OPTIMAL EXPRESSION OF CLONED NMDAR1 NMDAR2A HETEROMERIC GLUTAMATE RECEPTORS - A BIOCHEMICAL-CHARACTERIZATION
OPTIMAL EXPRESSION OF CLONED NMDAR1 NMDAR2A HETEROMERIC GLUTAMATE RECEPTORS - A BIOCHEMICAL-CHARACTERIZATION
复制标题
DOI：
10.1042/bj2960877
复制
发表时间：
1993-12-15
影响因子：
4.1
通讯作者：
STEPHENSON, FA
中科院分区：
文献类型：
--
作者：
CIK, M;CHAZOT, PL;STEPHENSON, FA
N-甲基-D-天冬氨酸R1（NMDAR 1）和NMDAR 2A亚基在人胚肾（HEK）293细胞中单独或组合瞬时表达。用新开发的抗NMDAR 2A-（1435-1445）抗体和[H-3] MK 801放射性配体结合活性的免疫学方法，将克隆受体的生化和药理学性质与成年大鼠脑NMDA受体的生化和药理学性质进行比较。抗NMDAR 2A-（1435-1445）抗体在大鼠脑中特异性识别四种免疫反应性物质，其M（r）分别为180000、122000、97000和54000，而在单独转染质粒pCISNMDAR 2A的HEK 293细胞中仅识别一条M（r）180000的条带。HEK细胞膜的N-去糖基化产生了165000-M的免疫反应性物质，这与从成熟NMDAR 2A亚基的cDNA序列预测的大小一致。NMDAR 1和NMDAR 2A亚基在HEK 293细胞中的共表达导致细胞死亡。因此，建立了异聚体受体在活细胞中最佳表达的条件，包括转染后培养基中需要DL-2-氨基-5-膦酰基戊酸（AP 5）。与单亚基表达相比，用pCISNMDAR 1和pCISNMDAR 2A组合转染的细胞产生了10倍的[H-3] MK 801结合位点的数量增加。MK 801对表达的受体具有与成年大鼠和小鼠脑中发现的受体相似的亲和力。这些结果表明，NMDAR 1和NMDAR 2A受体亚基共组装形成异聚复合物，其性质与成年哺乳动物前脑的天然受体的性质相似。此外，最大瞬时表达的条件报告提供了进一步的结构活性研究的基础。
The N-methyl-D-aspartate R1 (NMDAR1) and NMDAR2A subunits were expressed transiently either alone or in combination in human embryonic kidney (HEK) 293 cells. The biochemical and pharmacological properties of the cloned receptors were compared with those of adult rat brain NMDA receptors using both immunological methods with a newly developed anti-NMDAR2A-(1435-1445) antibody and [H-3]MK801 radioligand binding activity. Anti-NMDAR2A-(1435-1445) antibodies recognized specifically four immunoreactive species with M(r)s of 180000, 122000, 97000 and 54000 in rat brain, but only a single band of M(r) 180000 in HEK 293 cells singly transfected with plasmid pCISNMDAR2A. N-deglycosylation of HEK cell membranes yielded a 165000-M, immunoreactive species, which is in agreement with the size predicted from the cDNA sequence for the mature NMDAR2A subunit. Co-expression of NMDAR1 and NMDAR2A subunits in HEK 293 cells resulted in cell death. Thus conditions were established for the optimum expression of heteromeric receptors in viable cells, including a requirement for DL-2-amino-5-phosphonopentanoic acid (AP5) in the culture medium post-transfection. Cells transfected with pCISNMDAR1 and pCISNMDAR2A combined yielded a 10-fold increase in the number of [H-3]MK801 binding sites compared with single subunit expression. MK801 had similar affinity for the expressed receptors as for those found in adult rat and mouse brain. These results demonstrate that the NMDAR1 and NMDAR2A receptor subunits co-assemble to form a heteromeric complex with properties similar to those of the native receptors of adult mammalian forebrain. Furthermore, the conditions reported for maximal transient expression provide a basis for further structure-activity studies.