Neuropharmacological studies of clustering molecules expressing in the excitatory synapses

人类已经克隆并鉴定了一种与大圆盘(DLG)肿瘤抑制蛋白同源的NE-DLG(神经元和内分泌-DLG)(K.Makino等，Oncogene，14,2425,1997)。NE-DLG也被称为SAP102，它与NMDA受体2B(NR2B)相互作用，在突触后密度聚集受体。我们首次报道了NE-DLG在体外与钙调蛋白和PSD-95相互作用(J.Biol.化学，274,5782,1999)。我们还在大脑中发现了一种相互作用的蛋白质p51-Nedasin。P51-Nedasin干扰NE-DLG和NR2B之间的联系(J.Biol.化学。274、32204、1999年)。接下来，我们在体外和原位研究了突触后密度组分和培养的大鼠海马神经元中CaM激酶II对NE-DLG的磷酸化作用。在海马长时程增强后，也观察到NE-DLG的磷酸化增加。CaM激酶抑制剂KN93可阻止磷酸化增加，但蛋白激酶C抑制剂不能阻止这种增加。我们评估了NE-DLG磷酸化的功能相关性。依赖于CaM-Il的NE-DLG的磷酸化阻止了其与NR2B的结合，但促进了NR2B和NR1在突触后密度中的积聚。关于NE-DLG的胞浆定位，CaM激酶II对NE-DLG的磷酸化可能参与了NR2B和/或NR1向突触后密度的运输。我们最近发现，CaM激酶II还参与了多巴胺D2受体从高尔基体到神经元质膜的运输，在这一过程中，像NE-DLG这样的支架蛋白可能是其运输机制的基础。

A human homologue to the discs large (dlg) tumor suppresser protein, NE-dlg (neuronal and endocrine-dlg) has been cloned and characterized (K. Makino et al., Oncogene, 14, 2425, 1997). NE-dlg, also named SAP102, interacts with the NMDA receptor 2B (NR2B) to cluster the receptors in the postsynaptic density. We first documented that NE-dlg interacts with calmodulin and PSD-95 in vitro (J. Biol. Chem., 274, 5782, 1999). We also found an interacting protein, p51-Nedasin, in the brain. The P51-Nedasin interferes the association between NE-dlg and NR2B (J. Biol. Chem. 274, 32204, 1999). We next demonstrated in vitro and in situ phosphorylation of NE-dlg by CaM kinase II in the postsynaptic density fractions and in cultured rat hippocampal neurons. An increased phosphorylation of NE-dlg was also observed following hippocampal long-term potentiation. The increased phosphorylation was prevented by treatment with KN93, a CaM kinase inhibitor but not by protein kinase C inhibitors. We assessed the functional relevance of NE-dlg phosphorylation. CaM kinase Il-dependent NE-dlg phosphorylation prevented its binding to NR2B but promoted the accumulation of NR2B and NR1 in the postsynaptic density. As concerning the cytoplasmic localization of NE-dlg, the phosphorylation of NE-dlg by CaM kinase II is possibly involved in the transport of NR2B and/or NR1 into the postsynaptic density. We recently found that CaM kinase II is also involved in transport of dopamine D2 receptors from Golgi apparatus to the plasma membrane in the neurons, in which a scaffold protein like NE-dlg may underlie its transport mechanisms.