Cellular Neurobiology and Neurophysiology Laboratory, Center for Applied Medical Research and University of Navarra, 31008 Pamplona, Spain.
J Neurosci. 2013 Feb 27;33(9):4151-64. doi: 10.1523/JNEUROSCI.2721-12.2013.
Selective control of receptor trafficking provides a mechanism for remodeling the receptor composition of excitatory synapses, and thus supports synaptic transmission, plasticity, and development. GluN3A (formerly NR3A) is a nonconventional member of the NMDA receptor (NMDAR) subunit family, which endows NMDAR channels with low calcium permeability and reduced magnesium sensitivity compared with NMDARs comprising only GluN1 and GluN2 subunits. Because of these special properties, GluN3A subunits act as a molecular brake to limit the plasticity and maturation of excitatory synapses, pointing toward GluN3A removal as a critical step in the development of neuronal circuitry. However, the molecular signals mediating GluN3A endocytic removal remain unclear. Here we define a novel endocytic motif (YWL), which is located within the cytoplasmic C-terminal tail of GluN3A and mediates its binding to the clathrin adaptor AP2. Alanine mutations within the GluN3A endocytic motif inhibited clathrin-dependent internalization and led to accumulation of GluN3A-containing NMDARs at the cell surface, whereas mimicking phosphorylation of the tyrosine residue promoted internalization and reduced cell-surface expression as shown by immunocytochemical and electrophysiological approaches in recombinant systems and rat neurons in primary culture. We further demonstrate that the tyrosine residue is phosphorylated by Src family kinases, and that Src-activation limits surface GluN3A expression in neurons. Together, our results identify a new molecular signal for GluN3A internalization that couples the functional surface expression of GluN3A-containing receptors to the phosphorylation state of GluN3A subunits, and provides a molecular framework for the regulation of NMDAR subunit composition with implications for synaptic plasticity and neurodevelopment.
选择性控制受体运输为重塑兴奋性突触的受体组成提供了一种机制，从而支持突触传递、可塑性和发育。GluN3A（以前称为 NR3A）是非经典的 NMDA 受体（NMDAR）亚基家族的成员，与仅由 GluN1 和 GluN2 亚基组成的 NMDAR 相比，GluN3A 赋予 NMDAR 通道较低的钙离子通透性和降低的镁敏感性。由于这些特殊性质，GluN3A 亚基作为分子制动器，限制兴奋性突触的可塑性和成熟，表明 GluN3A 的去除是神经元回路发育的关键步骤。然而，介导 GluN3A 内吞去除的分子信号仍不清楚。在这里，我们定义了一个新的内吞基序（YWL），该基序位于 GluN3A 的细胞质 C 端尾部内，介导其与网格蛋白衔接蛋白 AP2 的结合。GluN3A 内吞基序中的丙氨酸突变抑制网格蛋白依赖性内化，并导致含有 GluN3A 的 NMDAR 在内质网表面积累，而模拟酪氨酸残基的磷酸化则促进内化并减少细胞表面表达，如通过免疫细胞化学和电生理学方法在重组系统和原代培养的大鼠神经元中所示。我们进一步证明，该酪氨酸残基被Src 家族激酶磷酸化，并且 Src 激活限制神经元表面的 GluN3A 表达。总之，我们的结果确定了 GluN3A 内化的新分子信号，该信号将含有 GluN3A 的受体的功能性表面表达与 GluN3A 亚基的磷酸化状态联系起来，并为 NMDAR 亚基组成的调节提供了一个分子框架，对突触可塑性和神经发育具有重要意义。
