Regulation of P2X4 receptors by lysosomal targeting, glycan protection and exocytosis
Regulation of P2X4 receptors by lysosomal targeting, glycan protection and exocytosis
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DOI：
10.1242/jcs.010348
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发表时间：
2007-11-01
影响因子：
4
通讯作者：
Murrell-Lagnado, Ruth D.
中科院分区：
文献类型：
--
作者：
Qureshi, Omar S.;Paramasivam, Anbalakan;Murrell-Lagnado, Ruth D.
P2 X（4）受体广泛分布于中枢神经系统和外周，在免疫细胞和血管系统的功能中发挥重要作用。其在小胶质细胞中的上调有助于神经损伤后的神经病理性疼痛。虽然我们以前已经证明，它是组成性地从质膜中检索，并主要驻留在细胞内隔室中，但其调节机制还不清楚。在这里，我们发现，在培养的大鼠小胶质细胞，血管内皮细胞和新鲜分离的腹腔巨噬细胞的内源性P2 X（4）受体主要定位于溶酶体。溶酶体靶向介导的N-末端内的二亮氨酸型基序，连同先前表征的酪氨酸为基础的内吞基序内的C-末端。P2 X（4）受体在溶酶体的蛋白水解环境中保持稳定，并凭借其N-连接聚糖抵抗降解。吞噬作用的刺激触发了P2 X（4）受体在吞噬体膜上的积累。刺激溶酶体胞吐作用，无论是通过与钙离子载体离子霉素孵育，对于正常大鼠肾（NRK）细胞和培养的大鼠小胶质细胞，或弱碱性甲胺，对于腹腔巨噬细胞，引起细胞表面的P2 X（4）受体和溶酶体蛋白LAMP-1的上调。巨噬细胞中的溶酶体胞吐作用增强ATP诱发的跨质膜P2 X（4）受体电流。综上所述，我们的数据表明，P2 X（4）受体保留其在溶酶体降解环境中的功能，随后可以运输出溶酶体，以上调其在细胞表面和吞噬体的暴露。
The P2X(4) receptor has a widespread distribution in the central nervous system and the periphery, and plays an important role in the function of immune cells and the vascular system. Its upregulation in microglia contributes to neuropathic pain following nerve injury. The mechanisms involved in its regulation are not well understood, although we have previously shown that it is constitutively retrieved from the plasma membrane and resides predominantly within intracellular compartments. Here, we show that the endogenous P2X(4) receptors in cultured rat microglia, vascular endothelial cells and freshly isolated peritoneal macrophages are localized predominantly to lysosomes. Lysosomal targeting was mediated through a dileucine-type motif within the N-terminus, together with a previously characterized tyrosine-based endocytic motif within the C-terminus. P2X(4) receptors remained stable within the proteolytic environment of the lysosome and resisted degradation by virtue of their N-linked glycans. Stimulation of phagocytosis triggered the accumulation of P2X(4) receptors at the phagosome membrane. Stimulating lysosome exocytosis, either by incubating with the Ca2+ ionophore ionomycin, for normal rat kidney (NRK) cells and cultured rat microglia, or the weak base methylamine, for peritoneal macrophages, caused an upregulation of both P2X(4) receptors and the lysosomal protein LAMP-1 at the cell surface. Lysosome exocytosis in macrophages potentiated ATP-evoked P2X(4) receptor currents across the plasma membrane. Taken together, our data suggest that the P2X(4) receptor retains its function within the degradative environment of the lysosome and can subsequently traffic out of lysosomes to upregulate its exposure at the cell surface and phagosome.