Cannabinoid Receptor Signaling is Dependent on Sub-Cellular Location.
Cannabinoid Receptor Signaling is Dependent on Sub-Cellular Location.
复制标题
大麻素受体信号传导取决于亚细胞位置。
DOI：
10.1101/2024.03.21.586146
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发表时间：
2024
期刊：
影响因子：
--
通讯作者：
Laguerre,Aurélien
中科院分区：
文献类型：
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作者：
Thomas,Alix;Lobingier,BradenT;Schultz,Carsten;Laguerre,Aurélien
G蛋白偶联受体（GPCR）是调节人类生理学的许多方面的膜结合信号分子。最近的进展表明，GPCR信号可以发生在细胞表面和细胞膜内部。我们的研究结果表明，大麻素受体1（CB 1）信号高度依赖于其亚细胞位置。我们发现细胞内CB 1受体主要与Gαi偶联，而质膜受体主要与Gαs偶联。在这里，我们显示CB 1的亚细胞位置，其信号，是视情况而定的启动子和受体标签的选择。具有强启动子或N-末端标签的异源表达导致CB 1主要定位于质膜并通过Gαs进行信号传导。相反，CB 1由低表达启动子驱动，缺乏N-末端遗传标签，主要定位于内膜，并通过Gαi发出信号。最后，我们证明，遗传编码的非规范氨基酸（ncAA）提供了一个解决方案的问题，非天然的N-末端标签破坏CB 1信号。我们鉴定了CB 1 R和CB 2 R中的位点，其可以用荧光团标记而不破坏CB信号传导或使用（连接至赖氨酸的反式环辛烯（TCO*A））和无铜点击化学在活细胞中连接荧光团的运输。总之，我们的数据证明了大麻素信号传导中位置偏差的起源，该信号传导可以通过启动子和新型CBR标记策略在活细胞中进行实验控制和跟踪。
G protein-coupled receptors (GPCRs) are membrane bound signaling molecules that regulate many aspects of human physiology. Recent advances have demonstrated that GPCR signaling can occur both at the cell surface and internal cellular membranes. Our findings suggest that cannabinoid receptor 1 (CB1) signaling is highly dependent on its subcellular location. We find that intracellular CB1 receptors predominantly couple to Gαi while plasma membrane receptors couple to Gαs. Here we show subcellular location of CB1, and its signaling, is contingent on the choice of promoters and receptor tags. Heterologous expression with a strong promoter or N-terminal tag resulted in CB1 predominantly localizing to the plasma membrane and signaling through Gαs. Conversely, CB1 driven by low expressing promoters and lacking N-terminal genetic tags largely localized to internal membranes and signals via Gαi. Lastly, we demonstrate that genetically encodable non-canonical amino acids (ncAA) offer a solution to the problem of non-native N-terminal tags disrupting CB1 signaling. We identified sites in CB1R and CB2R which can be tagged with fluorophores without disrupting CB signaling or trafficking using (trans-cyclooctene attached to lysine (TCO*A)) and copper-free click chemistry to attach fluorophores in live cells. Together, our data demonstrate the origin of location bias in cannabinoid signaling which can be experimentally controlled and tracked in living cells through promoters and novel CBR tagging strategies.