Spatiotemporal signaling and trafficking of the mu-opioid receptor

总结 阿片类药物是最有效的镇痛药，但与严重的副作用有关，包括呼吸道感染。 抑郁、耐受和成瘾。这些因素导致了美国阿片类药物滥用的流行， 药物过量是美国意外死亡的主要原因。因此，确定更安全的镇痛药， 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 主要通过其对μ-阿片受体（莫尔）的作用来发挥其镇痛和副作用。 虽然采取了几种方法来获得更安全的镇痛剂，但这些努力受到缺乏 理解由莫尔参与和激活的复杂的生化网络，以响应配体结合。 这一提议建立在最近的证据上，这些证据表明：（1）莫尔信号传导依赖于以下因素之间的相互作用： （2）莫尔显示配体- 依赖于其蛋白质相互作用网络及其激活的信号通路。因此，描绘 MOR启动的内源性肽和成瘾性阿片类药物的信号传导途径，以及这些信号传导途径如何 通过受体定位和运输的协调，为治疗方式提供了潜在的新策略， 更安全的止痛药该提案的总体目标是将联合收割机定量蛋白质组学、功能蛋白质组学和生物信息学相结合。 基因组学和阿片受体生物学来系统地发现和表征莫尔信号传导的调节剂 和人诱导多能干细胞衍生的神经元的运输。我们将联合收割机 光谱和定量磷酸蛋白质组学，系统地描绘相互作用网络， 参与并绘制它激活的信号通路。研究蛋白质组学靶点在莫尔中的功能作用 信号传导和贩运，我们将开发和应用受体信号传导和贩运的报告分析， CRISPRi基因调控筛选。最后，我们将测试从蛋白质组学和遗传学的机制假说， 筛选运输和信号传导的新型调节剂如何微调莫尔激活的细胞反应。 我们提出的方法将产生机制的见解MOR启动的信号通路，以及这些是如何 受受体运输的调控。确定莫尔激活的关键调节因子将填补设计的关键空白 更安全的，途径选择性镇痛药和阿片类药物成瘾的治疗。

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.