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首页> 外文期刊>Proceedings of the National Academy of Sciences of the United States of America >Visualizing odorant receptor trafficking in living cells down to the single-molecule level
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Visualizing odorant receptor trafficking in living cells down to the single-molecule level

机译：可视化嗅觉受体在活细胞中的运输，直至单分子水平

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摘要

Despite the importance of trafficking for regulating G protein-coupled receptor signaling, for many members of the seven trans-membrane helix protein family, such as odorant receptors, little is known about this process in live cells. Here, the complete life cycle of the human odorant receptor OR17-40 was directly monitored in living cells by ensemble and single-molecule imaging, using a double-labeling strategy. While the overall, intracellulartrafficking of the receptor was visualized continuously by using a GFP tag, selective imaging of cell surface receptors was achieved by pulse-labeling an acyl carrier protein tag. We found that OR17-40 efficiently translocated to the plasma membrane only at low expression, whereas at higher biosynthesis the receptor accumulated in intracellular compartments. Receptors in the plasma membrane showed high turnover resulting from constitutive internalization along the clathrin pathway, even in the absence of ligand. Single-molecule microscopy allowed monitoring of the early, dynamic processes in odorant receptor signaling. Although mobile receptors initially diffused either freely or within domains of various sizes, binding of an agonist or an antagonist increased partitioning of receptors into small domains of ≈190 nm, which likely are precursors of clathrin-coated pits. The binding of a ligand, therefore, resulted in modulation of the continuous, constitutive internalization. After endocytosis, receptors were directed to early endosomes for recycling. This unique mechanism of continuous internalization and recycling of OR17-40 might be instrumental in allowing rapid recovery of odor perception.
机译：尽管运输对于调节G蛋白偶联受体信号传导的重要性，但对于七个跨膜螺旋蛋白家族的许多成员，例如气味受体，在活细胞中对该过程知之甚少。在这里，人类气味受体OR17-40的完整生命周期是通过双标记策略通过集成和单分子成像直接在活细胞中监测的。虽然使用GFP标签连续观察受体的整体细胞内运输，但通过脉冲标记酰基载体蛋白标签可以实现细胞表面受体的选择性成像。我们发现，OR17-40仅在低表达时才有效地转移到质膜，而在更高的生物合成中，受体在细胞内区室中积累。即使在没有配体的情况下，质膜中的受体也显示出由于沿网格蛋白途径的组成型内在化而导致的高转换。单分子显微镜可以监测气味受体信号的早期动态过程。尽管移动受体最初自由扩散或在各种大小的区域内扩散，但激动剂或拮抗剂的结合增加了受体向≈190nm小区域内的分配，这可能是网格蛋白包被的凹坑的前体。因此，配体的结合导致对连续的组成型内在化的调节。内吞后，受体被导向早期的内体以循环利用。 OR17-40持续内在化和再循环的独特机制可能有助于快速恢复气味感知。

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