Assembly and Ligand Binding Properties of the Water-soluble Extracellular Domains of the Glutamate Receptor 1 Subunit*

Gregg B. WellsDagger §, Lin Lin, Elisabeth M. Jeanclos, and Rene Anand||

From the  Neuroscience Center of Excellence and the Department of Neurology, Louisiana State University Health Sciences Center, New Orleans, Louisiana 70112 and the Dagger  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



    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 alpha -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

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 alpha 7 subunit was demonstrated (28).

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 alpha -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

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 alpha  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.

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 beta -mercaptoethanol to avoid reduction of the disulfide linkage of the IgG chains) at 60 °C for 30 min, and then beta -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).

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 gamma  counter.

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 gamma  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), beta -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).

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.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 alpha  subunit and have been previously described (32). They have also been shown to be topogenically neutral when introduced into the human AChR alpha 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.



View larger version (23K):
[in this window]
[in a new window]
 
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.

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.



View larger version (71K):
[in this window]
[in a new window]
 
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.

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).



View larger version (33K):
[in this window]
[in a new window]
 
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.

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), beta -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).

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.



View larger version (16K):
[in this window]
[in a new window]
 
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), beta -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).

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).



View larger version (21K):
[in this window]
[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.

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.



View larger version (36K):
[in this window]
[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

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 alpha 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.


    ACKNOWLEDGEMENTS

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.


    FOOTNOTES

* 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


    ABBREVIATIONS

The abbreviations used are: GluR, glutamate receptor; AChR, nicotinic acetylcholine receptor; AMPA, alpha -amino-3-hydroxy-5-methyl-4-isoxazole propionic acid; mAb, monoclonal antibody; PAGE, polyacrylamide gel electrophoresis; TMD, transmembrane domain.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES


1. Headley, P. M., and Grillner, S. (1990) Trends Pharmacol. Sci. 11, 205-211[CrossRef][Medline] [Order article via Infotrieve]
2. Hollmann, M., and Heinemann, S. (1994) Annu. Rev. Neurosci. 17, 31-108[CrossRef][Medline] [Order article via Infotrieve]
3. Gasic, G. P., and Hollmann, M. (1992) Annu. Rev. Physiol. 54, 507-536[CrossRef][Medline] [Order article via Infotrieve]
4. Seeburg, P. H. (1993) Trends Neurosci. 16, 359-365[CrossRef][Medline] [Order article via Infotrieve]
5. Fletcher, E. J., and Lodge, D. (1996) Pharmacol. Ther. 70, 65-89[CrossRef][Medline] [Order article via Infotrieve]
6. Olney, J. W. (1990) Annu. Rev. Pharmacol. Toxicol. 30, 47-71[CrossRef][Medline] [Order article via Infotrieve]
7. Ultsch, M. H., Somers, W., Kossiakoff, A. A., and de Vos, A. M. (1994) J. Mol. Biol. 236, 286-299[CrossRef][Medline] [Order article via Infotrieve]
8. Somers, W., Ultsch, M., De Vos, A. M., and Kossiakoff, A. A. (1994) Nature 372, 478-481[CrossRef][Medline] [Order article via Infotrieve]
9. Muller, Y. A., Kelley, R. F., and de Vos, A. M. (1998) Protein Sci. 7, 1106-1115[Abstract/Free Full Text]
10. Walter, M. R., Windsor, W. T., Nagabhushan, T. L., Lundell, D. J., Lunn, C. A., Zauodny, P. J., and Narula, S. K. (1995) Nature 376, 230-235[CrossRef][Medline] [Order article via Infotrieve]
11. Brown, J. H., Jardetzky, T. S., Gorga, J. C., Stern, L. J., Urban, R. G., Strominger, J. L., and Wiley, D. C. (1993) Nature 364, 33-39[CrossRef][Medline] [Order article via Infotrieve]
12. Hubbard, S. R., Wei, L., Ellis, L., and Hendrickson, W. A. (1994) Nature 372, 746-754[CrossRef][Medline] [Order article via Infotrieve]
13. Fields, B. A., Ober, B., Malchiodi, E. L., Lebedeva, M. I., Braden, B. C., Ysern, X., Kim, J. K., Shao, X., Ward, E. S., and Mariuzza, R. A. (1995) Science 270, 1821-1824[Abstract]
14. Bentley, G. A., Boulot, G., and Mariuzza, R. A. (1995) Res. Immunol. 146, 277-290[CrossRef][Medline] [Order article via Infotrieve]
15. Colman, P. M., Laver, W. G., Varghese, J. N., Baker, A. T., Tulloch, P. A., Air, G. M., and Webster, R. G. (1987) Nature 326, 358-363[CrossRef][Medline] [Order article via Infotrieve]
16. Stern-Bach, Y., Bettler, B., Hartley, M., Sheppard, P. O., O'Hara, P. J., and Heinemann, S. F. (1994) Neuron 13, 1345-1357[Medline] [Order article via Infotrieve]
17. Kuusinen, A., Arvola, M., and Keinanen, K. (1995) EMBO J. 14, 6327-6332[Abstract]
18. Arvola, M., and Keinanen, K. (1996) J. Biol. Chem. 271, 15527-15532[Abstract/Free Full Text]
19. Armstrong, N., Sun, Y., Chen, G. Q., and Gouaux, E. (1998) Nature 395, 913-917[CrossRef][Medline] [Order article via Infotrieve]
20. Laube, B., Kuhse, J., and Betz, H. (1998) J. Neurosci. 18, 2954-2961[Abstract/Free Full Text]
21. Mano, I., and Teichberg, V. I. (1998) Neuroreport 9, 327-331[Medline] [Order article via Infotrieve]
22. Rosenmund, C., Stern-Bach, Y., and Stevens, C. F. (1998) Science 280, 1596-1599[Abstract/Free Full Text]
23. Wenthold, R. J., Yokotani, N., Doi, K., and Wada, K. (1992) J. Biol. Chem. 267, 501-507[Abstract/Free Full Text]
24. Ferrer-Montiel, A. V., and Montal, M. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 2741-2744[Abstract/Free Full Text]
25. Premkumar, L. S., and Auerbach, A. (1997) J. Gen. Physiol. 110, 485-502[Abstract/Free Full Text]
26. Brose, N., Huntley, G. W., Stern-Bach, Y., Sharma, G., Morrison, J. H., and Heinemann, S. F. (1994) J. Biol. Chem. 269, 16780-16784[Abstract/Free Full Text]
27. Wenthold, R. J., Petralia, R. S., Blahos, J., II, and Niedzielski, A. S. (1996) J. Neurosci. 16, 1982-1989[Abstract]
28. Wells, G. B., Anand, R., Wang, F., and Lindstrom, J. (1998) J. Biol. Chem. 273, 964-973[Abstract/Free Full Text]
29. Hollmann, M., O'Shea, G. A., Rogers, S. W., and Heinemann, S. (1989) Nature 342, 643-648[CrossRef][Medline] [Order article via Infotrieve]
30. Anand, R., Bason, L., Saedi, M. S., Gerzanich, V., Peng, X., and Lindstrom, J. (1993) Biochemistry 32, 9975-9984[Medline] [Order article via Infotrieve]
31. Wang, F., Gerzanich, V., Wells, G. B., Anand, R., Peng, X., Keyser, K., and Lindstrom, J. (1996) J. Biol. Chem. 271, 17656-17665[Abstract/Free Full Text]
32. Das, M. K., and Lindstrom, J. (1991) Biochemistry 30, 2470-2477[Medline] [Order article via Infotrieve]
33. Anand, R. (2000) Biochem. Biophys. Res. Commun. 276, 157-161[CrossRef][Medline] [Order article via Infotrieve]
34. Hollmann, M., Maron, C., and Heinemann, S. (1994) Neuron 13, 1331-1343[Medline] [Order article via Infotrieve]
35. Wo, Z. G., and Oswald, R. E. (1995) J. Biol. Chem. 270, 2000-2009[Abstract/Free Full Text]
36. Bennett, J. A., and Dingledine, R. (1995) Neuron 14, 373-384[Medline] [Order article via Infotrieve]
37. Leuschner, W. D., and Hoch, W. (1999) J. Biol. Chem. 274, 16907-16916[Abstract/Free Full Text]
38. Kuusinen, A., Abele, R., Madden, D. R., and Keinanen, K. (1999) J. Biol. Chem. 274, 28937-28943[Abstract/Free Full Text]


Copyright © 2001 by The American Society for Biochemistry and Molecular Biology, Inc.