Endocytosis of the Neuronal Glycine Transporter GLYT2: Role of Membrane Rafts and Protein Kinase C-Dependent Ubiquitination
Endocytosis of the Neuronal Glycine Transporter GLYT2: Role of Membrane Rafts and Protein Kinase C-Dependent Ubiquitination
复制标题
DOI：
10.1111/j.1600-0854.2011.01278.x
复制
发表时间：
2011-12-01
期刊：
影响因子：
4.5
通讯作者：
Aragon, Carmen
中科院分区：
文献类型：
--
作者：
de Juan-Sanz, Jaime;Zafra, Francisco;Aragon, Carmen
甘氨酸能神经传递由钠和氯依赖的质膜转运体终止。神经元甘氨酸转运蛋白2(GLYT2)为终末提供底物，以补充含有甘氨酸的突触小泡。这一关键过程在人类亢进中是有缺陷的，这种情况可能是由GLYT2突变引起的。抑制的甘氨酸能神经传递受GLYT2胞吐/内吞平衡的调节，尽管这种转运蛋白周转的机制仍然不清楚。我们研究了GLYT2的内化途径，以及转运蛋白的泛素化和膜筏结合在其内吞作用中的作用。利用药理学工具、显性-负性突变体和小干扰RNA，我们证明了笼蛋白介导的途径是异种细胞和神经元结构性和调节性GLYT2内吞的主要机制。我们发现GLYT2从细胞表面的脂筏中结构性地内化，在亚细胞循环结构中仍然与脂筏相关。蛋白激酶C(PKC)通过快速、动态地将GLYT2从RAFT重新分布到非RAFT膜亚区，增加泛素化的GLYT2内吞作用，对GLYT2进行负性调节。这种双相机制是调节GLYT2行为和抑制甘氨酸能神经传递的一种通用手段。这些发现可能揭示新的治疗靶点，以解决与GLYT2转运改变相关的甘氨酸能病理。
Glycinergic neurotransmission is terminated by sodium- and chloride-dependent plasma membrane transporters. The neuronal glycine transporter 2 (GLYT2) supplies the terminal with substrate to refill synaptic vesicles containing glycine. This crucial process is defective in human hyperekplexia, a condition that can be caused by mutations in GLYT2. Inhibitory glycinergic neurotransmission is modulated by the GLYT2 exocytosis/endocytosis equilibrium, although the mechanisms underlying the turnover of this transporter remain elusive. We studied GLYT2 internalization pathways and the role of ubiquitination and membrane raft association of the transporter in its endocytosis. Using pharmacological tools, dominant-negative mutants and small-interfering RNAs, we show that the clathrin-mediated pathway is the primary mechanism for constitutive and regulated GLYT2 endocytosis in heterologous cells and neurons. We show that GLYT2 is constitutively internalized from cell surface lipid rafts, remaining associated with rafts in subcellular recycling structures. Protein kinase C (PKC) negatively modulates GLYT2 via rapid and dynamic redistribution of GLYT2 from raft to non-raft membrane subdomains and increasing ubiquitinated GLYT2 endocytosis. This biphasic mechanism is a versatile means to modulate GLYT2 behavior and hence, inhibitory glycinergic neurotransmission. These findings may reveal new therapeutic targets to address glycinergic pathologies associated with alterations in GLYT2 trafficking.