Spatiotemporal signaling and trafficking of the mu-opioid receptor

摘要 阿片类药物是最有效的止痛药，但与包括呼吸道在内的严重副作用有关 抑郁、容忍和上瘾。这些因素导致了阿片类药物在美国的流行，使 吸毒过量是美国意外死亡的主要原因。因此，确定更安全的止痛药与 减少副作用和滥用的可能性对于解决目前的危机至关重要。临床使用的阿片类药物 主要通过对µ-阿片受体(MOR)的作用发挥其镇痛和不良反应。 虽然已经采取了几种更安全的止痛药的方法，但这些努力受到缺乏 了解MOR参与和激活的复杂生化网络对配体结合的反应。 这一建议建立在最近的证据表明：(1)MOR信号依赖于 配体特异性的亚细胞定位和膜转运；(2)MOR显示配体- 依赖于对其蛋白质相互作用网络及其激活的信号通路的影响。因此，描绘 MOR启动的内源性多肽和成瘾阿片类药物的信号通路及其机制 受体定位和贩运的协调提供了潜在的治疗方式和 更安全的止痛药。该提案的总体目标是将定量蛋白质组学、功能 基因组学和阿片受体生物学，系统地发现和表征MOR信号调节因子 以及人类诱导的多能干细胞来源的神经元的贩运。我们将结合邻近标记质量 光谱和定量磷蛋白质组学系统地描绘更多的相互作用网络 参与并绘制它激活的信号通路。蛋白质组学靶点在MOR中的功能研究 信号和贩运，我们将开发和应用受体信号和贩运的报告分析 CRISPRi基因调控筛选。最后，我们将从蛋白质组学和遗传学角度检验机械论假说 筛选新的运输和信号调节因子如何微调MOR激活的细胞反应。 我们建议的方法将产生对MOR启动的信号通路以及这些通路是如何 受受体交易的调节。确定MOR激活的关键调节器将填补设计的一个关键空白 更安全、选择性镇痛剂和阿片成瘾治疗。

SUMMARY Opioids are the most effective analgesics but are associated with severe side effects including respiratory depression, tolerance, and addiction. These factors helped cause the opioid abuse epidemic in the US, making drug overdose the leading cause of accidental death in the US. Thus, the identification of safer analgesics with diminished side effects and abuse potential is critical to address the ongoing crisis. Clinically used opioids predominantly exert both their analgesic and adverse effects through their action on the µ-opioid receptor (MOR). While several approaches were taken towards safer analgesics, these efforts are limited by a lack of understanding the complex biochemical networks engaged and activated by MOR in response to ligand binding. This proposal builds on recent evidence suggesting that (1) MOR signaling is dependent on the interplay between subcellular localization and membrane trafficking in a ligand-specific manner and (2) MOR shows ligand- dependent effects on its protein interaction network and the signaling pathways it activates. Thus, delineating the MOR-initiated signaling pathways for endogenous peptides and addictive opioids and how these are coordinated by receptor location and trafficking provides potential new strategies for therapeutic modalities and safer analgesics. The overarching goal of this proposal is to combine quantitative proteomics, functional genomics, and opioid receptor biology to systematically discover and characterize regulators of MOR signaling and trafficking in human induced pluripotent stem cell-derived neurons. We will combine proximity labeling mass spectrometry and quantitative phosphoproteomics to systematically delineate interaction networks that MOR engages and map the signaling pathways it activates. To study the functional role of proteomic targets in MOR signaling and trafficking, we will develop and apply reporter assays for receptor signaling and trafficking in CRISPRi gene regulation screens. Finally, we will test mechanistic hypotheses from proteomic and genetic screens on how novel regulators of trafficking and signaling fine tune the cellular response of MOR activation. Our proposed approach will yield mechanistic insights into MOR-initiated signaling pathways and how these are regulated by receptor trafficking. Identifying key regulators of MOR activation will fill a critical gap for designing safer, pathway selective analgesics and treatments for opioid addiction.