Appropriate NR1-NR1 disulfide-linked homodimer formation is requisite for efficient expression of functional, cell surface N-methyl-D-aspartate NR1/NR2 receptors
Appropriate NR1-NR1 disulfide-linked homodimer formation is requisite for efficient expression of functional, cell surface N-methyl-D-aspartate NR1/NR2 receptors
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DOI：
10.1074/jbc.m313446200
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发表时间：
2004-04-09
影响因子：
4.8
通讯作者：
Stephenson, FA
中科院分区：
文献类型：
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作者：
Papadakis, M;Hawkins, LM;Stephenson, FA
产生c-Myc表位标记的N-甲基-D-天冬氨酸受体NR 1 -2a亚基，NR 1 -2a（c-Myc），其中标签插入在氨基酸81之后。NR 1 -2a（c-Myc）/NR 2A受体当在哺乳动物细胞中表达时不被运输到细胞表面，也不产生转染后的细胞毒性。然而，通过免疫沉淀和[H-3] MK 801放射性配体结合试验显示NR 1 -2a（c-Myc）与NR 2A亚基组装。用野生型NR 1 -2a/NR 2A亚基共转染的细胞的免疫印迹产生了两种分子量为115和226 kDa的NR 1 -2a免疫反应性物质。在非还原和还原条件下的双向电泳显示，226-kDa的条带含有二硫键连接的NR 1 -2a亚基。对于NR 1 -2a（c-Myc）/NR 2A，仅检测到115-kDa NR 1 -2a种类。c-Myc表位插入到NR 1 -2a亚基的半胱氨酸79附近;因此，该标签可能会阻止NR 1二硫键的形成。产生了一系列半胱氨酸->丙氨酸NR 1 -2a突变体，并且NR 1 -2a突变体与NR 2A或NR 2B亚基在哺乳动物细胞中共表达，并在细胞表面表达、转染后的细胞毒性、通过免疫沉淀的共缔合以及在还原和非还原条件下SDS-PAGE后的免疫印迹方面进行表征。当在哺乳动物细胞中与NR 2A共表达时，NR 1 -2a（C79 A）/NR 2A显示出与NR 1 -2a（c-Myc）/NR 2A相似的性质，即226-kDa NR 1免疫反应性物质不可检测，并且与野生型NR 1/NR 2受体相比，向细胞表面的运输受损。这些结果提供了第一个生化证据的形成NR 1-NR 1亚基间二硫键连接的同源二聚体涉及半胱氨酸79。他们认为，NR 1 N-末端结构域内的二硫键桥接和结构完整性是细胞表面N-甲基-D-天冬氨酸受体表达所必需的。
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 [H-3] 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.