Immunochemical characterization of the non-NMDA glutamate receptor using subunit-specific antibodies. Evidence for a hetero-oligomeric structure in rat brain.
Antibodies were made to synthetic peptides corresponding to sequences specific to the glutamate receptor (GluR) subunits, GluR1-4. The specificity of the antibodies was established by Western blotting using membranes of simian kidney cells (COS-7) transfected with GluR subunit DNA. Four antibodies were found to be selective for each of the four GluR subunits, and a fifth antibody recognized both GluR2 and 3. All five antibodies immunoadsorbed Triton X-100-solubilized rat brain [3H]AMPA binding activity and labeled an Mr = 108,000 band in samples of rat brain. The structure of the Triton X-100-solubilized GluR was studied using subunit-specific antibodies covalently attached to protein A-agarose and analyzing GluR subunits bound to the antibodies by Western blotting. Each of the four subunit-specific antibodies immunoadsorbed its respective GluR subunit as well as the other three forms of GluR, showing that the detergent solubilized GluR exists as hetero-oligomers composed of two or more of the four subunits. Evidence supporting a similar structure for membrane bound GluR was obtained using synaptic membranes chemically cross-linked with dithiobis(succinimidylpropionate). GluR was immunoaffinity-purified using the GluR2 and 3-selective antibody. This antibody, covalently attached to protein A-agarose, adsorbed 55% of [3H]AMPA binding activity, and after elution with 1 M KSCN, 22-37% of the binding activity was recovered. Analysis of the purified product showed a major immunoreactive band at Mr = 108,000, and silver staining identified the same major band and no additional polypeptides. The GluR receptor complex, therefore, appears to be made up exclusively of GluR1-4. In the purified GluR preparation, in addition to the Mr = 108,000 band, three higher molecular weight immunoreactive components were also detected. These bands migrated at Mr = 325,000, 470,000, and 590,000. Similar sized proteins were seen in the cross-linked synaptic membrane sample, with the Mr = 590,000 component being substantially enriched after cross-linking. The Mr = 590,000 band is the largest component detected, and it has a size consistent with its being a pentamer of the Mr = 108,000 protein.
Wenthold
R J
RJ
Laboratory of Neurochemistry, National Institute on Deafness and Other Communication Disorders, National Institutes of Health, Bethesda, Maryland 20892.
Yokotani
N
N
Doi
K
K
Wada
K
K
D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies / Q000276:immunology*; D015153:Blotting, Western; D001923:Brain Chemistry; D002417:Cattle; D002460:Cell Line; D003432:Cross-Linking Reagents; D004247:DNA / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D005971:Glutamates / Q000378:metabolism*; D007120:Immunochemistry; D008969:Molecular Sequence Data; D016202:N-Methylaspartate / Q000378:metabolism*; D010455:Peptides / Q000276:immunology; D051381:Rats; D017470:Receptors, Glutamate; D017981:Receptors, Neurotransmitter / Q000737:chemistry* / Q000276:immunology / Q000378:metabolism; D014162:Transfection
D000906:Antibodies; D003432:Cross-Linking Reagents; D005971:Glutamates; D010455:Peptides; D017470:Receptors, Glutamate; D017981:Receptors, Neurotransmitter; D016202:N-Methylaspartate; D004247:DNA