配体如胰岛素,表皮生长因子,血小板源性生长因子,神经生长因子(NGF)通过与受体酪氨酸激酶(RTK)结合在细胞膜上启动信号。与G蛋白偶联受体一起,RTK是将细胞外信号转化为细胞内信号的主要平台。研究RTK信号一直是一个挑战,然而,由于RTK通常耦合的多个信号通路,包括MAP/ERK,PLCγ,和1A类磷酸肌醇3-激酶(PI3K)。多管齐下的RTK信号传导一直是隔离任何一个下游途径的作用的障碍。这里,我们使用PI3K的光遗传学激活将其激活与其他RTK信号通路分离。在这种情况下,我们使用遗传密码扩展将点击化学非规范氨基酸引入膜蛋白的胞外侧。应用细胞不渗透的点击化学荧光团允许我们实时可视化膜蛋白向质膜的递送。使用这些方法,我们证明PI3K的激活,不激活RTK信号下游的其他途径,足以将TRPV1离子通道和胰岛素受体输送到质膜。
Ligands such as insulin, epidermal growth factor, platelet-derived growth factor, and nerve growth factor (NGF) initiate signals at the cell membrane by binding to receptor tyrosine kinases (RTKs). Along with G-protein-coupled receptors, RTKs are the main platforms for transducing extracellular signals into intracellular signals. Studying RTK signaling has been a challenge, however, due to the multiple signaling pathways to which RTKs typically are coupled, including MAP/ERK, PLCγ, and Class 1A phosphoinositide 3-kinases (PI3K). The multi-pronged RTK signaling has been a barrier to isolating the effects of any one downstream pathway. Here, we used optogenetic activation of PI3K to decouple its activation from other RTK signaling pathways. In this context, we used genetic code expansion to introduce a click chemistry noncanonical amino acid into the extracellular side of membrane proteins. Applying a cell-impermeant click chemistry fluorophore allowed us to visualize delivery of membrane proteins to the plasma membrane in real time. Using these approaches, we demonstrate that activation of PI3K, without activating other pathways downstream of RTK signaling, is sufficient to traffic the TRPV1 ion channels and insulin receptors to the plasma membrane.