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

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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.