Ritter-Makinson Stefanie L, Paquet Maryse, Bogenpohl James W, Rodin Rachel E, Chris Yun C, Weinman Edward J, Smith Yoland, Hall Randy A
Department of Pharmacology, Emory University School of Medicine, Atlanta, GA 30322, USA.
Yerkes National Primate Research Center, Emory University, Atlanta, GA 30329, USA.
Neuroscience. 2017 Jun 14;353:58-75. doi: 10.1016/j.neuroscience.2017.03.060. Epub 2017 Apr 7.
The group II metabotropic glutamate receptors mGluR2 and mGluR3 are key modulators of glutamatergic neurotransmission. In order to identify novel Group II metabotropic glutamate receptor (mGluR)-interacting partners, we screened the C-termini of mGluR2 and mGluR3 for interactions with an array of PDZ domains. These screens identified the Na+/H+ exchanger regulatory factors 1 and 2 (NHERF-1 & -2) as candidate interacting partners. Follow-up co-immunoprecipitation studies demonstrated that both mGluR2 and mGluR3 can associate with NHERF-1 and NHERF-2 in a cellular context. Functional studies revealed that disruption of PDZ interactions with mGluR2 enhanced receptor signaling to Akt. However, further studies of mGluR2 and mGluR3 signaling in astrocytes in which NHERF expression was reduced by gene knockout (KO) and/or siRNA knockdown techniques revealed that the observed differences in signaling between WT and mutant mGluR2 were likely not due to disruption of interactions with the NHERF proteins. Electron microscopic analyses revealed that Group II mGluRs were primarily expressed in glia and unmyelinated axons in WT, NHERF-1 and NHERF-2 KO mice, but the relative proportion of labeled axons over glial processes was higher in NHERF-2 KO mice than in controls and NHERF-1 KO mice. Interestingly, our anatomical studies also revealed that loss of either NHERF protein results in ventriculomegaly, which may be related to the high incidence of hydrocephaly that has previously been observed in NHERF-1 KO mice. Together, these studies support a role for NHERF-1 and NHERF-2 in regulating the distribution of Group II mGluRs in the murine brain, while conversely the effects of the mGluR2/3 PDZ-binding motifs on receptor signaling are likely mediated by interactions with other PDZ scaffold proteins beyond the NHERF proteins.
II 型代谢型谷氨酸受体 mGluR2 和 mGluR3 是谷氨酸能神经传递的关键调节因子。为了鉴定与 II 型代谢型谷氨酸受体（mGluR）相互作用的新伙伴，我们筛选了 mGluR2 和 mGluR3 的 C 末端与一系列 PDZ 结构域的相互作用。这些筛选确定了钠/氢交换调节因子 1 和 2（NHERF-1 和 -2）作为候选相互作用伙伴。后续的共免疫沉淀研究表明，在细胞环境中，mGluR2 和 mGluR3 都可以与 NHERF-1 和 NHERF-2 结合。功能研究表明，破坏 PDZ 与 mGluR2 的相互作用会增强受体向 Akt 的信号传导。然而，通过基因敲除（KO）和/或 siRNA 敲低技术降低 NHERF 表达的星形胶质细胞中 mGluR2 和 mGluR3 信号传导的进一步研究表明，野生型和突变型 mGluR2 之间观察到的信号差异可能不是由于与 NHERF 蛋白相互作用的破坏。电子显微镜分析表明，在野生型、NHERF-1 和 NHERF-2 基因敲除小鼠中，II 型 mGluRs 主要表达于神经胶质细胞和无髓轴突，但与对照和 NHERF-1 基因敲除小鼠相比，NHERF-2 基因敲除小鼠中标记轴突与神经胶质细胞突起的相对比例更高。有趣的是，我们的解剖学研究还表明，任何一种 NHERF 蛋白的缺失都会导致脑室扩大，这可能与先前在 NHERF-1 基因敲除小鼠中观察到的高脑积水发生率有关。总之，这些研究支持 NHERF-1 和 NHERF-2 在调节小鼠脑中 II 型 mGluRs 分布方面的作用，而相反，mGluR2/3 PDZ 结合基序对受体信号传导的影响可能是由与 NHERF 蛋白以外的其他 PDZ 支架蛋白的相互作用介导的。
