Pharmacologie et Biochimie de la Synapse, CNRS UMR 8619, Institut de Biochimie et de Biophysique Moléculaire et Cellulaire, Univ. Paris-Sud, 91405 Orsay Cedex, France.
J Biol Chem. 2012 Jun 8;287(24):20176-86. doi: 10.1074/jbc.M112.347468. Epub 2012 Apr 23.
The eight pre- or/and post-synaptic metabotropic glutamatergic receptors (mGluRs) modulate rapid excitatory transmission sustained by ionotropic receptors. They are classified in three families according to their percentage of sequence identity and their pharmacological properties. mGluR4 belongs to group III and is mainly localized presynaptically. Activation of group III mGluRs leads to depression of excitatory transmission, a process that is exclusively provided by mGluR4 at parallel fiber-Purkinje cell synapse in rodent cerebellum. This function relies at least partly on an inhibition of presynaptic calcium influx, which controls glutamate release. To improve the understanding of molecular mechanisms of the mGluR4 depressant effect, we decided to identify the proteins interacting with this receptor. Immunoprecipitations using anti-mGluR4 antibodies were performed with cerebellar extracts. 183 putative partners that co-immunoprecipitated with anti-mGluR4 antibodies were identified and classified according to their cellular functions. It appears that native mGluR4 interacts with several exocytosis proteins such as Munc18-1, synapsins, and syntaxin. In addition, native mGluR4 was retained on a Sepharose column covalently grafted with recombinant Munc18-1, and immunohistochemistry experiments showed that Munc18-1 and mGluR4 colocalized at plasma membrane in HEK293 cells, observations in favor of an interaction between the two proteins. Finally, affinity chromatography experiments using peptides corresponding to the cytoplasmic domains of mGluR4 confirmed the interaction observed between mGluR4 and a selection of exocytosis proteins, including Munc18-1. These results could give indications to explain how mGluR4 can modulate glutamate release at parallel fiber-Purkinje cell synapses in the cerebellum in addition to the inhibition of presynaptic calcium influx.
八种类别（或前或后）的突触型代谢型谷氨酸受体（mGluRs）调节由离子型受体维持的快速兴奋传递。根据其序列同一性的百分比和药理学特性，它们被分为三个家族。mGluR4 属于第三类，主要定位于突触前。激活第三类 mGluRs 导致兴奋传递的抑制，这个过程仅由鼠类小脑浦肯野细胞平行纤维突触的 mGluR4 提供。该功能至少部分依赖于抑制突触前钙离子内流，这控制谷氨酸的释放。为了提高对 mGluR4 抑制剂作用的分子机制的理解，我们决定鉴定与该受体相互作用的蛋白质。用抗 mGluR4 抗体进行小脑提取物的免疫沉淀。鉴定出 183 种与抗 mGluR4 抗体共沉淀的假定伙伴，并根据其细胞功能进行分类。似乎天然的 mGluR4 与几种胞吐蛋白相互作用，如 Munc18-1、突触素和突触小泡相关蛋白。此外，天然 mGluR4 保留在与重组 Munc18-1 共价接枝的 Sepharose 柱上，免疫组织化学实验显示 Munc18-1 和 mGluR4 在 HEK293 细胞的质膜上共定位，这支持两种蛋白质之间的相互作用。最后，使用对应于 mGluR4 细胞质结构域的肽的亲和层析实验证实了观察到的 mGluR4 与包括 Munc18-1 在内的一系列胞吐蛋白之间的相互作用。这些结果可以为解释 mGluR4 如何除了抑制突触前钙离子内流外，还可以调节小脑浦肯野细胞平行纤维突触的谷氨酸释放提供线索。
