Abbreviations used : AMPA, α-amino-3-hydroxy-5-methyl-4-isoxaolepropionate ; BiP, immunoglobulin binding protein ; BSA, bovine serum albumin ; calnexin-C, C-terminus of calnexin ; calnexin-N, N-terminus of calnexin ; ER, endoplasmic reticulum ; PAGE, polyacrylamide gel electrophoresis ; PBS, phosphate-buffered saline ; SDS, sodium dodecyl sulfate ; TBS, Tris-buffered saline.
Address correspondence and reprint requests to Dr. M. E. Rubio at NIDCD, NIH, Bldg. 36, Rm. 5D08, 36 Convent Dr., Bethesda, MD 20892-4162, U.S.A.
Abstract : To identify proteins that interact with α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptors, we carried out coimmunoprecipitation analyses on detergent-solubilized rat forebrain membranes. Membranes were solubilized with Triton X-100, and immunoprecipitation was done using subunit-specific antibodies to GluR1, GluR2/3, and GluR4 attached to protein A-agarose. Proteins bound to the antibodies were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by silver staining and western blotting. With solubilization in low ionic strength buffer, several coimmunoprecipitating proteins, with Mr = 17,000-100,000, were identified in silver-stained gels. Western blots were then probed with antibodies to a series of candidate proteins that were chosen based on the molecular masses of the copurifying proteins. Two of these were identified as the molecular chaperones calnexin (90 kDa) and the immunoglobulin binding protein (BiP ; 78 kDa). Immunoprecipitation with antibodies to calnexin and BiP demonstrated that glycosylated AMPA receptor subunits were associated. The relationship between AMPA receptors and calnexin and BiP was further studied with immunocytochemistry of the hippocampus. Both calnexin and BiP labeling was present not only in the cell body but also in dendrites of hippocampal pyramidal neurons, where double-label immunofluorescence also showed the presence of AMPA receptor subunits.
The αamino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) subtype of glutamate receptor mediates most fast excitatory transmission in the CNS (Hollmann and Heinemann, 1994). Molecular cloning has identified four AMPA receptor subunits, GluR1-4 or GluRA-D (Hollmann and Heinemann, 1994), which assemble in different combinations to form functional heteromeric or homomeric complexes. Biochemical studies have suggested that the final mature form of AMPA receptor complexes is a pentamer (Wenthold et al., 1992 ; Ferrez-Montiel and Montal, 1995), although evidence supporting complexes of four subunits has been presented (Rosenmund et al., 1998). Analyzing the differential distribution of glutamate receptors in neurons, we and others recently showed that AMPA and other glutamate receptors can be selectively targeted to different synaptic populations in a single neuron (Landsend et al., 1997 ; Rubio and Wenthold, 1997 ; Zhao et al., 1997, 1998 ; Toth and McBain, 1998). The mechanisms by which neurons regulate the assembly of subunits to form functional receptor complexes and the targeting of these complexes to the appropriate synapses remain poorly understood but are though to involve interactions with a series of other proteins. The functions of these proteins may range from that of a molecular chaperone that aids in the folding and assembly of a receptor complex to one that anchors the receptor at the synapse.
A number of proteins that interact with AMPA receptors have been identified, including GRIP, ABP, PICK, and NSF (Dong et al., 1997 ; Nishimune et al., 1998 ; Osten et al., 1998 ; Song et al., 1998 ; Srivastava et al., 1998 ; Xia et al., 1999). All of these interact with the C-terminus of AMPA receptors, and it has been suggested that the function of some of these proteins is to anchor the receptors to the postsynaptic density (for review, see Ziff, 1997), although this remains to be demonstrated. Thus far, proteins involved in the assembly and targeting of glutamate receptors in neurons have not been identified.
Molecular chaperones such as immunoglobulin binding protein (BiP), calnexin, and calreticulin and enzymes such as protein disulfide isomerase have been shown to interact with various proteins such as vesicular stomatitis virus G protein influenza virus hemagglutinin, major histocompatibility complex class I antigen, and thyroglobulin (Hammond and Helenius, 1994 ; Kim and Arvan, 1995) and are thought to play critical roles in the folding of polypeptide chains and assembly of complex proteins. BiP and calnexin have been shown to interact with the ligand-gated ion channels, the nicotinic acetylcholine receptor (Forsayeth et al., 1992 ; Gelman et al., 1995 ; Keller et al., 1996), and the GABAA receptor (Connolly et al., 1996). For both receptors, the chaperones appear to associate with unassembled subunits but not with mature, correctly folded receptor complexes. A recent study demonstrated that the folding and assembly of nicotinic acetylcholine receptor subunits can be stimulated by cotransfection with calnexin and ultimately affect the amount of receptor expressed on the cell surface (Change et al., 1997). This is an important finding because it shows that the availability of chaperones could play a critical role in the number and composition of receptors expressed on the surface of a neuron.
In the present study, we used coimmunoprecipitation to investigate proteins that interact with AMPA receptors in mammalian brain. By polyacrylamide gel electrophoresis (PAGE) and silver staining, several proteins were found to coimmunoprecipitate ; two were identified as the molecular chaperones calnexin and BiP.
MATERIALS AND METHODS
Antibodies to GluR1, GluR2/3, and GluR4 have been previously characterized and described (Wenthold et al., 1992). An antibody to the C-terminus of calnexin (calnexin-C) was made using a synthetic peptide corresponding to the sequence Ala-Glu-Glu-Asp-Glu-Ile-Leu-Asn-Arg-Ser-Pro-Arg-Asn-Arg-Lys-Pro-Arg-Arg-Glu (Ou et al., 1993 ; Tjoelker et al., 1994). A cysteine residue was added to the N-terminus of the peptide to facilitate coupling to the carrier protein, bovine serum albumin (BSA). Antibodies were purified using the same peptide coupled to SulfoLink resin (Pierce, Rockford, IL, U.S.A.). The monoclonal antibody to BiP and a polyclonal antibody to the N-terminus of calnexin (calnexin-N) were obtained commercially (StressGen, Victoria, Canada). A monoclonal antibody to the C-terminus of GluR2/3 was kindly provided by Dr. Peter Streit (Nusser et al., 1994 ; Ottiger et al., 1995).
Detergent solubilization and immunoprecipitation
Brains were obtained from 30-to 40-day-old Sprague-Dawley rats (Pel Freez Biologicals, Rogers, AR, U.S.A.). Brains were thawed and the cerebellum and midbrain were removed. Forebrains were homogenized in 20 ml of 50 mM Tris-HCl (pH 7.5) and solubilized using Triton X-100 as detergent, under either high or low ionic strength conditions. Solubilization at high ionic strength was done with 1% (wt/vol) Triton X-100 in 0.5 M potassium phosphate (pH 7.0) containing 20% (wt/vol) glycerol, 0.5 mM phenylmethylsulfonyl fluoride, 2.5 mM EDTA, 10 μM leupeptin, and 10 μM pepstatin for 30 min at 37°C (Hunter et al., 1990). Solubilization at low ionic strength was done with 1% Triton X-100 in 50 mM Tris buffer (pH 7.5) containing 0.5 mM leupeptin, and 10 μM pepstatin for 30 min at 4°C. The protein concentration during solubilization was 4-6 mg/ml in both cases. After centrifugation at 45,000 g for 1 h, the supernatant was either used directly for immunoprecipitation (low ionic strength condition) or dialyzed against 50 mM Tris-HCl (pH 7.5) containing 20% glycerol and 0.1% Triton X-100. After dialysis, the material was centrifuged at 45,000 g for 1 h and the supernatant used for immunoprecipitation.
AMPA receptor subunits were immunoprecipitated using subunit-specific antibodies to GluR1, GluR2/3, and GluR4 attached to protein A-agarose ; BiP and calnexin were immunoprecipitated using the specific antibodies described above. For immunoprecipitation, 10 μg of antibody was incubated with 25 μl (volume of packed resin) of protein A-agarose (Pierce) in 50 mM Tris buffer (pH 7.5) containing 0.1% Triton X-100 for at least 4 h at 4°C. The resin was washed three times with 50 mM Tris buffer containing 0.1% Triton X-100 (pH 7.5) and mixed overnight with 10 ml of the detergent-solubilized fraction described above. The mixture was centrifuged briefly, and the unbound fraction was removed. The resin was washed three times in 50 mM Tris-buffered saline (TBS ; 0.1% Triton X-100, 150 mM NaCl, pH 7.4) and was boiled (3 min) in sodium dodecyl sulfate (SDS)-PAGE sample buffer (2× concentration). The resin was removed by centrifugation. Controls were performed using resin without the primary antibody.
Electrophoresis and immunoblotting
Analyses were performed on the immunoprecipitated fractions. Fifteen microliters was applied to a 4-20% acrylamide minigel (Novex, San Diego, CA, U.S.A.). Transfer to nitrocellulose and development of western blots were performed as described previously (Wenthold et al., 1992). In brief, membranes were treated with 5% instant powdered milk in TBS containing 0.05% Tween-20. Primary antibodies in TBS containing 0.05% Tween-20 were used in the following concentrations (in μg/ml) : GluR1, 0.5 ; GluR2/3, 0.15 ; GluR4, 0.75 ; and calnexin-C, 1-1.5. For the monoclonal antibodies, the following dilutions were used : GluR2/3, 1:2,000 ; and BiP, 1:500. Detection of bound antibody was done using an alkaline phosphatase-conjugated secondary antibody or by using the enhanced chemiluminescence detection system (Pierce) with horseradish peroxidase-conjugated secondary antibodies. Prestained standards from GibcoBRL (Life Technologies, Gaithersburg, MD, U.S.A.) were myosin (203 kDa), phosphorylase B (105 kDa), BSA (71 kDa), ovalbumin (44 kDa), carbonic anhydrase (28 kDa), and β-lactoglobulin (18 kDa).
For silver staining, gels were fixed in 50% ethanol and 10% glacial acetic acid for at least 1 h and then cross-linked with cold Fisher’s solution (10% glutaraldehyde) for 30 min with agitation (Giulian et al., 1983). Gels were washed extensively with H2O and transferred to an ammoniacal silver stain solution (90 mM NaOH, 14.8 M concentrated NH4OH, 1.14 M AgNO3) for 6 min. After two short washes, development of the reaction was done with a citric acid/formaldehyde solution (47.6 mM citric acid, 0.02% formaldehyde) until the bands began to appear. The reaction was stopped with a solution of 20% ethanol and 0.5% glacial acetic acid.
Five adult Sprague-Dawley rats (120-150 g) were anesthetized with a mixture of ketamine HCl (Ketaset, 100 mg/ml ; Forte Dodge Laboratories) and xylazine (Rompun, 20 mg/ml ; Miles) at 0.1 ml/100 g of body weight and perfused with 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4). The brain was sectioned sagittally (20-25 μm) with a vibratome (Pelco DTK-3000W microslicer). Sections were frozen at -78°C after 30% sucrose cryoprotection and stored at -80°C until use. Before the incubation with primary antibodies, the sections were thawed, washed extensively in phosphatebuffered saline (PBS), and preblocked with 10% normal goat serum in PBS. Sections were incubated with primary antibodies (GluR1 and GluR2/3 were used at 2 μg/ml, calnexin-C at 1.5 μg/ml, and GluR2/3 and BiP monoclonal antibodies were used at dilutions of 1 : 2,500 and 1 : 400, respectively) overnight at 4°C. Then, sections were washed three times with PBS at room temperature and incubated with the secondary antibodies [fluorescein anti-rabbit or anti-mouse antibody (Amersham ; 1 : 200) for the calnexin-C and BiP antibodies, respectively ; Texas Red anti-rabbit (Amersham ; 1 : 300) for GluR1 and GluR2/3 polyclonal antibodies or anti-mouse for GluR2/3 monoclonal antibody] in 2% BSA in PBS for 1 h at room temperature and washed with PBS before mounting on slides using Vectashield mounting medium (Vector, Burlingame, CA, U.S.A.). Control experiments were done omitting the primary antibody. Also, preadsorption controls were done by incubating the calnexin antibody plus 50 μg/ml (final concentration) of the specific BSA/peptide conjugated at 4°C for 24 h and then centrifuging and incubating them with the sections. Calnexin and BiP antibodies were characterized on western blots of hippocampus at concentrations of 0.5 and 0.1 μg/ml, respectively.
The use and care of the animals in this study were done following the guidelines of the NIH Animal Research Advisory Committee.
We have previously shown that the optimal conditions for solubilizing AMPA receptors that retain their ligand binding capabilities involve solubilization with 1% Triton X-100 at 37°C in 0.5 M phosphate buffer containing 20% glycerol (Hunter et al., 1990). Under these conditions, copurifying proteins, as determined by silver staining, were not detected (Wenthold et al., 1992). As these solubilization conditions may be too harsh to allow copurification of more weakly associated proteins, we used lower ionic strength buffer and a solubilization temperature of 4°C to facilitate identification of associated proteins. To immunoprecipitate AMPA receptors, we used antibodies to GluR1, GluR2/3, and GluR4 ; GluR1-3 are major subunits in rat forebrain, whereas GluR4 is only weakly expressed. We therefore expected that copurifying proteins would be found only with immunoprecipitation with antibodies to GluR1 and GluR2/3, whereas antibodies to GluR4 would immunoprecipitate few AMPA receptors and would serve as a control. After immunoprecipitation with antibodies to GluR1, GluR2/3, and GluR4, silver staining of membrane fractions solubilized with high and low ionic strength buffers showed the characteristic band at Mr = 108,000 corresponding to AMPA receptors subunits (Fig. 1). Whereas staining obtained with antibodies to GluR1 and GluR2/3 was intense, only a lightly stained band was obtained with GluR4. In addition, under the low ionic strength condition, several other bands migrating in the range of Mr = 17,000-100,000 in the GluR1 and GluR2/3 samples were present (Fig. 1). Those that were also immunoprecipitated with GluR4 antibodies and were equally abundant to those immunoprecipitated with GluR1 and GluR2/3 antibodies were assumed to be nonspecific, and we did not attempt to characterize them further. For the others, several candidates were identified based on their molecular masses. These included the endoplasmic reticulum (ER) proteins calreticulin (45 kDa), calnexin (90 kDa), BiP (78 kDa), and protein disulfide isomerase (57 kDa), the Golgi protein mannosidase II (135 kDa), calmodulin (18 kDa), and proteins related to vesicle fusion including SNAP-25 (25 kDa), syntaxin (35 kDa), and VAMP (18 kDa). All were tested for coimmunoprecipitation using selective antibodies. Of these, only BiP and calnexin were found to copurify with GluR1 and GluR2/3 (Fig. 1).
Western blotting analysis of immunoprecipitated AMPA receptor subunits, BiP, and calnexin
The interactions between AMPA receptor subunits and BiP and calnexin were characterized by coimmunoprecipitation. AMPA receptor subunits were immunoprecipitated with antibodies to GluR1, GluR2/3, and GluR4, and the immunoprecipitated product was stained with antibodies to calnexin and BiP. Antibodies to GluR1 and GluR2/3 coimmunoprecipitated calnexin and BiP, whereas little or no staining was seen with GluR4 immunoprecipitation (Fig. 2A). Similar results were obtained when a polyclonal antibody to calnexin-N was used (data not shown).
Immunoprecipitating with antibodies to calnexin-N or calnexin-C and to BiP produced similar results showing coimmunoprecipitation of GluR2/3 (Fig. 2B). GluR2/3 staining is eliminated when the antibody is first incubated with a peptide used to develop the antibody. Based on the size of the coimmunoprecipitating GluR2/3, the subunits appear to be fully glycosylated and no staining was associated with the nonglycosylated subunits that have been reported to migrate at Mr = 98,000 (Blackstone et al., 1992). Although coimmunoprecipitation of GluR2/3 with calnexin and BiP is clearly evident, only a small fraction of the subunits was coimmunoprecipitated, suggesting that only a subpopulation of AMPA receptors is associated with the chaperones.
Expression of AMPA receptors, BiP, and calnexin in hippocampus
Our demonstration of the interaction between AMPA receptors and calnexin and BiP raised the question as to where this interaction was occurring. Both calnexin and BiP are molecular chaperones that are associated with the ER, suggesting a cell body localization. The relationship between AMPA receptors and calnexin and BiP was investigated further in pyramidal cells of the hippocampus ; these neurons express AMPA receptor subunits GluR1-3 (Petralia and Wenthold, 1992 ; for review, see Petralia, 1997). Antibodies to BiP and calnexin-C recognize major bands migrating at 78 and 90 kDa, respectively, in hippocampus homogenates (Fig. 3). To investigate possible colocalization of receptor subunits and calnexin and BiP, the following combinations of antibodies were used : GluR1 (polyclonal) + BiP (monoclonal) ; GluR2/3 (polyclonal) + BiP (monoclonal) ; calnexin-C (polyclonal) + GluR2/3 (monoclonal).
Antibodies to GluR1 (polyclonal) and GluR2/3 (polyand monoclonal) showed AMPA receptors present in cell bodies and in proximal and distal dendrites of pyramidal cells in CA1, CA2, and CA3 regions (Figs. 4 and 5), confirming previous results using preembedding immunocytochemistry with 3,3′-diaminobenzidine as the chromogen (Petralia and Wenthold, 1992) or immunofluorescence on cultured neurons (Eshhar et al., 1993). In some cases, a punctate staining pattern corresponding to dendritic spines was observed (Figs. 4 and 5).
Calnexin and BiP were both found in cell bodies and in proximal and distal dendrites (Figs. 4 and 5). In cell bodies, the immunolabeling was always enriched in the juxtanuclear ER membranes and extended in a continuous reticular-like pattern from this cellular compartment to the most distal dendritic segment. In particular, zones corresponding to dendritic branches presented a strong signal. Comparing the labeling of the two molecular chaperones in dendrites, BiP immunolabeling was more widely distributed inside the dendritic processes and calnexin-C immunolabeling was more diffusely distributed and closer to the plasma membrane (Figs. 4 and 5). Double immunofluorescence showed that AMPA receptors and calnexin and BiP are present in the same subcellular locations, but a strict relationship was not seen.
Our results suggest that only a small percentage of GluR1 and GluR2/3 subunits copurify with BiP and calnexin. Although this may be due to technical factors such as incomplete immunoprecipitation or a loss of the copurifying protein during the immunoprecipitation, it is also consistent with BiP and calnexin associating with only a subpopulation of receptors. AMPA receptor subunits that coimmunoprecipitate with BiP and calnexin migrate on SDS-PAGE gels as fully glycosylated forms ; we do not detect unglycosylated forms of AMPA receptor subunits that have been described to migrate at ~98 kDa (Blackstone et al., 1992). As mature forms of AMPA receptor subunits interact with BiP and calnexin, this association occurs after translation of individual subunits has taken place in the ER. This raises the possibility that these chaperones, in addition to playing a role in protein folding, also have a more specialized function in the maturation and targeting of AMPA receptor subunits. Although it is known that both chaperones interact with individual receptor subunits, BiP and calnexin also facilitate the assembly of heterogeneous receptor complexes. In the case of nicotinic acetylcholine receptors, calnexin and BiP bind to immature individual subunits (Forsayeth et al., 1992 ; Gelman et al., 1995), but it was recently shown that calnexin facilitates acetylcholine receptor assembly and surface expression by promoting the correct folding of subunits for efficient oligomerization (Chang et al., 1997). In addition, there is evidence that calnexin coimmunoprecipitates with the trimeric (αβγ) and the tetrameric (αβγδ) structures of the acetylcholine receptor but not with its mature pentameric structure forms (α2βγδ). This interaction between calnexin and heteromeric acetylcholine receptors was shown to take place in the ER (Wanamaker and Green, 1997).
As it appears that only a small population of AMPA receptors is associated with BiP and calnexin, the interaction may be restricted to a specific location in the neuron. A likely site of interaction is the cell body, where BiP and calnexin are abundantly expressed and where most AMPA receptors are synthesized. However, this interaction could also occur in the dendrite. Three-dimensional reconstruction and experiments using dyes have shown the extension of the ER membranes from the cell body into dendrites, including dendritic spines (Martone et al., 1993 ; Terasaki et al., 1994 ; Krijnse-Locker et al., 1995 ; Spacek and Harris, 1997). Our present results on the distributions of calnexin and BiP, as well as a series of other studies (Villa et al., 1991 ; Krijnse-Locker et al., 1995 ; Torre and Steward, 1996 ; Gardiol et al., 1998), show that this population of membranes contains the proteins required for many of the functions of the ER. Whereas the presence of ER in dendrites has been used in support of the case for local synthesis and processing of proteins (Torre and Steward, 1992, 1996 ; Gardiol et al., 1998), it is also consistent with the idea that processing of proteins synthesized in the cell body can continue in the dendrite. The expression of BiP and calnexin throughout the dendrite tends to suggest a role in addition to an involvement in local protein synthesis. This membrane system in dendrites may represent a major conduit for the transport of membrane proteins from the cell body to their functional location. An analysis of glutamate receptors using immunogold labeling shows that they are often associated with the dendritic ER membranes that were immunoreactive for BiP and calnexin (Rubio and Wenthold, 1999).
This study was supported by the National Institute on Deafness and Other Communication Disorders Intramural Program. We thank Dr. R. S. Petralia for reading the manuscript and helpful comments and Drs. J. Fex and C. Vicario-Abejón for reviewing the manuscript. We thank Dr. P. Streit for kindly providing us with the monoclonal antibody for GluR2/3.