24 h. The surface
expression of GluR1-M1 was monitored by the binding of
125I-mAb 236 to whole oocytes expressing GluR1-M1. We found
that GluR1-WS was secreted as a water-soluble protein in the aqueous media (Fig. 3A) and that
GluR1-M1 was expressed on the surface membranes of oocytes (Fig.
3B).
The amount of secreted water-soluble protein was found to be at least
10-fold lower than the amount released after homogenization of the
membranes and at least 50-fold lower than the amount solubilized by
Triton X-100 as judged by the relative intensity of the immunoreactive bands. Two possible reasons could account for the relative difference in the yields of water-soluble GluR1-WS versus
Triton-soluble GluR1-WS. The first possibility is that the detergent
was required to release the GluR1-WS from membrane association that
arose through hydrophobic patches of the protein that were left exposed
by incomplete assembly of the truncated subunits. A second possibility
is that the detergent was required to release the bulk of the
water-soluble, nonmembrane associated GluR1-WS that was trapped inside
membrane vesicles. Because the overall yield of protein expression in
oocytes was low, we found it necessary to use Triton-solubilized
proteins for all our further studies.
Sedimentation Profile of GluR1-WS Suggests the Formation of
Homooligomeric GluR Species--
To determine whether the truncated
GluRs were capable of oligomerizing, we examined the velocity
sedimentation profiles of Triton X-100-solubilized proteins from
oocytes expressing GluR1-WS or GluR1-M1 on 5-20% (w/v) sucrose
gradients. To enable us to follow the sedimentation profiles of these
protein species across the gradient, we used either
125I-mAbs or chemiluminescent assays to detect them. We
followed the sedimentation profile of GluR1-WS across the gradient by
first immobilizing protein species using mAb 236 followed by
fractionation of the immobilized protein by SDS-PAGE and detection of
the tagged GluR1-WS protein by immunoblotting using mAb 236. The
sedimentation of four proteins of known molecular masses, bovine serum
albumin (66 kDa), alcohol dehydrogenase (150 kDa),
GluR1-WS sedimented near the top of the gradient as at least two
discernable protein species (Fig.
4A) defined by peaks on the
sedimentation profile between fractions 26-31 and 32-37,
respectively. The open arrows in Fig. 4A indicate
the fractions with the highest protein concentrations (fractions 30 and
34) that were used to estimate the apparent molecular masses of the two
species. The major species around fraction 34 had an apparent molecular
mass of 140 kDa, and the next most abundant species around fraction 30 had an apparent molecular mass of 255 kDa (Fig. 4B). The
calculated molecular mass of a GluR1-WS monomer is 78.5 kDa. Thus, the
molecular mass of the most abundant protein species (140 kDa) is close
in value to the calculated molecular mass of a GluR1 dimer (157 kDa). Similarly, the molecular mass of the second most abundant protein species (255 kDa) is close in value to the calculated molecular mass of
a GluR1-WS trimer (235.5 kDa). These results suggest that the GluR1-WS
formed dimers and trimers. A third, smaller peak around fractions
24-25 might represent a high order oligomer or mixture of oligomers.
Notably, very little of GluR1-WS was retained at the very top of the
gradient, where a monomeric form of the protein would be expected to
run relatively close to the position of the bovine serum albumin of
molecular mass of 66 kDa. Virtually no GluR1-WS was detected at the
bottom of the gradient (i.e. fast sedimentation), where
nonspecifically aggregated protein would be expected. In contrast,
GluR1 and GluR1-M1 showed broad sedimentation profiles (data not
shown). Anomalously fast sedimentation (i.e. faster than
GluR1-142/236) was observed for GluR1-M1, possibly because of
nonspecific aggregation.
GluR1-WS Binds [3H]AMPA--
To examine the
maturation of the ligand-binding site in glutamate receptor species
formed by GluR1-WS and GluR1-M1, we measured the ability of Triton
X-100-solubilized species to bind [3H]AMPA. We
solubilized oocytes with Triton X-100 to release all the intracellular
GluR1-WS species because the secreted fraction of GluR1-WS protein was
found to be too little to be used in ligand binding assays. Specific
high affinity radioligand binding was observed for both GluR1-WS and
GluR1-M1 species in the solid phase binding assays using 20 nM [3H] AMPA (data not shown). To more
precisely determine the affinity of GluR1-WS receptor species for
[3H]AMPA, binding assays were carried out with varying
concentrations of [3H]AMPA and yielded a
Kd of ~32 nM (Fig.
5). This value is within the nanomolar
range of Kd values reported for cloned AMPA
receptors (2, 5) as well as those reported for monomeric S1-S2 species
derived from GluR2 (18) and GluR4 subunits (17). The ability of
GluR1-WS to bind [3H]AMPA is consistent with the results
of other investigators, showing that only the S1-S2 portion of the
extracellular domains is sufficient for binding glutamatergic ligands
(17, 18).
GluR1-WS Assembles with Full-length GluR1 Subunits--
GluR1-WS
is water-soluble and as such is of significant interest for further
structural studies. Hence, to obtain additional evidence for the
ability of GluR1-WS to assemble into oligomers, we examined its ability
to assemble with full-length GluR1 subunits. GluR1-WS was coexpressed
with GluR1-142 in Xenopus oocytes and solubilized in
Triton X-100. Solubilized GluR species were immunopurified using mAb
236 beads and fractionated by SDS-PAGE. The presence of
coimmunopurified GluR1-142 subunits was examined by immunoblotting with mAb 142 to the engineered epitope in GluR1-142. As controls for
nonspecific binding of proteins to the mAb beads, we used beads coupled
to rat IgG. The coimmunopurification of GluR1-142 with GluR1-WS (Fig.
6) shows that GluR1-WS retained its
ability to coassemble with GluR1-142 subunits. This result indicated
that GluR1-WS appeared to have undergone sufficient conformational maturation to compete for assembly with the full-length GluR1 subunit.
In this study, we examined the assembly properties of two fusion
proteins consisting of the two extracellular domains of the GluR1
subunit without TMDs (GluR1-WS) or with a TMD (GluR1-M1) with velocity
sedimentation profiles on sucrose gradients, with the binding
properties of [3H]AMPA, and with coimmunoprecipitation.
We found that GluR1-WS appeared to be capable of assembling into
homooligomeric GluR species and bound [3H]AMPA with high
affinity. The GluR-WS protein was water-soluble, and the GluR1-M1
protein was membrane bound at the cell surface. Collectively, these
results demonstrate that GluR1-WS retains the ability to bind
glutamatergic ligands and suggest that it also assembles into specific
oligomers because of the inclusion of the N-terminal portion of the
GluR1 subunit.
Both GluR1-WS and GluR1-M1 appeared to be stably expressed in oocytes.
GluR1-WS was detected as a secreted water-soluble protein in keeping
with the newer topology proposed for GluR subunits in which the two
extracellular domain composing the GluR1-WS protein do not contain a
TMD. Because GluR1-WS was secreted as a water-soluble protein, it
provides a good starting point for exploring ways, in high level
expression systems, to circumvent problems associated with the use of
detergents to solubilize and crystallize GluRs. Our results extend the
work of others demonstrating that S1-S2 fusion proteins containing
shorter portions of the extracellular domains of the GluR2 subunit (18)
and the GluR4 (17) subunit are also processed as secreted water-soluble
protein in heterologous expression systems.
The velocity sedimentation profile of the Triton X-100-solubilized
GluR-WS on sucrose gradients was more compatible with that of a mixture
of specific higher order oligomers than with that of monomers alone. We
observed a close correlation between the apparent molecular masses (140 kDa and 255 kDa) of two peaks on the GluR1-WS sedimentation profile and
the calculated molecular masses of GluR1-WS dimers (157 kDa) and
trimers (235.5 kDa). Also, little GluR1-WS was detected at the bottom
of the gradient where nonspecifically aggregated protein would be
expected. These observations correlate well with a GluR1-WS dimer and
not a GluR1-WS monomer or nonspecifically aggregated protein, as the
dominant species in the most abundant peak near the top of the gradient
(fraction 34). The next most abundant peak with a faster sedimentation
velocity (fraction 30) is consistent with the next higher order
oligomer, i.e. a GluR1-WS trimer.
The ability of GluR1-WS to successfully compete for coassembly with the
full-length GluR1-142 subunit also supported our conclusion that
GluR-WS has the conformational maturation to form specific oligomers.
This result also further demonstrated that the extracellular domains
alone promote assembly of subunits. The ability of GluR1-WS to assemble
into higher order homooligomers is a novel finding because it
demonstrates that a fusion protein consisting of the complete
extracellular portion of the GluR1 subunit polypeptide chain is not
only water-soluble but also retains its ability to assemble. These
results are also consistent with the earlier finding that the most
distal N-terminal region of the GluR2 subunit promotes subunit
assembly (37).
We showed that Triton X-100-solubilized GluR1-WS was capable of binding
[3H]AMPA on solid phase radioimmunoassays, suggesting
that this protein, when synthesized in oocytes, exhibited maturation of the ligand-binding site. Ligand binding in this case, however, does not
distinguish between monomers and specific higher order oligomers of
GluR1-WS. These results complement those of other investigators who
have previously demonstrated that the S1-S2 portion of the
extracellular domain of the GluR4 subunit alone is sufficient to bind
several different glutamatergic ligands with high affinity (17).
Recently, while this work was in progress, a fusion protein similar in
design to the GluR1-WS construct but derived from the GluR4 subunit was
shown to assemble predominantly into dimers that exhibit high affinity
for glutamatergic ligands (38), when expressed at high levels in insect
cells. The authors speculated that the dimers might be an assembly
intermediate in the formation of oligomers (tetramers or pentamers) of
the GluR4 water-soluble fusion protein. Collectively, both these
results suggest that the ability of the extracellular domains to
oligomerize might be a general characteristic of all subunits within
the GluR family.
The GluR1-M1 subunit was designed with the hope that it might more
efficiently assemble than constructs lacking TMDs. This hope was not
clearly borne out by the results. The anomalously fast sedimentation of
GluR1-M1 as species that were apparently heavier than those of
GluR1-142/236 might reflect a large fraction of GluR1-M1 that formed
aggregates, perhaps in response to exposure of hydrophobic regions of
its transmembrane domain that normally associate with the other
transmembrane domains in the full-length receptor. Nonetheless, a
fraction of GluR1-M1 is transported to the surface membrane of oocytes.
This transport raises the possibility that a fraction of GluR1-M1
subunits also formed either monomers or specific oligomers and not
nonspecific aggregates, because it is unlikely that such aggregates
would be transported to the cell surface membrane. In addition, high
affinity [3H]AMPA binding was observed with
Triton-solubilized GluR1-M1 species.
The results presented in this paper along with those of others obtained
for water-soluble domains of GluRs (17-19, 38) and AChRs (28) are
encouraging starting points for producing a fully assembled,
water-soluble GluR. Our previous experience with the extracellular
domain of the AChR -amino-3-hydroxy-5-methyl-4-isoxazole propionic acid with high
affinity. These findings show that the extracellular domains of GluR1
that are sufficient for ligand binding apparently are sufficient for
subunit assembly and might be a suitable target for structural studies
of a water-soluble GluR1 oligomer.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
7 subunit was demonstrated (28).
-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA), and
competition for assembly with the full-length GluR1 subunit, we
conclude that GluR1-WS, like the previously crystallized S1-S2 GluR4
monomeric protein, not only retains the ability to bind glutamatergic
ligands but also possibly assembles into specific oligomers because of
the inclusion of the N-terminal portion of the GluR1 subunit. Thus, our
results suggest that N-terminal portions of the extracellular domains
not included in the crystallized monomeric S1-S2 fusion protein, are
important for assembly of GluR subunits, and might be important for
oligomeric structural models of GluRs.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
subunit (30). The sequence shown in
bold within an epitope corresponds to the minimal epitope sequence
necessary to bind the mAbs. The underlined sequences correspond to the
respective epitopes. The amino acid sequence
SSQVTGEVIFQTPLIKNPSS, corresponding to the
insert containing the epitope (for mAb 142), was used to create the
C-terminal tagged GluR1-142 subunit. The amino acid sequence
SVSISPESDRPDLSTFVSISPESDRPDLSTFL, corresponding to the insert containing two tandem epitopes (for mAb 236), was used to
create the C-terminal tagged GluR1-236 subunit. The truncated epitope-tagged GluR1 subunits were created using a similar strategy. GluR1-M1 was constructed by inserting a double-stranded synthetic DNA
cassette corresponding to the amino acid sequence
SSVSISPESDRPDLSTFSR between the BglII
site immediately preceding the putative first TMD of the GluR1 subunit
and the BglI site immediately after the putative last TMD of
the GluR1-142 subunit. GluR1-WS was constructed by inserting a
double-stranded synthetic DNA cassette encoding the amino acid sequence
LSLSNVAG* between the HaeII site preceding the last putative
TMD of the GluR1-M1 subunit and the EcoRI cloning site, thus
eliminating the putative last TMD. The asterisk represents the absence of an amino acid because of the introduced stop codon.
-mercaptoethanol to avoid reduction of the disulfide linkage of the
IgG chains) at 60 °C for 30 min, and then
-mercaptoethanol was
added to the eluted sample prior to analysis by SDS-PAGE. The
proteins were electroblotted onto polyvinylidene difluoride membrane
(IMMUN-BLOT; Bio-Rad).
counter.
counting. For the
GluR1-WS protein, the immunoisolated protein was fractionated by
SDS-PAGE and detected by immunoblotting in conjunction with a
chemiluminescence kit (SuperSignal; Pierce). A nonsaturating exposure
of the chemiluminescent bands captured on x-ray film was scanned using
the Gel Doc 1000 system, and the digitized bands were quantified using
the Multi-Analyst software program (Bio-Rad). To calibrate the sucrose
gradient, ~200 µg of each of bovine serum albumin (66 kDa), alcohol
dehydrogenase (150 kDa),
-amylase (200 kDa), and apoferritin (443 kDa) were also sedimented on the sucrose gradients. The sedimentation
of each of these proteins on the gradients was detected using a
colorimetric assay in conjunction with the Dc protein assay
kit (Bio-Rad). The optical density of the colorimetric reaction was
measured in microwells using the MR700 microtiter plate
reader (Dynatech Laboratories).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
subunit and have been previously described (32). They have also been shown to be
topogenically neutral when introduced into the human AChR
1 subunit
(30) and the GluR1 subunit (33). The GluR1-M1 subunit was derived from
the GluR1-142 subunit by linking the entire first extracellular domain
(Ala1-Glu520) of the GluR1 subunit to the
second extracellular domain (Asn615-Leu889)
via a 32-amino acid linker sequence containing epitopes for mAb 236, thus eliminating its first TMD, the reentrant loop and the second TMD
but retaining the last TMD. The GluR1-WS is derived from GluR1-M1 and
truncates at Gly790, thus eliminating the putative third
TMD and the intracellular C-terminal domain.
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Fig. 1.
Schematic representations of various GluR1
protein constructs. GluR1-142 represents the GluR1 subunit tagged
with an epitope for mAb 142 (142t) after its
C-terminal residue 889. Similarly, GluR1-236 represents GluR1 tagged
with an epitope for mAb 236 (236t) after residue
889. GluR1-M1 represents linkage of the residues
Ala1-Glu520 via a linker containing the
epitope for mAb 236 to residues Asn615-Leu889
and includes the C-terminal tag for mAb 142. GluR1-WS is derived from
GluR1-M1 but truncates at residue Gly790.
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Fig. 2.
Expression of the GluR1 protein constructs in
oocytes. 2% Triton X-100-solubilized proteins from oocytes
expressing the various GluR1 subunit constructs were immunopurified on
mAb coated Immulon 4 wells and then eluted and fractionated by
SDS-PAGE. The expression of GluR1-142, GluR1-236, and GluR1-M1 was
detected by immunoblotting with the appropriate 125I-mAbs
to the engineered epitopes tags, and mAb 236 binding to GluR-WS was
detected using an horseradish peroxidase-conjugated secondary antibody
in conjunction with a chemiluminescence detection method.
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Fig. 3.
GluR1-WS is water-soluble, and GluR1-M1 is
membrane-bound. A, the water-soluble nature of GluR1-WS
was studied by immunopurifying proteins using mAb 236 beads from the
aqueous oocyte incubation medium, the aqueous supernatant obtained
after homogenization and centrifugation of oocyte membranes, and from
2%Triton X-100-solubilized oocyte proteins expressing GluR1-WS, and
then detecting them by immunoblotting with 125I-mAb 236. The controls correspond to proteins from noninjected oocytes treated
similarly. The arrow indicates the GluR1-WS band. The amount
of GluR1-WS detected on the immunoblot was isolated from 40 oocytes.
B, surface expression of GluR1-M1 was detected using
125I-mAb 236 to individual oocytes. Nonspecific binding was
determined using noninjected oocytes. The error bars
represent the standard error of binding to six oocytes. Approximately 1 fmol equivalent of 125I-mAb 236 binding sites to the
GluR1-M1 protein is detected on the surface of each oocyte.
-amylase (200 kDa), and apoferritin (433 kDa), was used to calibrate the gradient. The smaller fraction numbers correspond to the first fractions collected from the bottom of the gradient tube (i.e. faster
sedimentation), and the larger fractions numbers correspond to later
fractions from the top of the gradient tube (i.e. slower sedimentation).
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Fig. 4.
Velocity sedimentation profile of
GluR1-WS. A, the normalized sedimentation profile
(top) of Triton X-100-solubilized GluR1-WS on 5-20%
sucrose gradients containing 0.5% Triton X-100 was derived from
quantitating the intensities of bands obtained for GluR1-WS by
immunoblotting the fractions (bottom). The smaller fraction
numbers corresponded to the first fractions collected from the bottom
of the gradient tube (i.e. faster sedimentation), and the
larger fractions numbers corresponded to later fractions from the top
of the gradient tube (i.e. slower sedimentation). The
open arrows at fractions 30 and 34 highlight two relatively
abundant peaks in the sedimentation profile that correspond to apparent
molecular masses of 140 and 255 kDa. The apparent molecular masses
corresponding to these two peaks suggest that the peaks could represent
dimers and trimers, respectively, of GluR1-WS. The solid
arrows above the profile of GluR1-WS indicate the positions of the
peaks of each of the four proteins used to calibrate the gradient.
B, the peak fraction number of each of the standard
proteins, bovine serum albumin (66 kDa), alcohol dehydrogenase (150 kDa), -amylase (200 kDa), and apoferritin (443 kDa) is plotted
against the log (molecular mass) of each of the standard proteins
(filled circles). The line is a linear best fit of the
points. The apparent molecular masses for the two peaks as calculated
from this plot at fractions 30 and 34 are also shown (open
circles).
View larger version (21K):
[in a new window]
Fig. 5.
Binding of [3H]AMPA to Triton
X-100-solubilized GluR1-WS solubilized in Triton X-100. Triton
X-100-solubilized proteins from 10 oocytes expressing GluR1-WS tethered
to mAb-coated microwells were incubated with various concentrations of
[3H]AMPA in the presence of 100 mM NaSCN for
2 h. Nonspecific binding at each concentration was determined from
wells containing Triton X-100-solubilized proteins from an equivalent
number of uninjected oocytes. Following washing, the amount of bound
radioactivity was determined by scintillation counting. The points
shown correspond to the mean specific binding of duplicate
determinations from one experiment after subtraction of the mean
nonspecific binding from each value at each of the concentrations. The
error bars correspond to the standard error. The
Kd and Bmax were determined
by least squares, nonlinear fitting of the binding data to a Hill-type
equation.
View larger version (36K):
[in a new window]
Fig. 6.
Assembly of GluR1-WS with GluR1-142.
2% Triton X-100-solubilized proteins from oocytes coexpressing
GluR1-142 and GluR1-WS were immunopurified (I.P.) on mAb
236 beads and rat IgG beads (control), eluted, fractionated by
SDS-PAGE, and sequentially immunoblotted (I.B.) with mAb 236 followed by mAb 142. Beads coupled to rat IgG were used as controls for
nonspecific binding. The upper band corresponds to
GluR1-142, and the lower band corresponds to GluR1-WS. The
GluR1-WS band weakly reappears in the right panel because
the blot was not stripped prior to immunoblotting with mAb 142.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
7 subunit and the work described in this paper on
the GluR1 subunit suggest that modifications in design are needed to
successfully use this strategy for structural studies beyond a
monomeric form of GluR. We suggest that water-soluble domains bridged
by a protease-cleavable site to a TMD anchor derived from an integral
membrane protein that contains only a single TMD or membrane tethers
such as a glycosylphosphatidyl inositol moiety, and regulated
expression of the extracellular domains in high expression systems
might improve the efficiency of subunit assembly and thus yield larger
amounts of higher order oligomers. These strategies are likely to yield
intermediate milestones in the challenging task of obtaining high
resolution structural information for neurotransmitter-gated ion channels.
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ACKNOWLEDGEMENTS |
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We thank Dr. Stephen Heinemann (Salk Institute, San Diego) for providing the GluR1 cDNA clone, Dr. Jon Lindstrom (University of Pennsylvania, Philadelphia, PA) for allowing the generous use of his laboratory's facilities during the initial phase of this work, and John Cooper for generously radioiodinating mAbs for our use.
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FOOTNOTES |
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* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Supported by National Institutes of Health Grant NS01903 and Grant MCB940011P from the Pittsburgh Supercomputing Center, which was supported by several federal agencies, the Commonwealth of Pennsylvania, and private industry.
Supported by National Institutes of Health Grant NS33625 and
generous start-up funds from the Louisiana State University Health Sciences Center Neuroscience Center. To whom correspondence should be
addressed: Neuroscience Center of Excellence and Dept. of Neurology, 2020 Gravier St., Suite D, Louisiana State University Health Sciences Center, New Orleans, LA 70112. Tel.: 504-599-0847; Fax:
504-599-0891; E-mail: ranand@lsuhsc.edu.
Published, JBC Papers in Press, November 13, 2000, DOI 10.1074/jbc.M006668200
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ABBREVIATIONS |
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The abbreviations used are:
GluR, glutamate
receptor;
AChR, nicotinic acetylcholine receptor;
AMPA, -amino-3-hydroxy-5-methyl-4-isoxazole propionic acid;
mAb, monoclonal antibody;
PAGE, polyacrylamide gel electrophoresis;
TMD, transmembrane domain.
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REFERENCES |
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