Yan Sheng, Sanders James M, Xu Jian, Zhu Yongling, Contractor Anis, Swanson Geoffrey T
Department of Pharmacology and Toxicology, University of Texas Medical Branch, Galveston, Texas 77555, USA.
J Neurosci. 2004 Jan 21;24(3):679-91. doi: 10.1523/JNEUROSCI.4985-03.2004.
Intracellular trafficking of ionotropic glutamate receptors is regulated predominantly by determinants in the cytoplasmic C-terminal domain of the subunit proteins. Although AMPA receptors are found at the vast majority of excitatory synapses, synaptic kainate receptors exhibit a much more restricted distribution, suggesting that specific mechanisms exist for selective trafficking of these receptor proteins. In this report, we define a critical forward trafficking motif that is necessary for surface expression of the glutamate receptor 6 (GluR6) kainate receptor as well as chimeric proteins containing only the GluR6 C-terminal domain. The trafficking determinant was identified by tracking surface expression of green fluorescent protein-tagged GluR6 receptors with confocal immunofluorescence in COS-7 cells and cultured neurons and patch-clamp electrophysiology in human embryonic kidney 293 cells. Serial truncation and alanine site mutagenesis of the GluR6 subunit C terminus localized the critical motif to a seven amino acid stretch of predominantly basic residues. Alanine mutation of the trafficking motif reduced kainate receptor current amplitudes by >90% and resulted in retention of the mutated receptors in the endoplasmic reticulum. This forward trafficking domain is the first such identified for kainate receptors.
离子型谷氨酸受体的细胞内运输主要受亚基蛋白胞质C末端结构域中的决定因素调控。虽然在绝大多数兴奋性突触中都能发现AMPA受体，但突触海人酸受体的分布则更为局限，这表明存在特定机制来选择性运输这些受体蛋白。在本报告中，我们定义了一个关键的正向运输基序，它对于谷氨酸受体6（GluR6）海人酸受体以及仅包含GluR6 C末端结构域的嵌合蛋白的表面表达是必需的。通过共聚焦免疫荧光在COS-7细胞和培养神经元中追踪绿色荧光蛋白标记的GluR6受体的表面表达，并在人胚肾293细胞中进行膜片钳电生理实验，确定了运输决定因素。对GluR6亚基C末端进行系列截短和丙氨酸位点诱变，将关键基序定位到一段主要由碱性残基组成的七个氨基酸片段。运输基序的丙氨酸突变使海人酸受体电流幅度降低>90%，并导致突变受体在内质网中滞留。这个正向运输结构域是首次为海人酸受体鉴定出的此类结构域。
