Al-Hallaq Rana A, Conrads Thomas P, Veenstra Timothy D, Wenthold Robert J
Laboratory of Neurochemistry, National Institute on Deafness and Other Communication Disorders, National Institutes of Health, Bethesda, Maryland 20892, USA.
J Neurosci. 2007 Aug 1;27(31):8334-43. doi: 10.1523/JNEUROSCI.2155-07.2007.
Subunit composition of NMDA receptors (NMDARs) determines a range of physiological properties, downstream signaling effects, and binding partners. Differential localization of NR2A- or NR2B-containing NMDARs within the neuron and subunit-specific protein associations may explain differences in NR2A and NR2B contributions to synaptic plasticity and excitotoxic cell death. This question is complicated by the existence of tri-heteromeric complexes (NR1/NR2A/NR2B). To date, no quantitative biochemical determinations have been made of the relative abundance of different NMDAR populations in intact hippocampus, the region extensively correlated with NMDAR-dependent long-term potentiation. We investigated subunit composition and subunit-specific interactions in CA1/CA2 of rat hippocampus. Using sequential immunoprecipitations to deplete either NR2B or NR2A, di-heteromeric NR1/NR2A and NR1/NR2B receptor populations were isolated from postnatal day 7 (P7) hippocampus and P42 and 6-month-old CA1/CA2. Quantitative Western blot analysis revealed that 60-70% of NR2A and 70-85% of NR2B subunits were associated in NR1/NR2A or NR1/NR2B di-heteromeric complexes. Isolated di-heteromeric receptor fractions were used to examine NR2A- or NR2B-specific interactions with synapse-associated proteins. Our results indicate that NR2A- or NR2B-containing NMDARs associate similarly with postsynaptic density-95 (PSD-95), synapse-associated protein 102, and PSD-93 at P42. However, NR2A-containing receptors coimmunoprecipitated a greater proportion of the synaptic proteins neuronal nitric oxide synthase, Homer, and beta-catenin. Finally, mass spectrometry analysis of isolated di-heteromeric receptors identified a novel NMDAR interactor, collapsin response mediator protein 2, which preferentially associates with NR2B-containing di-heteromeric NMDARs. In summary, in rat hippocampus, NR2A and NR2B exist primarily in di-heteromeric complexes that interact similarly with PSD-95-related proteins but are associated with different protein complexes.
N-甲基-D-天冬氨酸受体（NMDARs）的亚基组成决定了一系列生理特性、下游信号转导效应及结合伴侣。含NR2A或NR2B的NMDARs在神经元内的差异定位以及亚基特异性蛋白质关联，可能解释了NR2A和NR2B在突触可塑性和兴奋性毒性细胞死亡中作用的差异。由于三杂聚体复合物（NR1/NR2A/NR2B）的存在，这个问题变得复杂。迄今为止，尚未对完整海马体中不同NMDAR群体的相对丰度进行定量生化测定，海马体是与NMDAR依赖的长时程增强广泛相关的区域。我们研究了大鼠海马体CA1/CA2区的亚基组成和亚基特异性相互作用。利用连续免疫沉淀法去除NR2B或NR2A，从出生后第7天（P7）的海马体以及P42和6月龄的CA1/CA2区中分离出二聚体NR1/NR2A和NR1/NR2B受体群体。定量蛋白质免疫印迹分析显示，60 - 70%的NR2A亚基和70 - 85%的NR2B亚基存在于NR1/NR2A或NR1/NR2B二聚体复合物中。分离出的二聚体受体组分用于检测NR2A或NR2B与突触相关蛋白的特异性相互作用。我们的结果表明，在P42时，含NR2A或NR2B的NMDARs与突触后致密蛋白95（PSD - 95）、突触相关蛋白102和PSD - 93的结合情况相似。然而，含NR2A的受体共免疫沉淀出更大比例的突触蛋白神经元型一氧化氮合酶、Homer和β-连环蛋白。最后，对分离出的二聚体受体进行质谱分析，鉴定出一种新型的NMDAR相互作用蛋白——塌陷反应介导蛋白2，它优先与含NR2B的二聚体NMDARs结合。总之，在大鼠海马体中，NR2A和NR2B主要存在于二聚体复合物中，它们与PSD - 95相关蛋白的相互作用相似，但与不同的蛋白质复合物相关。
