Li Haiyan, Santos Magda S, Park Chihyung K, Dobry Yuriy, Voglmaier Susan M
Department of Psychiatry, School of Medicine, Weill Institute for Neurosciences, Kavli Institute for Fundamental Neuroscience, University of California, San Francisco, San Francisco, CA, United States.
Front Cell Neurosci. 2017 Oct 26;11:324. doi: 10.3389/fncel.2017.00324. eCollection 2017.
Release of the major excitatory neurotransmitter glutamate by synaptic vesicle exocytosis depends on glutamate loading into synaptic vesicles by vesicular glutamate transporters (VGLUTs). The two principal isoforms, VGLUT1 and 2, exhibit a complementary pattern of expression in adult brain that broadly distinguishes cortical (VGLUT1) and subcortical (VGLUT2) systems, and correlates with distinct physiological properties in synapses expressing these isoforms. Differential trafficking of VGLUT1 and 2 has been suggested to underlie their functional diversity. Increasing evidence suggests individual synaptic vesicle proteins use specific sorting signals to engage specialized biochemical mechanisms to regulate their recycling. We observed that VGLUT2 recycles differently in response to high frequency stimulation than VGLUT1. Here we further explore the trafficking of VGLUT2 using a pHluorin-based reporter, VGLUT2-pH. VGLUT2-pH exhibits slower rates of both exocytosis and endocytosis than VGLUT1-pH. VGLUT2-pH recycling is slower than VGLUT1-pH in both hippocampal neurons, which endogenously express mostly VGLUT1, and thalamic neurons, which endogenously express mostly VGLUT2, indicating that protein identity, not synaptic vesicle membrane or neuronal cell type, controls sorting. We characterize sorting signals in the C-terminal dileucine-like motif, which plays a crucial role in VGLUT2 trafficking. Disruption of this motif abolishes synaptic targeting of VGLUT2 and essentially eliminates endocytosis of the transporter. Mutational and biochemical analysis demonstrates that clathrin adaptor proteins (APs) interact with VGLUT2 at the dileucine-like motif. VGLUT2 interacts with AP-2, a well-studied adaptor protein for clathrin mediated endocytosis. In addition, VGLUT2 also interacts with the alternate adaptors, AP-1 and AP-3. VGLUT2 relies on distinct recycling mechanisms from VGLUT1. Abrogation of these differences by pharmacological and molecular inhibition reveals that these mechanisms are dependent on the adaptor proteins AP-1 and AP-3. Further, shRNA-mediated knockdown reveals differential roles for AP-1 and AP-3 in VGLUT2 recycling.
通过突触小泡胞吐作用释放主要兴奋性神经递质谷氨酸，这依赖于囊泡谷氨酸转运体（VGLUTs）将谷氨酸装载到突触小泡中。两种主要的亚型，VGLUT1和VGLUT2，在成人大脑中呈现互补的表达模式，这种模式大致区分了皮质（VGLUT1）和皮质下（VGLUT2）系统，并且与表达这些亚型的突触中不同的生理特性相关。有人提出VGLUT1和VGLUT2的差异运输是它们功能多样性的基础。越来越多的证据表明，单个突触小泡蛋白利用特定的分选信号参与专门的生化机制来调节它们的循环利用。我们观察到，与VGLUT1相比，VGLUT2在高频刺激下的循环利用方式不同。在这里，我们使用基于pHluorin的报告基因VGLUT2-pH进一步探究VGLUT2的运输。VGLUT2-pH的胞吐作用和内吞作用速率都比VGLUT1-pH慢。在主要内源性表达VGLUT1的海马神经元和主要内源性表达VGLUT2的丘脑神经元中，VGLUT2-pH的循环利用都比VGLUT1-pH慢，这表明是蛋白质特性，而非突触小泡膜或神经元细胞类型，控制着分选。我们对C末端双亮氨酸样基序中的分选信号进行了表征，该基序在VGLUT2运输中起关键作用。破坏这个基序会消除VGLUT2的突触靶向作用，并基本消除该转运体的内吞作用。突变和生化分析表明，网格蛋白衔接蛋白（APs）在双亮氨酸样基序处与VGLUT2相互作用。VGLUT2与AP-2相互作用，AP-2是一种研究充分的用于网格蛋白介导的内吞作用的衔接蛋白。此外，VGLUT2还与替代衔接蛋白AP-1和AP-3相互作用。VGLUT2依赖于与VGLUT1不同的循环利用机制。通过药理学和分子抑制作用消除这些差异表明，这些机制依赖于衔接蛋白AP-1和AP-3。此外，shRNA介导的敲低揭示了AP-1和AP-3在VGLUT2循环利用中的不同作用。
