Blahos J, Wenthold R J
Laboratory of Neurochemistry, NIDCD, National Institutes of Health, Bethesda, Maryland 20892, USA.
J Biol Chem. 1996 Jun 28;271(26):15669-74. doi: 10.1074/jbc.271.26.15669.
The N-methyl-D-aspartate (NMDA) subtype of ionotropic glutamate receptor is assembled from one NR1 subunit, expressed in eight splice variants, and four NR2 subunits (NR2A-D). The combination of subunits and splice variants determines the pharmacological and physiological properties of the receptor. In the present study we investigated the relationship between NR1 splice variants and NR2 subunits in rat brain using a series of antibodies selective for the four NR1 cassettes, which vary in the NR1 splice variants, and for NR2A and NR2B. Sodium deoxycholate at pH 9.0 solubilized about 35% of the receptor, which was intact based on co-immunoprecipitation of NR1 and NR2 subunits and chemical cross-linking of the solubilized receptor. The cross-linked product contained three high molecular weight components, Mr = 603,000, 700,000, and 750,000, which were immunolabeled with antibodies to NR1 and to NR2 subunits. Immunoprecipitation analyses using antibodies selective for NR2A and NR2B showed no preferential assembly between NR2 subunits and NR1 splice variants. There was little co-immunoprecipitation of NR2A and NR2B, suggesting that most NMDA receptor complexes contain only one of these subunits. However, receptor complexes can contain at least two different NR1 splice variants. In developing conditions for the solubilization of intact NMDA receptor complexes, we observed a differential solubilization of NR1 and NR2 subunits. NR2 was nearly insoluble in Triton X-100 in both microsomal and synaptic membrane fractions, while NR1 was readily soluble in the microsomal fraction but insoluble in the synaptic membrane fraction. These results suggest that the NR1 subunit is modified when it is incorporated into the synaptic membrane, possibly by strengthening its interaction with NR2 or another synaptic protein.
离子型谷氨酸受体的N-甲基-D-天冬氨酸（NMDA）亚型由一个以八种剪接变体形式表达的NR1亚基和四个NR2亚基（NR2A-D）组装而成。亚基和剪接变体的组合决定了受体的药理和生理特性。在本研究中，我们使用一系列对四个NR1结构域具有选择性的抗体（这些结构域在NR1剪接变体中有所不同）以及针对NR2A和NR2B的抗体，研究了大鼠脑中NR1剪接变体与NR2亚基之间的关系。pH 9.0的脱氧胆酸钠溶解了约35%的受体，基于NR1和NR2亚基的共免疫沉淀以及溶解受体的化学交联，该受体是完整的。交联产物包含三个高分子量组分，分子量分别为603,000、700,000和750,000，它们用针对NR1和NR2亚基的抗体进行免疫标记。使用针对NR2A和NR2B的选择性抗体进行的免疫沉淀分析表明，NR2亚基与NR1剪接变体之间没有优先组装。NR2A和NR2B几乎没有共免疫沉淀，这表明大多数NMDA受体复合物仅包含这些亚基中的一个。然而，受体复合物可以包含至少两种不同的NR1剪接变体。在完整NMDA受体复合物溶解的条件下，我们观察到NR1和NR2亚基的溶解差异。在微粒体和突触膜组分中，NR2在Triton X-100中几乎不溶，而NR1在微粒体组分中易于溶解，但在突触膜组分中不溶。这些结果表明，NR1亚基在整合到突触膜中时会发生修饰，可能是通过加强其与NR2或另一种突触蛋白的相互作用。
