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

对于许多G蛋白偶联受体（gpcr），已经证明细胞内信号可以通过G蛋白和阻滞蛋白介导。配体可以更好地刺激一个或另一个信号通路的发现，导致了功能选择性或偏置配体的概念。然而，对于大多数gpcr来说，目前尚不清楚哪些生理反应是通过与G蛋白的相互作用介导的，哪些体内效应是通过与抑制蛋白的相互作用介导的。尽管如此，目前人们对开发功能选择性GPCR配体越来越感兴趣，希望能生产出更有效、副作用更小的药物。这些努力的一个关键驱动力是对arrestin2敲除小鼠的早期研究，这些研究显示吗啡应用后镇痛效果增强并延长，同时减少了呼吸抑制和便秘等副作用。这些结果导致假设阿片类镇痛完全通过G蛋白介导，而呼吸抑制和便秘主要通过抑制蛋白依赖的信号通路介导。然而，仍有许多悬而未决的问题。一方面，在体内被认为介导呼吸抑制和便秘的μ -阿片受体的抑制蛋白依赖信号通路在很大程度上尚未得到解释。另一方面，g蛋白偏向性mu配体的靶向开发，如TRV130和PZM21，令人惊讶地导致了具有非常不同药理特征的物质，而不是减少人类副作用的镇痛药。因此，我们提出一种遗传方法来检验上述假设。在以前的研究中，只能在体内测试arrestin1或arrestin2的缺失，因为这两种基因的全基因敲除都是胚胎致死的。我们已经通过创建一个莫尔- creer鼠标解决了这个问题。使用该模型，现在可以在无骤停条件下研究多阿片受体介导的效应。相反，现在也有可能在规定的时间点诱导所有mu-阿片受体细胞中的百日咳毒素，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对体内g蛋白和抑制蛋白依赖的mu-阿片受体效应进行详细分析，因此将为实现功能选择性（偏倚）配体的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.