Turnover Analysis of Glutamate Receptors Identifies a Rapidly Degraded Pool of the N-Methyl-d-aspartate Receptor Subunit, NR1, in Cultured Cerebellar Granule Cells
1999; Elsevier BV; Volume: 274; Issue: 1 Linguagem: Inglês
10.1074/jbc.274.1.151
ISSN1083-351X
AutoresKyung-Hye Huh, Robert J. Wenthold,
Tópico(s)Photochromic and Fluorescence Chemistry
ResumoThe number, composition, and location of receptors in neurons are critically important factors in determining the neuron's response to neurotransmitters. The functional expression of receptors appears to be regulated both generally, at the level of transcription or translation, and locally, at the level of the individual synapse. A key component in the regulation of any protein is its turnover rate, which, measured in half-lives, ranges from a few minutes to several days. In the present study, we measured the turnover rates of subunits of N-methyl-d-aspartate (NMDA) and α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptors, the two major ionotropic glutamate receptors, using cultured cerebellar granule cells. Turnover rates for NR1, NR2A/B, GluR2/3, and GluR4 subunits were determined by pulse-chase labeling of cells with [35S]methionine. Half-lives were found to be 18 ± 5 h and 23 ± 8 h for the AMPA receptor subunits GluR2/3 and GluR4, respectively, and 16 ± 5 h for NR2A. The NR1 subunit showed a biphasic decay with half-lives of 2 and 34 h for the rapidly and slowly degraded populations, respectively. Splice variants of the NR1 subunit with different carboxyl-terminal cassettes, C2 and C2′, showed similar biphasic degradation patterns. To further characterize the rapidly degraded pool of NR1, surface receptors were labeled by biotinylation, and half-lives of the biotinylated proteins were determined. All surface NR1 was slowly degraded with a pattern similar to that of NR2A, GluR2/3, and GluR4, suggesting that the rapidly degraded pool is confined to the cytoplasm and not assembled with NR2 subunits. A significant amount of NR1 was not immunoprecipitated by NR2 subunit-specific antibodies after solubilization with deoxycholate. This unassembled pool, but not the assembled one, was greatly diminished following treatment of cycloheximide for 5 h, indicating that the rapidly degraded pool of NR1 is not assembled with NR2. These results show that NMDA and AMPA receptors have similar turnover rates, but NMDA receptors have a separate pool of NR1 subunits that is rapidly degraded and accounts for most of the intracellular pool. The number, composition, and location of receptors in neurons are critically important factors in determining the neuron's response to neurotransmitters. The functional expression of receptors appears to be regulated both generally, at the level of transcription or translation, and locally, at the level of the individual synapse. A key component in the regulation of any protein is its turnover rate, which, measured in half-lives, ranges from a few minutes to several days. In the present study, we measured the turnover rates of subunits of N-methyl-d-aspartate (NMDA) and α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptors, the two major ionotropic glutamate receptors, using cultured cerebellar granule cells. Turnover rates for NR1, NR2A/B, GluR2/3, and GluR4 subunits were determined by pulse-chase labeling of cells with [35S]methionine. Half-lives were found to be 18 ± 5 h and 23 ± 8 h for the AMPA receptor subunits GluR2/3 and GluR4, respectively, and 16 ± 5 h for NR2A. The NR1 subunit showed a biphasic decay with half-lives of 2 and 34 h for the rapidly and slowly degraded populations, respectively. Splice variants of the NR1 subunit with different carboxyl-terminal cassettes, C2 and C2′, showed similar biphasic degradation patterns. To further characterize the rapidly degraded pool of NR1, surface receptors were labeled by biotinylation, and half-lives of the biotinylated proteins were determined. All surface NR1 was slowly degraded with a pattern similar to that of NR2A, GluR2/3, and GluR4, suggesting that the rapidly degraded pool is confined to the cytoplasm and not assembled with NR2 subunits. A significant amount of NR1 was not immunoprecipitated by NR2 subunit-specific antibodies after solubilization with deoxycholate. This unassembled pool, but not the assembled one, was greatly diminished following treatment of cycloheximide for 5 h, indicating that the rapidly degraded pool of NR1 is not assembled with NR2. These results show that NMDA and AMPA receptors have similar turnover rates, but NMDA receptors have a separate pool of NR1 subunits that is rapidly degraded and accounts for most of the intracellular pool. α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid N-methyl-d-aspartate polyacrylamide gel electrophoresis synaptosomal associated protein of 25 kDa sulfosuccinimidyl-6-(biotinamido)hexanoate sulfosuccinimidyl-2-(biotinamido)ethyl-1,3-dithiopropionate Hanks' buffered saline solution. Glutamate is the major excitatory neurotransmitter in the mammalian central nervous system and three distinct subtypes of ionotropic glutamate receptors mediate fast excitatory transmission (1Monaghan D.T. Bridges R.J. Cotman C.W. Annu. Rev. Pharmacol. Toxicol. 1989; 29: 365-402Crossref PubMed Scopus (2014) Google Scholar). AMPA1 receptors and NMDA receptors are the principle ionotropic receptors, while kainate receptors are widely distributed but less abundant (2Hollmann M. Heinemann S. Annu. Rev. 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Therefore, if glutamate receptors have short half-lives, changes in synthesis, resulting from either changes in transcription or translation, as well as changes in degradation could be effective in rapid regulation of levels of synaptic receptors. In the present study, we investigated the turnover characteristics of two key ionotropic receptors, AMPA and NMDA receptors, in cultured cerebellar granule cells. These cultures have the advantage of representing a nearly homogenous population of neurons that express several glutamate receptors, including functional AMPA and NMDA receptors. We demonstrate that subunits of both receptors have similar and relatively long half-lives, with values of about 20 h, as measured both by pulse-chase and surface biotinylation. However, a pool of the NR1 subunit, which is not assembled with NR2 and represents about half of the total NR1, is rapidly degraded with a half-life of about 2 h. Tissue culture media, sera, and supplies were purchased from Life Technologies, Inc.; cytosine arabinoside from Sigma; methionine and cysteine-free medium from NIH Media Service branch; [35S]methionine and Enlightening from NEN Life Science Products; protein A-agarose beads, Ultralink Plus immobilized streptavidin beads, Sulfo-NHS-LC-biotin, and NHS-SS-biotin from Pierce; Kodak X-Omat AR films from Eastman Kodak Co.; autoradiographic14C-labeled microscales and horseradish peroxidase-conjugated secondary antibodies from Amersham Pharmacia Biotech; SDS electrophoresis gels from Novex; and horseradish peroxidase-conjugated streptavidin from Southern Biotechnology Associates. Cultures of cerebellar granule neurons were prepared as described by Gallo et al.(33Gallo W. Ciotti M.T. Aloisi F. Levi G. Proc. Natl. Acad. Sci. U. S. A. 1982; 79: 7919-7923Crossref PubMed Scopus (487) Google Scholar). Briefly, cerebella were obtained from 7–8-day-old Sprague-Dawley rat pups. They were chopped by using a tissue slicer and treated with 0.025% trypsin for 15 min at 37 °C. Following trypsinization, tissue was dissociated by passage through fire-polished pipettes, and tissue debris was separated from dissociated cells by sedimentation. Cells were centrifuged and suspended in the plating medium, which consisted of basal medium Eagle, 25 mm KCl, 10 μg/ml gentamycin, 2 mm glutamine, 10% fetal calf serum, and were plated on 3.5-cm culture plates coated with poly-l-lysine at a density of 2.5 × 106 cells/dish. After 19–20 h, proliferation of glial cells was inhibited by treatment with 10 μm cytosine arabinoside. All experiments were performed on 8–9-day in vitro cells. Cells were washed twice with prewarmed Hanks' balanced salt solution (HBSS) containing 25 mm KCl, incubated for 30 min at 37 °C in depletion media, which consisted of methionine- and cysteine-free basal medium Eagle, 25 mm KCl, 2 mm glutamine, 10 μg/ml gentamycin, 5% dialyzed fetal calf serum, and then pulse-labeled with 250 μCi of [35S]methionine (1175 Ci/mmol) in depletion medium for 20 min at 37 °C. Some experiments were performed in medium lacking glutamine. After 20 min, cells were washed once with basal medium Eagle containing 2 mm methionine and then were incubated with conditioned medium containing 2 mmmethionine and 2.5 mm HEPES for variable times. KCl (25 mm) was included in all media throughout the experiment. To block glycosylation, tunicamycin was added to a final concentration of 1 μg/ml to the medium 12 h before pulse labeling and was included during pulse labeling. To harvest, cells were washed twice with cold Dulbecco's phosphate-buffered saline, scraped into phosphate-buffered saline containing protease inhibitor mixture (1 mm EDTA, 1 mm 4-(2-aminoethyl)benzenesulfonyl fluoride, 20 μm leupeptin, 2 μg/ml pepstatin, 2 μg/ml aprotinin), and centrifuged at 2000 × g for 15 min. Pellets were stored frozen at −70 °C until use. Monoclonal antibodies (54.2) to NR1 subunits and NR2B subunits were purchased from Pharmingen (San Diego, CA) and Transduction Laboratories (Lexington, KY), respectively. Antibodies specific to NR2A, NR2B, and NR2C subunits were generated against polyhistidine fusion proteins containing the C-terminal region of each subunit encompassing 934–1203 for NR2A, 935–1856 for NR2B (34Leonard A.S. Hell J.W. J. Biol. Chem. 1997; 272: 12107-12115Abstract Full Text Full Text PDF PubMed Scopus (252) Google Scholar), and 1110–1242 for NR2C subunits. The NR2C antibody was specific to NR2C subunits as tested by Western blot analysis using human embryonic kidney (HEK293) cells transfected with different NR2 subunits. Other antibodies to splice variants of NR1, NR2A/B, GluR1, GluR2/3, and GluR4 subunits were generated against synthetic peptides which correspond to the sequences at the C termini, and characterization and demonstration of the specificity of the antibodies was made in previous studies (5Wenthold R.J. Yokotani N. Doi K. Wada K. J. Biol. Chem. 1992; 267: 501-507Abstract Full Text PDF PubMed Google Scholar,35Petralia R.S. Wang Y.-X. Wenthold R.J. J. Neurosci. 1994; 14: 6102-6120Crossref PubMed Google Scholar, 36Blahos II, J. Wenthold R.J. J. Biol. Chem. 1996; 271: 15669-15674Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar). Cell pellets were suspended in buffer A (50 mm Tris, pH 7.5, 150 mm NaCl, 0.02% NaN3, 1 mm EDTA, 20 μm leupeptin, 1 mm4-(2-aminoethyl)benzenesulfonyl fluoride, 2 μg/ml pepstatin, 2 μg/ml aprotinin) by trituration and solubilized with 2% SDS in buffer A by incubating for 3 min at 90 °C. The soluble extract was obtained by centrifugation at 100,000 × g for 30 min at 15 °C, and 100 μl of the soluble extract was diluted 6-fold with 2% Triton X-100 in buffer A. To retain NMDA receptor subunit associations, cells were solubilized with 1% deoxycholate at pH 9 in buffer A (36Blahos II, J. Wenthold R.J. J. Biol. Chem. 1996; 271: 15669-15674Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar) (200 μm phenylmethylsulfonyl fluoride was used instead of 1 mm ABESF). After centrifugation, the soluble extract was diluted 10-fold with 0.1% Triton X-100. SDS or deoxycholate soluble fractions were incubated overnight at 4 °C with 50 μl of protein A-agarose beads to which 10 μg of polyclonal antibodies or 2.5 μg of monoclonal antibodies were preattached. Immunoprecipitated pellets were washed three times with buffer A containing 2% Triton X-100, 10% glycerol, and the above protease mixture, followed by two washes with buffer A containing 0.1% Triton X-100 and protease inhibitor mixture. Immunoprecipitated proteins were eluted with SDS-sample buffer (60 μl) by incubating at 90 °C for 3 min and subjected to SDS-PAGE on 4–20% gradient gels. For fluorography, electrophoresed gels were fixed with the solution containing 30% methanol and 10% glacial acetic acid for 30 min and treated with fluorophore (Enlightening) for 30 min. Gels were dried onto Whatman filter papers and exposed to films (Kodak X-Omat AR) at −70 °C. Fluorograms were scanned with a Molecular Dynamics densitometer, and densities were normalized using standard curves generated with autoradiographic 14C-labeled microscales. Membrane homogenates from cultured cells or cerebellum were suspended in 10 mmNaH2PO4 buffer containing 10 mmEDTA, 0.2 mm leupeptin, and 10 μg/ml pepstatin and solubilized with 1% SDS containing 5% β-mercaptoethanol by incubating at 90 °C for 2–3 min. The soluble fraction was diluted with 1% β-octyl glucopyranoside in 10 mmNaH2PO4 containing the above protease mixture to a final concentration of 0.1% SDS and incubated with endoglycosidase H (15 milliunits) or N-glycosidase F (3 units) overnight at 37 °C. An equal volume of 2× SDS-sample buffer was added for SDS-PAGE analysis. Membrane homogenates or soluble extracts of granule cells or adult rat cerebella were subjected to SDS-PAGE using 4–20% gradient gels. Proteins were transferred to nitrocellulose membranes, and the membranes were blocked with Tris-buffered saline containing 0.1% Tween (TBST) and 5% nonfat dry milk overnight at 4 °C, incubated with primary antibodies in TBST for 1.5 h, and washed three times for 15 min each. Membranes were incubated with horseradish peroxidase-conjugated secondary antibodies for 1 h and washed three times, and bound antibodies were visualized by the chemiluminescence detection method. Cultured granule cells were washed four times with HBSS, and surface proteins were biotinylated with Sulfo-NHS-LC-biotin or NHS-SS-biotin (1 mg/ml) in HBSS for 30 min at 4 °C. Cells were washed with HBSS four times and incubated in conditioned medium for 15 min at 37 °C in a humidified incubator before harvest. For determination of the cytoplasmic pool of the receptors, cells were harvested immediately after washing. KCl (25 mm) was included in all HBSS solutions for washing and biotinylation. Cells were harvested at various times as described above, by using phosphate-buffered saline containing 0.1 mglycine and protease mixture. Cells were stored at −70 °C. Pellets were solubilized with 2% SDS, diluted with 2% Triton X-100, immunoprecipitated using appropriate antibodies as described above, and subjected to SDS-PAGE. After transferring the gel to nitrocellulose membranes, membranes were blocked with 5% nonfat dry milk in TBST, incubated for 1.5 h with horseradish peroxidase-conjugated streptavidin (1:10,000) in TBST containing 0.5% milk, and washed three times for 15 min each. Biotinylated proteins were visualized by chemiluminescence. When NHS-SS-biotin was used, Ultralink-immobilized streptavidin beads were used to precipitate biotinylated proteins, and subunit proteins were detected by Western blot analysis. Cultured cerebellar granule cells, 8–9 days in vitro, were chosen for this study because it has been shown previously that these cells express functional AMPA and NMDA receptors (37Bessho Y. Nawa H. Nakanishi S. Neuron. 1994; 12: 87-95Abstract Full Text PDF PubMed Scopus (151) Google Scholar, 38Didier M. Mienville J.M. Soubrie P. Bockaert J. Berman S. Bursztajn S. Pin J.P. Eur. J. Neurosci. 1994; 6: 1536-1543Crossref PubMed Scopus (21) Google Scholar, 39Hack N.J. Sluiter A.A. Balázs R. Dev. Brain Res. 1995; 87: 55-61Crossref PubMed Scopus (39) Google Scholar, 40Resink A. Villa M. Benke D. Möhler H. Balázs R. J. Neurochem. 1995; 64: 558-565Crossref PubMed Scopus (69) Google Scholar, 41Vallano M.L. Lambolez B. Audinat E. Rossier J. J. Neurosci. 1996; 16: 631-639Crossref PubMed Google Scholar). Using antibodies selective for AMPA and NMDA receptor subunits and NR1 splice variants, Western blot analysis shows that multiple subunits and splice variants are expressed in these cultures (Fig. 1). It has been previously shown that NR2A and NR2C are the predominant NR2 subunits in granule cellsin vivo in the adult animal, while NR2B is the major subunit in granule cells at early developmental stages (20Watanabe M. Inoue Y. Sakimura K. Mishina M. Neuroreport. 1992; 3: 1138-1140Crossref PubMed Scopus (617) Google Scholar, 23Monyer H. Burnashev N. Laurie D.J. Sakmann B. Seeburg P.H. Neuron. 1994; 12: 529-540Abstract Full Text PDF PubMed Scopus (2897) Google Scholar). This developmental change of the subunit expression also occurs in cultured granule cells (42Condorelli D.F. Dell'Albani P. Aronica E. Genazzani A.A. Casabona G. Corsaro M. Balázs R. Nicoletti F. J. Neurochem. 1993; 61: 2133-2139Crossref PubMed Scopus (66) Google Scholar). The pattern of NR2 subunit expression in the present study is consistent with previous findings of mRNA analysis, where NR2A was the major subunit expressed and NR2B and NR2C were expressed in a lower abundance at 7–8 days in vitro(37Bessho Y. Nawa H. Nakanishi S. Neuron. 1994; 12: 87-95Abstract Full Text PDF PubMed Scopus (151) Google Scholar, 41Vallano M.L. Lambolez B. Audinat E. Rossier J. J. Neurosci. 1996; 16: 631-639Crossref PubMed Google Scholar). Since all four alternatively spliced cassettes of NR1 are found in cultured granule cells, it is possible that they are differentially assembled and that multiple functionally distinct NMDA receptors are expressed. NR1 C2-containing receptors appear to be more abundant than those containing the other C-terminal cassette, C2′, as determined by using different antibodies selective for the two cassettes. This was confirmed in a separate experiment where NR1 PAN antibodies were used to probe receptors immunoprecipitated with NR1 C2 and C2′ antibodies (data not shown). At least three AMPA receptor subunits were expressed in granule cells, GluR1, GluR2/3, and GluR4 (the antibody to GluR2/3 recognizes both GluR2 and GluR3 (5Wenthold R.J. Yokotani N. Doi K. Wada K. J. Biol. Chem. 1992; 267: 501-507Abstract Full Text PDF PubMed Google Scholar); mRNA for GluR2 and GluR3 subunits have been shown to be expressed (42Condorelli D.F. Dell'Albani P. Aronica E. Genazzani A.A. Casabona G. Corsaro M. Balázs R. Nicoletti F. J. Neurochem. 1993; 61: 2133-2139Crossref PubMed Scopus (66) Google Scholar)). GluR1 is not expressed in granule cells in vivo in adult rats but is expressed in cultured cells, as previously reported by Hacket al. (39Hack N.J. Sluiter A.A. Balázs R. Dev. Brain Res. 1995; 87: 55-61Crossref PubMed Scopus (39) Google Scholar), who showed a greater than 3-fold increase in GluR1 between 2 and 9 days in culture, while GluR2/3 and GluR4 remained relatively constant. Because of these rapidly changing levels of GluR1, this subunit was not included in the turnover analyses. To measure the degradation rates of receptor subunits, cells were pulse-labeled with [35S]methionine and chased in conditioned medium containing unlabeled methionine. Sufficient radioactivity was incorporated under these conditions to permit quantitation of receptor subunits immunoprecipitated with antibodies to NR1, NR2A, GluR2/3, and GluR4. Incubation with 2% SDS at 90 °C was used to ensure optimal solubilization of subunits; NMDA receptor subunits, in particular, have been reported to be only partially solubilized from the brain with nonionic or weaker ionic detergents (36Blahos II, J. Wenthold R.J. J. Biol. Chem. 1996; 271: 15669-15674Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar, 43Lau L.-F. Mammen A. Ehlers M.D. Kindler S. Chung W.J. Garner C.C. Huganir R.L. J. Biol. Chem. 1996; 271: 21622-21628Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar). Under the conditions used in the present study, about 90% of NR1 and 80% of NR2A immunoreactivities were present in the soluble fraction (data not shown). After a 20-min incubation with [35S]methionine, the predominant species of NMDA and AMPA receptor subunits appeared to be mature, fully glycosylated forms (Fig.2), since molecular weights of pulse-labeled subunits are similar to those of subunits that are expressed in cultured cells and the brain as determined by Western blotting (data not shown). Treatment of the culture with tunicamycin before and during pulse labeling resulted in the complete loss of the mature subunit and the appearance of lower molecular weight bands that migrate at a position consistent with that of the deglycosylated forms of subunits (44Brose N. Gasic G.P. Vetter D.E. Sullivan J.M. Heinemann S.F. J. Biol. Chem. 1993; 268: 22663-22671Abstract Full Text PDF PubMed Google Scholar, 45Hall R.A. Hansen A. Andersen P.H. Soderling T.R. J. Neurochem. 1997; 68: 625-630Crossref PubMed Scopus (62) Google Scholar, 46Chazot P.L. Cik M. Stephenson A. Mol. Membr. Biol. 1995; 12: 331-337Crossref PubMed Scopus (44) Google Scholar, 47Portera-Cailliau C. Price D.L. Martin L.J. J. Neurochem. 1996; 66: 692-700Crossref PubMed Scopus (152) Google Scholar). The absence of significant lower molecular weight unglycosylated forms indicates a rapid glycosylation of the subunits, and this appears to occur for nicotinic acetylcholine receptor subunits as well (48Claudio T. Paulson H.L. Green W.N. Ross A.F. Hartman D.S. Hayden D. J. Cell Biol. 1989; 108: 2277-2290Crossref PubMed Scopus (52) Google Scholar, 49Ross A.F. Green W.N. Hartman D.S. Claudio T. J. Cell Biol. 1991; 113: 623-636Crossref PubMed Scopus (56) Google Scholar). To characterize the glycosylation properties of NMDA and AMPA receptor subunits, membrane homogenates of cultured cells and the cerebellum were analyzed by Western blotting after treatment with endoglycosidase H, which recognizes N-linked high mannose carbohydrates or N-glycosidase F (Fig.3). Treatment with endoglycosidase H generated low molecular weight species of NR1 subunits, which comigrate with those that were treated with N-glycosidase F, indicating glycosylation of NR1 subunits is of the high mannose type. NR2A subunits showed significant sensitivity but were not completely sensitive to endoglycosidase H. GluR2/3 and GluR4 subunits appeared to be resistant; however, there was a slight shift in molecular weights, indicating that some glycosylation moieties have high mannose structures (Fig. 3).Figure 3Glycosylation properties of glutamate receptor subunits. Membrane homogenates from cultured cells (gr) and cerebellum (cb) were solubilized with 1% SDS in the presence of 5% β-mercaptoethanol. Soluble extracts, after dilution with 1% β-octyl glucoside, were incubated with endoglycosidase H or N-glycosidase F and analyzed by SDS-PAGE and Western blotting. Migration of glycosidase-treated samples is compared with that of the control samples, which were treated identically but without enzymes.View Large I
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