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, NR1-2ac-Myc, where the tag was inserted after amino acid 81. NR1-2ac-Myc /NR2A receptors when expressed in mammalian cells are not trafficked to the cell surface nor do they yield cell cytotoxicity post-transfection. NR1-2ac-Myc was, however, shown to assemble with NR2A subunits by immunoprecipitation and [3H]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 NR1-2ac-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, NR1-2aC79A/NR2A displayed similar properties to NR1-2ac-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. A c-Myc epitope-tagged N-methyl-d-aspartate receptor NR1-2a subunit was generated, NR1-2ac-Myc, where the tag was inserted after amino acid 81. NR1-2ac-Myc /NR2A receptors when expressed in mammalian cells are not trafficked to the cell surface nor do they yield cell cytotoxicity post-transfection. NR1-2ac-Myc was, however, shown to assemble with NR2A subunits by immunoprecipitation and [3H]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 NR1-2ac-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, NR1-2aC79A/NR2A displayed similar properties to NR1-2ac-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. N-Methyl-d-aspartate (NMDA) 1The abbreviations used are: NMDA, N-methyl-d-aspartate; AMPA, amino α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid; HEK, human embryonic kidney; LAMP, lysosomal-associated membrane protein; MDL105,519, (E)-3-(2)-phenyl-2-carboxyethenyl)-4,6-dichloro-1-indole-2-carboxylic acid; MK801, (+)-5-methyl-10,11-dihydro-dibenzo-[a,d]cyclohepten-5,10-imine; NR1, NR2, etc., NMDA receptor subunit NR1, NR2; PSD-95, postsynaptic density-95; ELISA, enzyme-linked immunosorbent assay; LIVBP, leucine-isoleucine-valine-binding protein; PBS, phosphate-buffered saline. receptors are a subclass of excitatory, ionotropic glutamate neurotransmitter receptors. They are unique in that they require the binding of co-agonists, i.e. l-glutamate and glycine, together with the alleviation of a voltage-dependent blockade by magnesium ions for channel activation. NMDA receptors are critical mediators of excitatory neurotransmission in the brain, being pivotal for long term potentiation. They are also important as a therapeutic target post-ischemia. NMDA receptor channels are highly permeable to calcium ions, and thus overactivation leads to excitotoxic neuronal cell death (reviewed in Ref. 1Dingledine R. Borges L. Bowie D. Traynelis S.F. Pharmacol. Rev. 1999; 51: 7-61PubMed Google Scholar). Seven genes encode NMDA receptor subunits NR1, NR2A–NR2D, and NR3A–NR3B. The NR1 subunit undergoes extensive splicing to yield eight variants NR1-1a,1b to NR1-4a,4b. Functional NMDA receptors are formed from the co-assembly of the obligatory NR1 glycine-binding subunit with NR2 and/or NR3 subunits, although it was recently shown (2Chatterton J.E. Awobuluyi M. Premkumar L.S. Takahashi H. Talantova M. Shin Y. Cui J. Tu S. Sevarino K.A. Nakanishi N. Tong G. Lipton S.A. Zhang D. Nature. 2002; 415: 793-798Crossref PubMed Scopus (536) Google Scholar) that NR1/NR3 subunits formed a novel, glycine-gated receptor. The quaternary structure of NMDA receptors is still not yet established. Experimental data support either a tetrameric structure comprising two NR1 and two NR2 subunits or a pentamer with reports of either three NR1 subunits co-assembled with two NR2s or two NR1s with three NR2s (1Dingledine R. Borges L. Bowie D. Traynelis S.F. Pharmacol. Rev. 1999; 51: 7-61PubMed Google Scholar). All NMDA receptor subunits share the same transmembrane organization. They have an extracellular N-terminal domain of ∼550 amino acids, 3 transmembrane domains, 1 re-entrant membrane domain M2 that is thought to form the inner lining of the cation channel, and an intracellular C-terminal tail. The N-terminal region can be subdivided into two discrete domains based on the amino acid sequence homology between the NMDA receptor subunits and amino acid-binding proteins of bacteria. Thus, the first ∼400 amino acids have homology with the bacterial periplasmic leucine-isoleucine-valine-binding protein (LIVBP), whereas amino acids 420–550 show similarity with the bacterial lysine-arginine-ornithine and glutamine-binding protein (reviewed in Refs. 1Dingledine R. Borges L. Bowie D. Traynelis S.F. Pharmacol. Rev. 1999; 51: 7-61PubMed Google Scholar and 3Lipton S.A. Choi Y.-B. Takahashi H. Zhang D. Li W. Godzik A. Bankston L.A. Trends Neurosci. 2002; 25: 474-480Abstract Full Text Full Text PDF PubMed Scopus (328) Google Scholar). The glycine- and glutamate-binding sites of the NR1 and NR2 subunits, respectively, have been localized to the N-terminal regions, i.e. amino acids 420–550 (S1), that are proximal to the membrane and the S2 extracellular loop found between TM3 and TM4 by a combination of site-directed mutagenesis studies (4Kuryatov A. Laube B. Betz H. Kuhse J. Neuron. 1994; 12: 1291-1300Abstract Full Text PDF PubMed Scopus (337) Google Scholar, 5Hirai H. Kirsch J. Laube B. Betz H. Kuhse J. Proc. Natl. Acad. Sci. U. S. A. 1994; 93: 6031-6036Crossref Scopus (195) Google Scholar, 6Sandhu S. Grimwood S. Mortishire-Smith R.J. Whiting P.J. le Bourdelles B. J. Neurochem. 1999; 72: 1694-1698Crossref PubMed Scopus (2) Google Scholar, 7Laube B. Hirai H. Sturgess M. Betz H. Hukse J. Neuron. 1997; 18: 493-503Abstract Full Text Full Text PDF PubMed Scopus (411) Google Scholar, 8Anson L.E. Chen P.E. Wyllie D.J. Colquhoun D. Schoepfer R. J. Neurosci. 1998; 18: 581-589Crossref PubMed Google Scholar) and by analogy with the crystal structure of the soluble extracellular domain of the non-NMDA, amino α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) GluR2 glutamate receptor subunit (9Armstrong N. Gouaux E. Neuron. 2000; 28: 165-181Abstract Full Text Full Text PDF PubMed Scopus (799) Google Scholar). Most interesting, the N-terminal region distal to the membrane has been shown to be important for the assembly of non-NMDA receptors (10Leuschner W.D. Hoch W. J. Biol. Chem. 1999; 274: 16907-16916Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar, 11Ayalon G. Stern-Bach Y. Neuron. 2001; 31: 103-113Abstract Full Text Full Text PDF PubMed Scopus (210) Google Scholar). This domain is requisite for GluR-GluR dimer formation, an initial step in the assembly pathway of functional non-NMDA receptors (11Ayalon G. Stern-Bach Y. Neuron. 2001; 31: 103-113Abstract Full Text Full Text PDF PubMed Scopus (210) Google Scholar). For the assembly of functional heteromeric NR1/NR2 NMDA it has been shown that NR2A subunits are localized to the membrane when co-expressed with an NR1 subunit le Bourdelles B. N. E. Whiting P.J. 1998; PubMed Scopus Google Scholar) and that is important for subunit E. Bourdelles B. Grimwood S. S. Whiting J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). it was recently that NR1-NR1 are an initial step in NMDA receptor assembly S. Colquhoun D. J. Neurosci. PubMed Google Scholar). E. Bourdelles B. Grimwood S. S. Whiting J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar) the of NR1-NR1 following of in mammalian have an epitope-tagged NR2B subunit J. Biol. Chem. 1999; 274: Full Text Full Text PDF PubMed Scopus Google Scholar). The or c-Myc were between amino acids and of the NR2B subunit to yield and J. Biol. Chem. 1999; 274: Full Text Full Text PDF PubMed Scopus Google Scholar). as wild-type NR2B with NR1 subunits to form functional cell surface NMDA receptors following in mammalian cells or in J. Biol. Chem. 1999; 274: Full Text Full Text PDF PubMed Scopus Google Scholar, G. J. R.J. S. J. Neurosci. 2002; PubMed Google Scholar). The c-Myc epitope tag was into the N-terminal domain of the NR1-2a a acid similar to that used for the of the NR2B Most interesting, epitope-tagged form of the NR1-2a not in cell that it was to form functional cell surface NMDA receptors. the biochemical properties of NR1-2ac-Myc epitope-tagged NMDA receptor show that of the NR1-2a subunit into the of both NR1 disulfide bridging and the integrity of the N-terminal domain for both the assembly and cell surface of functional NMDA receptors. and NMDA receptor and were as in J. Biol. Chem. 1999; 274: Full Text Full Text PDF PubMed Scopus Google Scholar). The amino acid i.e. the c-Myc epitope tag sequence with an N-terminal was inserted between by the unique of the NR1-2a amino acid and sequence to i.e. the is was by with and was by The with and were and into the of by a The was by The cysteine → alanine NR1-2a and the cysteine → alanine 79 and and the cysteine → alanine and were from by the to The following were used to NR1-2a → NR1-2a → NR1-2a → were by the was a from M. that NR1 that NR1 with the i.e. NR1-1a,1b and and were as J. Biol. Chem. 1999; 274: Full Text Full Text PDF PubMed Scopus Google Scholar, M. J. Neurochem. PubMed Scopus Google Scholar, J. Neurochem. 1997; PubMed Scopus Google Scholar, M. Biol. 12: PubMed Scopus Google Scholar). and were from membrane were from the the of were from embryonic cells were and with either subunit NR1 or NR2 NMDA receptor co-transfected with both NR1/NR2 subunit the calcium with a of of and a of for respectively, or with NR1, NR2, and a of of and a of NMDA receptor of M. J. PubMed Scopus (104) Google Scholar, J. Neurochem. 2002; PubMed Scopus Google Scholar). were in the of 1 post-transfection. They were by and were to and for [3H]MK801 and radioligand binding or and by cells were co-transfected with with or post-transfection, cell was the cytotoxicity to the and as M. J. PubMed Scopus (104) Google Scholar). was as of the and SDS-PAGE under both reducing and non-reducing conditions as in J. Biol. Chem. 1999; 274: Full Text Full Text PDF PubMed Scopus Google Scholar). and were used of were used a of and were used a of and were detected by the were by either molecular a with in the of the J. Biol. Chem. 1999; 274: Full Text Full Text PDF PubMed Scopus Google Scholar) or the and Two-dimensional electrophoresis was as by and N. R.J. Proc. Natl. Acad. Sci. U. S. A. 1999; PubMed Scopus Google Scholar) that after the first of SDS-PAGE under non-reducing the band of was from the and the protein was into glycine, by electrophoresis for by electrophoresis for with The protein was to a of SDS-PAGE with and reducing proteins were to and proteins were detected by immunoblotting as NMDA by of cell surface NMDA receptors was by an from that in J. Neurochem. 2002; PubMed Scopus Google Scholar). cells were to in and of was used were in the of 1 for post-transfection. was and was with 1 of phosphate-buffered were by the of for with of PBS, and sites by with in for were for 1 with the was NR2 subunits are found on the cell surface when co-expressed with an NR1 subunit le Bourdelles B. N. E. Whiting P.J. 1998; PubMed Scopus Google Scholar). were for with of in was for 1 in in PBS, were 3 for with of in PBS, 1 with of for and was were for and the was For the of in the cells, cells were for with 1 with of PBS, and by with in for All were as for the of cell surface NMDA receptor subunit expression. For the of binding of receptor to cells, cells were with the and the were to NMDA receptor and cell surface radioligand binding to cell of cells were as 1 for the of binding B. J. Pharmacol. 2000; Scopus Google Scholar). radioligand binding to cell of cells were as J. Neurochem. 2000; PubMed Scopus Google Scholar) in the of glycine, and for the of cells were co-transfected with either with of or wild-type and were after and cell were for 1 with and were to 1 of and the was by were with or protein A was and were for 1 were by for with as 1 with SDS-PAGE and by of from were as and used J. Neurochem. 1997; PubMed Scopus Google Scholar). cells were co-transfected with NR1-2a/NR2A or NR1-2ac-Myc/NR2A and for in the of 1 and cells were by for The was by a by a in of and The was for The was to a were an for 3 were and by of NR1-2ac-Myc/NR2A NMDA in the of Functional NMDA have that of wild-type NR1/NR2 heteromeric receptors results in cell cytotoxicity M. J. PubMed Scopus (104) Google Scholar). This NMDA cell death was by the of NMDA receptor in the cell post-transfection. was to of functional cell surface NMDA receptors by l-glutamate and in the cell with a of The cell cytotoxicity is a biochemical to functional cell surface NMDA receptors. Most interesting, in to and NR1-2ac-Myc/NR2A not yield cell cytotoxicity The of cell death was not of a of the NR1-2ac-Myc of cell that when expressed was in molecular and between wild-type and epitope-tagged subunits and When NR1-2ac-Myc was co-expressed with however, the subunit was compared with wild-type NR1-2a/NR2A and surface the cell cytotoxicity in that cell surface was detected following the of receptors in cells This with cell cytotoxicity and cell surface for wild-type NR1-2a/NR2A and NR1-2ac-Myc with NR2A by and the it was that although the c-Myc NR1-2a subunit was it was not trafficked to the cell surface when co-expressed with an NR2 were to the properties of the the first immunoprecipitation studies were used to NR1-2ac-Myc subunits were to with NR2 subunits. and NR1-2a were co-expressed with NR2A in cells were and immunoprecipitation were with The results are shown in A and B. can be that both NR1-2a and NR1-2ac-Myc subunits were by that NR1-2a subunits do with of co-association was by radioligand binding [3H]MK801 is a of NMDA receptors. with to NR1/NR2 receptors Pharmacol. 1994; Google Scholar, D.J. J. Pharmacol. 1994; PubMed Scopus Google Scholar). [3H]MK801 radioligand binding were on cells with NR1-2a/NR2A and NR1-2ac-Myc/NR2A. The binding are shown in were by a with in between wild-type and are as and was, however, a in the of binding are as protein and protein This with that found by immunoblotting for the of NR1-2ac-Myc compared with NR1-2a in NR1/NR2 and These results that the of the c-Myc epitope not the integrity of the NMDA receptor is a and with to NR1 subunits expressed for binding to NR1-2a and The binding are as protein and protein was thus in the and the between wild-type and receptors. These binding studies are in with the of by and they that the of the c-Myc epitope amino acid NR1-2a not the NR1 binding of NR1-2ac-Myc/NR2A NR1-2ac-Myc/NR2A receptors are not expressed the cell studies were to The results are shown in For wild-type both NR1 and NR2 subunit are the of the for the epitope-tagged both NR1 and NR2 subunit are to the of the where they with a molecular and in the as be from of the epitope of NR1-2ac-Myc it was shown that although both wild-type NR1 and NR2 subunit were with of with NR1 in cells with subunits was NMDA receptor NR1 not NR2 subunits have been to with 2001; PubMed Scopus Google Scholar). of wild-type NR2 subunits may be to the in mammalian cells compared with NR2 in NR1-2ac-Myc/NR2A with is a protein that is in of where it is to with NMDA receptors the found the of the NR2 is thought to a in the of NMDA receptor (reviewed in Ref. M. Neuron. Full Text Full Text PDF Scopus Google Scholar). NR1-2ac-Myc/NR2A receptors are not to the cell it was of to they with NR1-2a/NR2A and NR1-2ac-Myc/NR2A receptors were both co-expressed with cell with and the by The results in show that is by NR1 subunits were also in the as found by immunoblotting with not and of NR1-2ac-Myc Thus, with NR2A subunit receptors the that they are within the This with the of N. R. S. R.J. Biol. PubMed Scopus Google Scholar) that that NMDA proteins in the pathway the NR1-2a/NR2A and NR1-2ac-Myc/NR2A by SDS-PAGE and of cell NR1-2a/NR2A and NR1-2ac-Myc/NR2A receptors with in to the 115 immunoreactive i.e. the NR1 a immunoreactive band with 226 was for wild-type not for the NR1-2a and This molecular is with an NR1-NR1 was also detected for the not The c-Myc epitope tag is inserted is a cysteine NR1-2a thus it was that NR1-NR1 subunits form homodimers that are the of the tag may prevent disulfide formation involving The 226-kDa immunoreactive band for wild-type NR1-2a may thus be NR1-NR1 cell of NR1-2a/NR2A and NR1-2ac-Myc/NR2A and were by immunoblotting following SDS-PAGE under both reducing and non-reducing conditions. The results are shown in with as under reducing conditions for cells, two immunoreactive with 115 and 226 were for wild-type with band for non-reducing conditions for immunoreactive with 226 and were as as that not the NR1-NR1 subunits are disulfide-linked as the of the 115-kDa it has been shown that in and in the brain, a of NR1 subunits J. Neurochem. 1997; PubMed Scopus Google Scholar, R.J. J. Biol. Chem. 1999; 274: Full Text Full Text PDF PubMed Scopus Google under non-reducing the species is For NMDA the 115-kDa NR1 immunoreactive species was detected under reducing conditions. non-reducing immunoreactive species were This may be by both the of NR1 in compared with cells, and the molecular of NMDA i.e. that they not the For NR1-2ac-Myc under non-reducing the immunoreactive band with a molecular to that found for wild-type are as The in molecular between wild-type NR1-2a and NR1-2ac-Myc is to in to as to formation in NR1-NR1 homodimers and in SDS-PAGE of the NR1-2a subunits for were found in the properties of NR2A subunits under reducing and non-reducing conditions not that the 226-kDa species in NR1-2a/NR2A receptors contained NR1 disulfide-linked subunits, SDS-PAGE was The first SDS-PAGE was under non-reducing conditions to the of the 226-kDa This 226-kDa band was and and a SDS-PAGE under reducing and non-reducing conditions was The results shown in that following the 226-kDa immunoreactive species with a molecular 115 thus evidence for disulfide bridging of NR1 subunits. NR1-NR1 homodimers have been detected in cell of both NR1 or by E. Bourdelles B. Grimwood S. S. Whiting J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). disulfide bridging between subunits, however, was not of the of in the NR1 in the of NR1/NR2 NMDA to the of NR1 is to be the first amino acids of the NR1 M. R. N. S. Nature. PubMed Scopus Google Scholar). are thus three in the domain of the NR1 and of was to a and the were also NR1-2a/NR2A or subunit were expressed in cells, and the following were the cell cytotoxicity post-transfection, the of NR1-2a subunits in cell from NMDA receptor cell surface and the co-association of NR1-2a and NR2 subunits and NR1-2a/NR2A receptors and by The results are shown in and is a of the properties of the mutants compared with wild-type NR1-2a/NR2A and NR1-2ac-Myc/NR2A expressed by immunoprecipitation that NR1-2a → subunits with NR2A and cells were co-transfected with NR1-2a cysteine to alanine mutants and NR2A cell were and and were with and by immunoblotting either or as under The is the same in both A and where 1 is wild-type is NR1-2a 3 is is NR1-2a and in cells were with and immunoprecipitation was with The of molecular are shown on the of the properties of wild-type NR1-2ac-Myc, and NR1-2a cysteine following with NR2A subunits in conditions conditions cell cell cytotoxicity is the for where the cytotoxicity for NR1-2a/NR2A receptors in the of cell surface cell surface is the of NR2B subunit expressed the cell surface to NR2B in cell for 3 1 1 3 1 cell cytotoxicity is the for where the cytotoxicity for NR1-2a/NR2A receptors in the of cell surface is the of NR2B subunit expressed the cell surface to NR2B in cell for in a it can be that NR1-2a/NR2A in cytotoxicity post-transfection. was in cell death between wild-type and NR1-2a/NR2A receptors. it was found that of either or in the of trafficking of receptors to the cell For wild-type and and respectively, of was expressed the cell surface compared with for and for receptors was in cell surface between and Thus, although the of cell cytotoxicity is a of functional NMDA is not an with the of functional NMDA receptors expressed in the following SDS-PAGE under reducing the 226-kDa NR1-NR1 was not in NR1-2a/NR2A receptors that contained the a 226-kDa species was detected for under non-reducing conditions the molecular of the NR1-2a subunits was similar to that found for the NR1-2ac-Myc i.e. compared with for non-reducing a band was for receptors and These suggest the is requisite for NR1-NR1 intersubunit disulfide both cysteine 79 and cysteine are requisite for the of the NR1 subunit; in the of leads to intersubunit disulfide and and subunit disulfide bridging is requisite for the trafficking of NR1/NR2 NMDA receptors to the The band for also for NR1-2ac-Myc under non-reducing conditions may be to NR1-NR1 dimer formation with a to NR1 subunits therefore, to wild-type NR1-NR1 may disulfide bridging between within the lysine-arginine-ornithine and glutamine-binding protein and/or S2 impaired trafficking to the cell and were shown to with NR2A subunits by immunoprecipitation the of the 226-kDa species in NR1-2a subunits was found to with and co-expressed with NR2A The of with NR1-2a with cysteine of in the domain NR1-2a subunits that similar properties to the epitope-tagged have the properties of an NR1-2a NMDA receptor subunit with the c-Myc epitope in the N-terminal This NR1-2ac-Myc subunit when co-expressed with NR2A was not to the cell surface was not to an into the cysteine within the NR1 was that cysteine and are for into the Thus, trafficking of functional NMDA receptors to the membrane of NR1 and NR2 subunits in the le Bourdelles B. N. E. Whiting P.J. 1998; PubMed Scopus Google the formation of NR1-NR1 disulfide-linked homodimers and by the of the NR1 domain E. Bourdelles B. Grimwood S. S. Whiting J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar, S. Colquhoun D. J. Neurosci. PubMed Google and assembly of NR1-NR1 with NR2 subunits formation S. Colquhoun D. J. Neurosci. PubMed Google by into the pathway and in the NMDA receptors are by of (reviewed in Ref. 1Dingledine R. Borges L. Bowie D. Traynelis S.F. Pharmacol. Rev. 1999; 51: 7-61PubMed Google Scholar). in extensive studies of the of wild-type NR1/NR2 and NR1/NR2 where were to either alanine or Lipton and that in the NR1 subunit two of cysteine were important for the of NMDA receptors. in the S2 extracellular i.e. and is for the of the of and receptors Traynelis S.F. Chen W. S.F. Lipton S.A. Neuron. 1994; Full Text PDF PubMed Scopus Google Scholar). A and not to form an within NR1, was to be for an of Y.-B. Chen Lipton S.A. J. Neurosci. 2001; PubMed Google Scholar). the studies that two are they in that that NR1 subunits are disulfide bridging involving studies involving of were the for was of wild-type receptors. This with the in cell surface of receptors was that in cell surface of the NR1-2a cysteine was in the cell cytotoxicity This may be by the that the cytotoxicity was it may be possible that of the 79 and binding or channel to and an cytotoxicity post-transfection. Choi Y.-B. Chen Lipton S.A. J. Neurosci. 2001; PubMed Google however, that the NMDA and for of were either not or were Thus, although the was to alanine as in binding or channel are The of between cell surface and cell cytotoxicity may also be by an in cell surface to a of used to cell surface of and to possible in by cysteine → alanine ionotropic glutamate to the same as the NMDA receptors. Most interesting, when under non-reducing it was that molecular species were that were in reducing conditions. These subunits to receptor subunit disulfide-linked J. PubMed Scopus Google Scholar). the N-terminal domain of non-NMDA, are and The crystal structure of the binding domain of the GluR2 receptor subunit has been (9Armstrong N. Gouaux E. Neuron. 2000; 28: 165-181Abstract Full Text Full Text PDF PubMed Scopus (799) Google Scholar, Y. R. M. N. M. Gouaux E. Nature. 2002; PubMed Scopus Google Scholar). This however, to thus structural the N-terminal region of GluR2 and is not the crystal structure of the NMDA receptor NR1 domain was recently H. Gouaux E. J. PubMed Scopus Google the of disulfide formation is important in that it both the of and it the integrity of soluble proteins and cell surface receptors. have been shown to for the protein M. J. Nature. 2001; PubMed Scopus Google Scholar) and the channel 2000; PubMed Scopus Google Scholar). the integrity of disulfide formation was shown to be important for the trafficking to the cell surface of human channels R.J. Pharmacol. 2002; PubMed Scopus Google Scholar). The results NR1-NR1 intersubunit disulfide formation involving cysteine 79 is requisite for the trafficking of NR1/NR2 NMDA receptors to the cell is as yet disulfide bridging is a step in the formation of NR1-NR1 it the NR1-NR1 dimer formation, it in to NR1-NR1 to assembly with as by and Colquhoun S. Colquhoun D. J. Neurosci. PubMed Google or disulfide formation as a in the of functional NMDA receptors from the S. Nakanishi and M. for the of the NMDA receptor M. for the of the and of of for of the