In 2020, drug overdoses jumped by 30% with 190 people in the United States dying every day from opioid
overdose. Despite their significant risks, opioids analgesics, including morphine, codeine, Vicodin and
oxycontin, are still regarded as the gold standard for alleviating severe pain. All of these drugs exert their
analgesic effects through activation of a G protein coupled receptor (GPCR), specifically the Gi coupled µ-
Opioid receptor (MOR). Repeated use of opioids leads to the development of tolerance to the analgesic effects
of these drugs. This tolerance necessitates dose escalation to maintain sufficient pain control, which, in turn,
increases both the likelihood of adverse events including enhanced respiratory depression and death as well
as the liability to develop opioid dependence, manifested as physical and emotional distress in the absence of
drug. Importantly, dependence is a key driver of the transition from opioid use to opioid abuse yet there is little
understanding of the cellular signaling properties of the MOR that promote this transition. The signaling
cascade induced by endogenous opioid ligands, such as beta-endorphin, from the MOR in response provide
insight, as they produce analgesia without causing the severe tolerance and dependence observed in
response to the small molecule opioid drugs. One key difference between the endogenous and exogenous
opioids are their ability to recruit ß-arrestin-2 following G-protein activation. Counter-intuitively, the endogenous
ligands are better recruiters of ß-arrestin-2 and subsequently drive substantially more receptor endocytosis and
recycling than opioid drugs, even though they produce less tolerance. As such, we propose that facilitating
endocytosis will titrate signal transduction at MOR, emulating endorphin signal transduction, thereby reducing
tolerance and dependence. To test this hypothesis, we have manipulated the ‘phosphorylation barcode’ of
MOR and created an engineered receptor that recruits ß-arrestin-2 and undergoes endocytosis in response to
morphine. Here we will thoroughly characterize this mutant receptor in a cell-based model, including a cell-
based model of tolerance and dependence. We will also assess the phenotypes of WT and ß-arrestin-2-KO
mice in response to morphine and methadone, the only small molecule drug that significantly recruits ß-
arrestin-2. We will assess tolerance and dependence in ß-arrestin-2-KO in response to morphine and more
importantly, methadone. We will use a novel oral operant self-administration paradigm for 16 weeks to model
the transition from impulsive to compulsive drug use in both WT and ß-arrestin-2-KO animals. For decades the
opioid field has focused, unsuccessfully, on developing drugs that show reduced side effects. We propose that
these efforts have failed because they were based on the assumption that recruitment of ß-arrestin-2 produces
tolerance and dependence. The studies proposed here are designed to provide substantial evidence that
recruitment of ß-arrestin-2 and subsequent endocytosis and recycling is beneficial. As such, they could inform
drug development of novel opioid with reduced abuse liability.