Department of Physiology, Johns Hopkins University School of Medicine, Baltimore MD, USA.
PLoS One. 2013 Jul 1;8(7):e68758. doi: 10.1371/journal.pone.0068758. Print 2013.
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 G(αolf)) 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 G(αolf)), 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.
嗅觉受体（ORs）是 G 蛋白偶联受体，可检测嗅上皮中的气味物质，是基因组中最大的基因家族。OR 配体的鉴定通常需要在异源细胞中表达 OR 表面；然而，当外源性表达时，OR 很少转运到细胞表面。因此，大多数 OR 是没有已知配体的孤儿受体。迄今为止，研究已经利用不可切割的视紫红质（Rho）标签和/或伴侣蛋白（即受体转运蛋白、RTP1S、Ric8b 和 G（αolf））来提高表面表达。然而，即使使用这些工具，许多 OR 仍然无法到达细胞表面。我们使用了一组十五个 OR 来检查可切割亮氨酸丰富信号肽序列（Lucy 标签）对 HEK293T 细胞中 OR 表面表达的影响。我们在这里报告，将 Lucy 标签添加到 N 端可将到达细胞表面的 OR 数量增加到 15 个 OR 中的 7 个（与没有 Rho 或 Lucy 标签的 3/15 相比）。此外，当 OR 与之前报道的伴侣蛋白（RTP1S、Ric8b 和 G（αolf））共表达时，带有 Lucy 和 Rho 标签的 OR 都能观察到表面表达。实际上，即使去除 Rho 标签，Lucy 标记的 OR 中有三分之二能够与伴侣蛋白协同到达细胞表面（10/15 ORs），这使得仅在成熟蛋白上带有 8 个氨基酸的 Flag 标签就有可能评估 OR 功能。Lucy 标签作为信号肽被切割，不改变 OR-配体结合和信号传导。我们的研究表明，OR 可以在 HEK293T 细胞中实现广泛的表面表达，为未来的大规模去孤儿化研究提供了希望。
