Real-time imaging of μ-opioid receptor signaling, phosphorylation and desensitization

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G protein receptor kinases (GRKs) and β-arrestins are key regulators of μ-opioid receptor (MOR) signaling and trafficking. In the previous funding period, we showed that GRK2/3 is recruited more quickly than β-arrestins. β-Arrestin recruitment required GRK2 activity and MOR phosphorylation, but GRK recruitment also depended on phosphorylation sites in the C-terminal tail. Consistent with this, phosphorylation-deficient MORs mediate strongly enhanced acute analgesia in vivo and upon chronic administration analgesic tolerance to fentanyl is abrogated. In this proposal, we will (I) delineate the role of PKC in non-canonical MOR desensitization and tolerance, (II) evaluate the physiological role of unconventional β-arrestin signaling in vivo and in vitro, and (III) utilize advanced imaging techniques to monitor agonist-dependent changes in endogenous MOR receptor organization in living neurons.