Characterization of Fyn-mediated tyrosine phosphorylation sites on GluRε2 (NR2B) subunit of the N-methyl-D-aspartate receptor
Characterization of Fyn-mediated tyrosine phosphorylation sites on GluRε2 (NR2B) subunit of the N-methyl-D-aspartate receptor
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DOI：
10.1074/jbc.m008085200
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发表时间：
2001-01-05
影响因子：
4.8
通讯作者：
Yamamoto, T
中科院分区：
文献类型：
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作者：
Nakazawa, T;Komai, S;Yamamoto, T
N-甲基-D-天冬氨酸（NMDA）受体在突触可塑性、神经元发育和兴奋性毒性中起关键作用，Src家族酪氨酸激酶如Fyn对NMDA受体的酪氨酸磷酸化与突触可塑性有关。为了更精确地阐明NMDA受体酪氨酸磷酸化的作用，我们在NMDA受体的GluR ε 2（NR 2B）亚基上鉴定了Fyn介导的磷酸化位点。在体外，GluR ε 2的C-末端胞质区域的25个酪氨酸残基中有7个被Fyn磷酸化。在这7个残基中，GluR ε 2中的Tyr-1252、Tyr-1336和Tyr-1472在与活性Fyn共表达时在人胚肾成纤维细胞中被磷酸化，并且Tyr-1472是该系统中的主要磷酸化位点。然后，我们产生了特异于Tyr-1472-磷酸化GluR ε 2的兔多克隆抗体，并使用该抗体显示GluR ε 2的Tyr-1472确实在小鼠脑中被磷酸化。重要的是，Tyr-1472磷酸化在fyn突变小鼠中大大降低。此外，当海马长时程增强开始观察时，Tyr-1472磷酸化变得明显，并且其幅度在小鼠脑中变得更大。最后，在海马CA 1区诱导长时程增强后，Tyr-1472磷酸化显著增强。这些数据表明GluR ε 2的Tyr-1472磷酸化对于突触可塑性是重要的。
The N-methyl-D-aspartate (NMDA) receptors play critical roles in synaptic plasticity, neuronal development, and excitotoxicity, Tyrosine phosphorylation of NMDA receptors by Src-family tyrosine kinases such as Fyn is implicated in synaptic plasticity. To precisely address the roles of NMDA receptor tyrosine phosphorylation, we identified Fyn-mediated phosphorylation sites on the GluR epsilon2 (NR2B) subunit of NMDA receptors, Seven out of 25 tyrosine residues in the C-terminal cytoplasmic region of GluR epsilon2 were phosphorylated by Fyn in vitro. Of these 7 residues, Tyr-1252, Tyr-1336, and Tyr-1472 in GluR epsilon2 were phosphorylated in human embryonic kidney fibroblasts when co-expressed with active Fyn, and Tyr-1472 was the major phosphorylation site in this system. We then generated rabbit polyclonal antibodies specific to Tyr-1472-phosphorylated GluR epsilon2 and showed that Tyr-1472 of GluR epsilon2 was indeed phosphorylated in murine brain using the antibodies. Importantly Tyr-1472 phosphorylation was greatly reduced in fyn mutant mice. Moreover, Tyr-1472 phosphorylation became evident when hippocampal long term potentiation started to be observed, and its magnitude became larger in murine brain. Finally, Tyr-1472 phosphorylation was significantly enhanced after induction of long term potentiation in the hippocampal CA1 region. These data suggest that Tyr-1472 phosphorylation of GluR epsilon2 is important for synaptic plasticity.