A cleavable N-terminal signal peptide promotes widespread olfactory receptor surface expression in HEK293T cells.
A cleavable N-terminal signal peptide promotes widespread olfactory receptor surface expression in HEK293T cells.
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DOI：
10.1371/journal.pone.0068758
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发表时间：
2013
期刊：
影响因子：
3.7
通讯作者：
Pluznick JL
中科院分区：
文献类型：
--
作者：
Shepard BD;Natarajan N;Protzko RJ;Acres OW;Pluznick JL
嗅觉受体（ORs）是G蛋白偶联受体，能在嗅上皮中检测气味分子，并且是基因组中最大的基因家族。OR配体的鉴定通常需要OR在异源细胞中的表面表达；然而，当外源表达时，ORs很少运输到细胞表面。因此，大多数ORs是孤儿受体，没有已知的配体。到目前为止，研究已经利用不可切割的视紫红质（Rho）标签和/或分子伴侣（即受体转运蛋白，RTP1S，Ric8b和Gαolf）来改善表面表达。然而，即使有这些工具，许多ORs仍然无法到达细胞表面。我们使用了一组15个ORs来检测一种可切割的富含亮氨酸的信号肽序列（Lucy标签）对HEK293T细胞中OR表面表达的影响。我们在此报告，在N端添加Lucy标签使到达细胞表面的ORs数量在15个ORs中增加到7个（相比之下，没有Rho或Lucy标签时为3/15）。此外，当同时带有Lucy和Rho标签的ORs与先前报道的分子伴侣（RTP1S、Ric8b和Gαolf）共表达时，我们观察到所检测的所有15个受体都有表面表达。事实上，即使去除Rho标签，三分之二带有Lucy标签的ORs也能够与分子伴侣协同到达细胞表面（10/15个ORs），这使得仅在成熟蛋白上带有一个8个氨基酸的Flag标签就有可能评估OR的功能。正如对一个信号肽所预期的那样，Lucy标签从成熟蛋白上被切割下来，并且没有改变OR - 配体的结合和信号传导。我们的研究表明，在HEK293T细胞中可以实现ORs的广泛表面表达，为未来大规模的去孤儿化研究提供了希望。
Olfactory receptors (ORs) are G protein-coupled receptors that detect odorants in the olfactory epithelium, and comprise the largest gene family in the genome. Identification of OR ligands typically requires OR surface expression in heterologous cells; however, ORs rarely traffic to the cell surface when exogenously expressed. Therefore, most ORs are orphan receptors with no known ligands. To date, studies have utilized non-cleavable rhodopsin (Rho) tags and/or chaperones (i.e. Receptor Transporting Protein, RTP1S, Ric8b and Gαolf) to improve surface expression. However, even with these tools, many ORs still fail to reach the cell surface. We used a test set of fifteen ORs to examine the effect of a cleavable leucine-rich signal peptide sequence (Lucy tag) on OR surface expression in HEK293T cells. We report here that the addition of the Lucy tag to the N-terminus increases the number of ORs reaching the cell surface to 7 of the 15 ORs (as compared to 3/15 without Rho or Lucy tags). Moreover, when ORs tagged with both Lucy and Rho were co-expressed with previously reported chaperones (RTP1S, Ric8b and Gαolf), we observed surface expression for all 15 receptors examined. In fact, two-thirds of Lucy-tagged ORs are able to reach the cell surface synergistically with chaperones even when the Rho tag is removed (10/15 ORs), allowing for the potential assessment of OR function with only an 8-amino acid Flag tag on the mature protein. As expected for a signal peptide, the Lucy tag was cleaved from the mature protein and did not alter OR-ligand binding and signaling. Our studies demonstrate that widespread surface expression of ORs can be achieved in HEK293T cells, providing promise for future large-scale deorphanization studies.
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影响因子：
11.8
作者：
Griffin CA;Kafadar KA;Pavlath GK
通讯作者：
Pavlath GK
DOI：
10.1016/s0006-291x(03)00863-5
发表时间：
2003-06-13
影响因子：
3.1
作者：
Katada, S;Nakagawa, T;Touhara, K
通讯作者：
Touhara, K
影响因子：
4.5
作者：
Lu, M;Echeverri, F;Moyer, BD
通讯作者：
Moyer, BD
DOI：
10.1073/pnas.0307882100
发表时间：
2004-02-24
影响因子：
11.1
作者：
Malnic, B;Godfrey, PA;Buck, LB
通讯作者：
Buck, LB
影响因子：
64.5
作者：
Saito, H;Kubota, M;Matsunami, H
通讯作者：
Matsunami, H