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.
Papadakis
Michalis
M
Department of Pharmaceutical and Biological Chemistry, School of Pharmacy, University of London, 29/39 Brunswick Square, London WC1N 1AX, United Kingdom.
Hawkins
Lynda M
LM
Stephenson
F Anne
FA
D000818:Animals; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism*; D019281:Dimerization; D004220:Disulfides; D004305:Dose-Response Relationship, Drug; D015180:Electrophoresis, Gel, Two-Dimensional; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes; D006801:Humans; D015151:Immunoblotting; D009154:Mutation; D011233:Precipitin Tests; D016548:Prosencephalon / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D016271:Proto-Oncogene Proteins c-myc / Q000378:metabolism; D011869:Radioligand Assay; D051381:Rats; D016194:Receptors, N-Methyl-D-Aspartate / Q000096:biosynthesis* / Q000737:chemistry*; D013347:Subcellular Fractions; D014162:Transfection
D004220:Disulfides; D000939:Epitopes; C409466:NR1 NMDA receptor; C120997:NR2A NMDA receptor; D016271:Proto-Oncogene Proteins c-myc; D016194:Receptors, N-Methyl-D-Aspartate