Abstract
N-Methyl-D-aspartate (NMDA) receptor subunits were characterized with seven polyclonal antibodies. The antibodies were directed against NR1-A, NR2A-N1, and NR2C-N1, representing N-terminal sequences of the NR1, NR2A, and NR2C subunits, and against NR1-E, NR2A-C1, and NR2C-C1, derived from C-terminal sequences of these subunits. The anti-NR1-D antibody was raised against the putative internal loop of NR1. A size of 118 kDa was found in sodium dodecyl sulfate-polyacrylamide gel electrophoresis for NR1 (from rat brain) detected by anti-NR1-D and -NR1-E, but not anti-NR1-A. With the anti-NR1-A antibody, a 125-kDa protein was discovered that may represent a glutamate receptor not yet characterized. NR2A and NR2C were identified as proteins with sizes of 175 and 140 kDa, respectively. Enzymatic N-deglycosylation generated a 97-kDa protein from NR1, a 105-kDa protein from the 125-kDa protein, a 162-kDa protein from NR2A, and a 127-kDa protein from NR2C. In contrast to the deglycosylation product of the NR2A, the 97- and 127-kDa proteins derived from NR1 and NR2C, respectively, were found significantly smaller than the molecular masses of 103 and 141 kDa, respectively, predicted on the basis of DNA data. These products may represent truncated proteins. The tissue content of the NR1 and NR2A was high in bovine hippocampus and cortex but lower in the cerebellum. In contrast, NR2C was solely found in the cerebellum. The 125-kDa protein was highest in the cerebellum and cortex.
Full Text
The Full Text of this article is available as a PDF (6.2 MB).
Selected References
These references are in PubMed. This may not be the complete list of references from this article.
- Anantharam V., Panchal R. G., Wilson A., Kolchine V. V., Treistman S. N., Bayley H. Combinatorial RNA splicing alters the surface charge on the NMDA receptor. FEBS Lett. 1992 Jun 22;305(1):27–30. doi: 10.1016/0014-5793(92)80648-z. [DOI] [PubMed] [Google Scholar]
- Bliss T. V., Lomo T. Long-lasting potentiation of synaptic transmission in the dentate area of the anaesthetized rabbit following stimulation of the perforant path. J Physiol. 1973 Jul;232(2):331–356. doi: 10.1113/jphysiol.1973.sp010273. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Chazot P. L., Cik M., Stephenson F. A. Immunological detection of the NMDAR1 glutamate receptor subunit expressed in embryonic kidney 293 cells and in rat brain. J Neurochem. 1992 Sep;59(3):1176–1178. doi: 10.1111/j.1471-4159.1992.tb08364.x. [DOI] [PubMed] [Google Scholar]
- Chu G., Hayakawa H., Berg P. Electroporation for the efficient transfection of mammalian cells with DNA. Nucleic Acids Res. 1987 Feb 11;15(3):1311–1326. doi: 10.1093/nar/15.3.1311. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Dieckmann C. L., Tzagoloff A. Assembly of the mitochondrial membrane system. CBP6, a yeast nuclear gene necessary for synthesis of cytochrome b. J Biol Chem. 1985 Feb 10;260(3):1513–1520. [PubMed] [Google Scholar]
- Fling S. P., Gregerson D. S. Peptide and protein molecular weight determination by electrophoresis using a high-molarity tris buffer system without urea. Anal Biochem. 1986 May 15;155(1):83–88. doi: 10.1016/0003-2697(86)90228-9. [DOI] [PubMed] [Google Scholar]
- Graham F. L., Smiley J., Russell W. C., Nairn R. Characteristics of a human cell line transformed by DNA from human adenovirus type 5. J Gen Virol. 1977 Jul;36(1):59–74. doi: 10.1099/0022-1317-36-1-59. [DOI] [PubMed] [Google Scholar]
- Hollmann M., Boulter J., Maron C., Beasley L., Sullivan J., Pecht G., Heinemann S. Zinc potentiates agonist-induced currents at certain splice variants of the NMDA receptor. Neuron. 1993 May;10(5):943–954. doi: 10.1016/0896-6273(93)90209-a. [DOI] [PubMed] [Google Scholar]
- Hollmann M., O'Shea-Greenfield A., Rogers S. W., Heinemann S. Cloning by functional expression of a member of the glutamate receptor family. Nature. 1989 Dec 7;342(6250):643–648. doi: 10.1038/342643a0. [DOI] [PubMed] [Google Scholar]
- Hunter C., Wheaton K. D., Wenthold R. J. Solubilization and partial purification of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid binding sites from rat brain. J Neurochem. 1990 Jan;54(1):118–125. doi: 10.1111/j.1471-4159.1990.tb13290.x. [DOI] [PubMed] [Google Scholar]
- Huntley G. W., Rogers S. W., Moran T., Janssen W., Archin N., Vickers J. C., Cauley K., Heinemann S. F., Morrison J. H. Selective distribution of kainate receptor subunit immunoreactivity in monkey neocortex revealed by a monoclonal antibody that recognizes glutamate receptor subunits GluR5/6/7. J Neurosci. 1993 Jul;13(7):2965–2981. doi: 10.1523/JNEUROSCI.13-07-02965.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Ishii T., Moriyoshi K., Sugihara H., Sakurada K., Kadotani H., Yokoi M., Akazawa C., Shigemoto R., Mizuno N., Masu M. Molecular characterization of the family of the N-methyl-D-aspartate receptor subunits. J Biol Chem. 1993 Feb 5;268(4):2836–2843. [PubMed] [Google Scholar]
- Kutsuwada T., Kashiwabuchi N., Mori H., Sakimura K., Kushiya E., Araki K., Meguro H., Masaki H., Kumanishi T., Arakawa M. Molecular diversity of the NMDA receptor channel. Nature. 1992 Jul 2;358(6381):36–41. doi: 10.1038/358036a0. [DOI] [PubMed] [Google Scholar]
- Köpke A. K., Leggatt P. A., Matheson A. T. Structure function relationships in the ribosomal stalk proteins of archaebacteria. J Biol Chem. 1992 Jan 15;267(2):1382–1390. [PubMed] [Google Scholar]
- Monyer H., Sprengel R., Schoepfer R., Herb A., Higuchi M., Lomeli H., Burnashev N., Sakmann B., Seeburg P. H. Heteromeric NMDA receptors: molecular and functional distinction of subtypes. Science. 1992 May 22;256(5060):1217–1221. doi: 10.1126/science.256.5060.1217. [DOI] [PubMed] [Google Scholar]
- Moriyoshi K., Masu M., Ishii T., Shigemoto R., Mizuno N., Nakanishi S. Molecular cloning and characterization of the rat NMDA receptor. Nature. 1991 Nov 7;354(6348):31–37. doi: 10.1038/354031a0. [DOI] [PubMed] [Google Scholar]
- Nakanishi N., Axel R., Shneider N. A. Alternative splicing generates functionally distinct N-methyl-D-aspartate receptors. Proc Natl Acad Sci U S A. 1992 Sep 15;89(18):8552–8556. doi: 10.1073/pnas.89.18.8552. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Sugihara H., Moriyoshi K., Ishii T., Masu M., Nakanishi S. Structures and properties of seven isoforms of the NMDA receptor generated by alternative splicing. Biochem Biophys Res Commun. 1992 Jun 30;185(3):826–832. doi: 10.1016/0006-291x(92)91701-q. [DOI] [PubMed] [Google Scholar]
- Tingley W. G., Roche K. W., Thompson A. K., Huganir R. L. Regulation of NMDA receptor phosphorylation by alternative splicing of the C-terminal domain. Nature. 1993 Jul 1;364(6432):70–73. doi: 10.1038/364070a0. [DOI] [PubMed] [Google Scholar]