Lau L F, Huganir R L
Department of Neuroscience, Howard Hughes Medical Institute, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA.
J Biol Chem. 1995 Aug 25;270(34):20036-41. doi: 10.1074/jbc.270.34.20036.
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 were 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-fold 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.
最近有人提出，蛋白酪氨酸磷酸化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。在本研究中，用抗磷酸酪氨酸抗体进行免疫细胞化学染色表明，在培养的海马神经元的兴奋性突触处，存在高水平的磷酸酪氨酸，其与谷氨酸受体共定位。此外，使用亚基特异性抗体检测了谷氨酸受体各亚型的酪氨酸磷酸化。谷氨酸受体是中枢神经系统中的主要兴奋性神经递质受体，根据其电生理和药理特性可分为三大类：α-氨基-3-羟基-5-甲基-4-异恶唑丙酸、海人酸和N-甲基-D-天冬氨酸（NMDA）受体。NMDA受体在突触可塑性、突触形成和兴奋毒性中起核心作用，被认为是由两种亚基组成的异聚体复合物：NR1和NR2（A-D）亚基。用抗磷酸酪氨酸抗体-琼脂糖对大鼠突触质膜的去污剂提取物进行免疫亲和层析表明，NR2A和NR2B亚基而非NR1亚基发生了酪氨酸磷酸化。相反，用亚基特异性抗体对NR1、NR2A和NR2B亚基进行免疫沉淀，然后用抗磷酸酪氨酸抗体进行免疫印迹，证实NR2A和NR2B亚基而非NR1亚基在酪氨酸残基上发生了磷酸化。未检测到AMPA（GluR1-4）和海人酸（GluR6/7、KA2）受体亚基的酪氨酸磷酸化。据估计，体内2.1±1.3%的NR2A亚基和3.6±2.4%的NR2B亚基发生了酪氨酸磷酸化。此外，突触质膜中的内源性蛋白酪氨酸激酶在体外使NR2A亚基磷酸化，使其磷酸化增加6-8倍，但未使NR1或NR2B磷酸化。这些研究表明，NMDA受体亚基的酪氨酸磷酸化存在差异，提示NR2亚基的酪氨酸磷酸化可能对调节NMDA受体功能很重要。
