Genetic dissection of arrestin-mediated µ-opioid receptor signaling in vivo

对于许多G蛋白偶联受体（GPCR），已经表明细胞内信号可以通过G蛋白以及通过抑制蛋白介导。可以优选地刺激一个或另一个信号通路的配体的发现导致了功能选择性或偏向性配体的概念。然而，对于大多数GPCR，仍不清楚哪些生理反应是由与G蛋白的相互作用介导的，哪些体内效应是由与抑制蛋白的相互作用介导的。然而，目前人们对功能选择性GPCR配体的开发越来越感兴趣，这是由于希望产生具有减少的副作用的更有效的药物。这些努力的一个关键驱动力是对arrestin 2基因敲除小鼠的早期研究，这些小鼠在应用吗啡后表现出增强和延长的镇痛作用，同时减少了呼吸抑制和便秘等副作用。这些结果导致了阿片类镇痛完全通过G蛋白介导的假设，而呼吸抑制和便秘主要通过抑制蛋白依赖性信号通路介导。然而，许多悬而未决的问题仍然存在。一方面，μ-阿片受体的抑制蛋白依赖性信号传导途径，其被认为在体内介导呼吸抑制和便秘，在很大程度上是无法解释的。另一方面，G蛋白偏向的μ配体（例如TRV 130和PZM 21）的靶向开发令人惊讶地导致具有非常不同的药理学特征的物质，而不是在人体中具有降低的副作用的镇痛剂。因此，我们提出了一个遗传的方法来测试上述假设。在以前的研究中，只有arrestin 1或arrestin 2的缺失可以在体内进行测试，因为这两种arrestins的整体敲除是胚胎致死的。我们通过创建MOR-CreER鼠标解决了这个问题。使用这种模型，现在可以在无抑制素的条件下研究μ阿片受体介导的作用。相反，现在也可以在确定的时间点在所有μ阿片受体细胞中诱导百日咳毒素，以特异性地阻止Gi介导的信号传递。这些新的小鼠模型将首次允许详细分析G蛋白和抑制蛋白依赖性μ阿片受体在体内的作用，因此将为功能选择性（偏倚）配体的可实现治疗效果提供科学框架。

For many G-protein-coupled receptors (GPCRs) it has been shown that intracellular signals can be mediated via G proteins as well as via arrestins. The discovery of ligands which can preferably stimulate one or the other signal pathway, has led to the concept of functionally selective or biased ligands. However, for most GPCRs it is still unclear which physiological responses are mediated by interaction with G proteins and which in vivo effects are mediated by interaction with arrestins. Nevertheless, there is currently a growing interest in the development of functionally selective GPCR ligands facilitated by hopes of producing more effective drugs with reduced side effects. A key driving force for these efforts were early studies on arrestin2 knockout mice, which showed enhanced and prolonged analgesic effects after morphine application with simultaneously reduced side effects such as respiratory depression and constipation. These results led to the hypothesis that opioid analgesia is mediated exclusively via G proteins, while respiratory depression and constipation are mediated predominantly via arrestin-dependent signaling pathways. However, many open questions remain. On the one hand, the arrestin-dependent signaling pathways of the mu-opioid receptor, which are supposed to mediate respiratory depression and constipation in vivo, are largely unexplained. On the other hand, the targeted development of G-protein biased mu ligands, e.g. TRV130 and PZM21, have surprisingly led to substances with very different pharmacological profiles and not to analgesics with reduced side effects in humans. We therefore propose a genetic approach to test the above hypothesis. In previous studies, only the deletion of arrestin1 or arrestin2 could be tested in vivo, because global knockout of both arrestins is embryonic lethal. We have solved this problem by creating a MOR-CreER mouse. Using this model, it is now possible to study mu-opioid receptor-mediated effects under arrestin-free conditions. Conversely, it is now also possible to induce pertussis toxin in all mu-opioid receptor cells at a defined time point in order to specifically prevent Gi-mediated signal transmission. These new mouse models will allow for the first time a detailed analysis of G-protein- and arrestin-dependent mu-opioid receptor effects in vivo and will therefore provide a scientific framework for the achievable therapeutic effects of functionally-selective (biased) ligands.