Appropriate NR1-NR1 disulfide-linked homodimer formation is requisite for efficient expression of functional, cell surface N-methyl-D-aspartate NR1/NR2 receptors.
A c-Myc epitope-tagged N-methyl-D-aspartate receptor NR1-2a subunit was generated, NR1-2a(c-Myc), where the tag was inserted after amino acid 81. NR1-2a(c-Myc) /NR2A receptors when expressed in mammalian cells are not trafficked to the cell surface nor do they yield cell cytotoxicity post-transfection. NR1-2a(c-Myc) was, however, shown to assemble with NR2A subunits ... by immunoprecipitation and [(3)H]MK801 radioligand binding assays. Immunoblots of cells co-transfected with wild-type NR1-2a/NR2A subunits yielded two NR1-2a immunoreactive species with molecular masses of 115 and 226 kDa. Two-dimensional electrophoresis under non-reducing and reducing conditions revealed that the 226-kDa band contained disulfide-linked NR1-2a subunits. Only the 115-kDa NR1-2a species was detected for NR1-2a(c-Myc)/NR2A. The c-Myc epitope is inserted adjacent to cysteine 79 of the NR1-2a subunit; therefore, it is possible that the tag may prevent the formation of NR1 disulfide bridges. A series of cysteine --> alanine NR1-2a mutants was generated, and the NR1-2a mutants were co-expressed with NR2A or NR2B subunits in mammalian cells and characterized with respect to cell surface expression, cell cytotoxicity post-transfection, co-association by immunoprecipitation, and immunoblotting following SDS-PAGE under both reducing and non-reducing conditions. When co-expressed with NR2A in mammalian cells, NR1-2a(C79A)/NR2A displayed similar properties to NR1-2a(c-Myc)/NR2A in that the 226-kDa NR1 immunoreactive species was not detectable, and trafficking to the cell surface was impaired compared with wild-type NR1/NR2 receptors. These results provide the first biochemical evidence for the formation of NR1-NR1 intersubunit disulfide-linked homodimers involving cysteine 79. They suggest that disulfide bridging and structural integrity within the NR1 N-terminal domain is requisite for cell surface N-methyl-D-aspartate receptor expression.
Mesh Terms:
Animals, Cell Line, Cell Membrane, Dimerization, Disulfides, Dose-Response Relationship, Drug, Electrophoresis, Gel, Two-Dimensional, Electrophoresis, Polyacrylamide Gel, Enzyme-Linked Immunosorbent Assay, Epitopes, Humans, Immunoblotting, Mutation, Precipitin Tests, Prosencephalon, Protein Binding, Protein Structure, Tertiary, Proto-Oncogene Proteins c-myc, Radioligand Assay, Rats, Receptors, N-Methyl-D-Aspartate, Subcellular Fractions, Transfection
Animals, Cell Line, Cell Membrane, Dimerization, Disulfides, Dose-Response Relationship, Drug, Electrophoresis, Gel, Two-Dimensional, Electrophoresis, Polyacrylamide Gel, Enzyme-Linked Immunosorbent Assay, Epitopes, Humans, Immunoblotting, Mutation, Precipitin Tests, Prosencephalon, Protein Binding, Protein Structure, Tertiary, Proto-Oncogene Proteins c-myc, Radioligand Assay, Rats, Receptors, N-Methyl-D-Aspartate, Subcellular Fractions, Transfection
J. Biol. Chem.
Date: Apr. 09, 2004
PubMed ID: 14732708
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