DIFFERENTIAL TYROSINE PHOSPHORYLATION OF N-METHYL-D-ASPARTATE RECEPTOR SUBUNITS
DIFFERENTIAL TYROSINE PHOSPHORYLATION OF N-METHYL-D-ASPARTATE RECEPTOR SUBUNITS
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DOI：
10.1074/jbc.270.34.20036
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发表时间：
1995-08-25
影响因子：
4.8
通讯作者：
HUGANIR, RL
中科院分区：
文献类型：
--
作者：
LAU, LF;HUGANIR, RL
蛋白质酪氨酸磷酸化最近被认为在神经肌肉接头的突触传递中起重要作用。然而，酪氨酸磷酸化在中枢神经系统突触功能调节中的作用尚不清楚。在这项研究中，用抗磷酸酪氨酸抗体的免疫细胞化学染色表明，有高水平的磷酸酪氨酸，它与谷氨酸受体共定位在兴奋性突触培养的海马神经元。此外，使用亚基特异性抗体检查了谷氨酸受体的各种亚型的酪氨酸磷酸化。谷氨酸受体是中枢神经系统中主要的兴奋性神经递质受体，根据其电生理学和药理学特性，分为三大类：α-氨基-3-羟基-5-甲基-4-异恶唑丙酸盐、红藻氨酸盐和N-甲基-D-天冬氨酸（NMDA）受体。NMDA受体在突触可塑性、突触发生和兴奋性毒性中起核心作用，并且被认为是两种类型的亚基：NR 1和NR 2（A-D）亚基的异聚复合物。大鼠突触质膜洗涤剂提取物在抗磷酸酪氨酸抗体-琼脂糖上的免疫亲和层析表明，NR 2A和NR 2B亚基，而不是NR 1亚基是酪氨酸磷酸化的。相反，NR 1，NR 2A和NR 2B亚基与亚基特异性抗体的免疫沉淀，然后用抗磷酸酪氨酸抗体进行免疫印迹，证实了NR 2A和NR 2B亚基，而不是NR 1亚基在酪氨酸残基上磷酸化。未检测到AMPA（GluR 1 -4）和红藻氨酸（GluR 6/7，KA 2）受体亚基的酪氨酸磷酸化。据估计，2.1 +/- 1.3%的NR 2A亚基和3.6 +/- 2.4%的NR 2B亚基在体内仅仅是酪氨酸磷酸化。此外，突触质膜中的内源性蛋白酪氨酸激酶在体外磷酸化NR 2A亚基，使其磷酸化增加6-8倍，但不磷酸化NR 1或NR 2B。这些研究表明，NMDA受体亚基是差异酪氨酸磷酸化，并建议酪氨酸磷酸化的NR 2亚基可能是重要的调节NMDA受体功能。
Protein-tyrosine phosphorylation has recently been suggested to play an important role in synaptic transmission at the neuromuscular junction. The role of tyrosine phosphorylation in the modulation of synaptic function in the central nervous system, however, is not clear. In this study, immunocytochemical staining with an anti-phosphotyrosine antibody demonstrates that there are high levels of phosphotyrosine, which co localizes with glutamate receptors at excitatory synapses on cultured hippocampal neurons. In addition, the tyrosine phosphorylation of various subtypes of glutamate receptors were examined using subunit-specific antibodies. Glutamate receptors are the major excitatory neurotransmitter receptors in the central nervous system and are classified into three major classes: alpha-amino-3-hydroxy-5-methyl-4-isoxazole proprionate, kainate, and N-methyl-D-aspartate (NMDA) receptors, based on their electrophysiological and pharmacological properties. NMDA receptors play a central role in synaptic plasticity, synaptogenesis, and excitotoxicity and are thought to be heteromeric complexes of the two types of subunits: NR1 and NR2(A-D) subunits. Immunoaffinity chromatography of detergent extracts of rat synaptic plasma membranes on anti-phosphotyrosine antibody-agarose showed that the NR2A and NR2B subunits but not the NR1 subunit are tyrosine-phosphorylated. Conversely, immunoprecipitation of the NR1, NR2A, and NR2B subunits with subunit specific antibodies followed by immunoblotting with anti-phosphotyrosine antibodies confirmed that the NR2A and NR2B subunits but not the NR1 subunit were phosphorylated on tyrosine residues. No tyrosine phosphorylation of the AMPA (GluR1-4) and kainate (GluR6/7, KA2) receptor subunits was detected. It was estimated that 2.1 +/- 1.3% of the NR2A subunits and 3.6 +/- 2.4% of the NR2B subunits mere tyrosine-phosphorylated in vivo. In addition, endogenous protein-tyrosine kinases in synaptic plasma membranes phosphorylated the NR2A subunit in vitro, increasing its phosphorylation 6-8-foId but did not phosphorylate NR1 or NR2B. These studies demonstrate that NMDA receptor subunits are differentially tyrosine-phosphorylated and suggest that tyrosine phosphorylation of the NR2 subunits may be important for regulating NMDA receptor function.