Lin Da-Ting, Huganir Richard L
Department of Neuroscience, Johns Hopkins University School of Medicine, Howard Hughes Medical Institute, Baltimore, Maryland 21205, USA.
J Neurosci. 2007 Dec 12;27(50):13903-8. doi: 10.1523/JNEUROSCI.1750-07.2007.
Changes in surface trafficking of AMPA receptors play an important role in synaptic plasticity. Phosphorylation of the C terminus of the AMPA receptor (AMPAR) subunit glutamate receptor 2 (GluR2) and the binding of GluR2 to the PDZ [postsynaptic density-95/Discs large/zona occludens-1]-domain containing protein, protein interacting with protein kinase C (PICK1), have been proposed to play an important role in NMDA receptor dependent internalization of GluR2. However, the fate of internalized GluR2 after NMDA receptor (NMDAR) activation is still unclear. Both recycling and degradation of GluR2 after the activation of NMDAR have been reported. Here, we used a pH-sensitive green fluorescent protein variant, pHluorin, tagged to the N terminus of GluR2 (pH-GluR2) to study the dynamic internalization and recycling of GluR2 after NMDAR activation. Using fluorescence recovery after photobleach (FRAP), we directly demonstrate that internalized pH-GluR2 subunits recycle back to the cell surface after NMDAR activation. We further demonstrate that changing the phosphorylation state of the S880 residue at the C terminus of GluR2 does not affect NMDAR-dependent GluR2 internalization, but alters the recycling of GluR2 after NMDAR activation. In addition, mutation of the N-ethylmaleimide-sensitive fusion protein (NSF) binding site in the pH-GluR2 slows receptor recycling. Finally, neurons lacking PICK1 display normal NMDAR dependent GluR2 internalization compared with wild-type neurons, but demonstrate accelerated GluR2 recycling after NMDAR activation. These results indicate that phosphorylation of GluR2 S880 and NSF and PICK1 binding to GluR2 dynamically regulate GluR2 recycling.
AMPA 受体的表面转运变化在突触可塑性中起重要作用。有人提出，AMPA 受体（AMPAR）亚基谷氨酸受体 2（GluR2）的 C 末端磷酸化以及 GluR2 与含 PDZ[突触后致密物 95/盘状大蛋白/紧密连接蛋白 1]结构域的蛋白（即与蛋白激酶 C 相互作用的蛋白，PICK1）的结合，在 NMDA 受体依赖性的 GluR2 内化过程中起重要作用。然而，NMDA 受体（NMDAR）激活后内化的 GluR2 的去向仍不清楚。已有报道称 NMDAR 激活后 GluR2 会发生再循环和降解。在此，我们使用一种 pH 敏感的绿色荧光蛋白变体 pHluorin，将其标记在 GluR2 的 N 末端（pH-GluR2），以研究 NMDAR 激活后 GluR2 的动态内化和再循环。通过光漂白后荧光恢复（FRAP）技术，我们直接证明了 NMDAR 激活后内化的 pH-GluR2 亚基会再循环回到细胞表面。我们进一步证明，改变 GluR2 C 末端 S880 残基的磷酸化状态不会影响 NMDAR 依赖性的 GluR2 内化，但会改变 NMDAR 激活后 GluR2 的再循环。此外，pH-GluR2 中 N-乙基马来酰亚胺敏感融合蛋白（NSF）结合位点的突变会减缓受体的再循环。最后，与野生型神经元相比，缺乏 PICK1 的神经元在 NMDAR 依赖性的 GluR2 内化方面表现正常，但在 NMDAR 激活后显示出加速的 GluR2 再循环。这些结果表明，GluR2 S880 的磷酸化以及 NSF 和 PICK1 与 GluR2 的结合动态调节 GluR2 的再循环。
