Src‐dependent phosphorylation of μ‐opioid receptor at Tyr336 modulates opiate withdrawal

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Abstract
Opiate withdrawal/negative reinforcement has been implicated as one of the mechanisms for the progression from impulsive to compulsive drug use. Increase in the intracellular cAMP level and protein kinase A (PKA) activities within the neurocircuitry of addiction has been a leading hypothesis for opiate addiction. This increase requires the phosphorylation of μ‐opioid receptor (MOR) at Tyr336 by Src after prolonged opiate treatment in vitro. Here, we report that the Src‐mediated MOR phosphorylation at Tyr336 is a prerequisite for opiate withdrawal in mice. We observed the recruitment of Src in the vicinity of MOR and an increase in phosphorylated Tyr336 (pY336) levels during naloxone‐precipitated withdrawal. The intracerebroventricular or stereotaxic injection of a Src inhibitor (AZD0530), or Src shRNA viruses attenuated pY336 levels, and several somatic withdrawal signs. This was also observed in Fyn−/− mice. The stereotaxic injection of wild‐type MOR, but not mutant (Y336F) MOR, lentiviruses into the locus coeruleus of MOR−/− mice restored somatic withdrawal jumping. Regulating pY336 levels during withdrawal might be a future target for drug development to prevent opiate addictive behaviors. Synopsis The μ‐opioid receptor (MOR) non‐canonical signaling pathway involves its phosphorylation at Tyr336 by Src kinase within the MOR signal complex. The focal event of MORY336 phosphorylation may be the key for adenylyl cyclase superactivation during the withdrawal/negative stage of opiate addiction. The phosphorylation levels of MORY336 and SrcY416 increased during naloxone‐precipitated withdrawal in wild‐type mice. The intracerebroventricular or stereotaxic injection of a Src inhibitor (AZD0530), or Src shRNA viruses reduced pMORY336 and pSrcY416 levels, and several somatic withdrawal signs. Increases of pMORY336 levels as well as some somatic withdrawal signs after naloxone‐precipitated withdrawal were not observed in Fyn−/− mice. The stereotaxic injection of wild‐type MOR lentiviruses into the locus coeruleus of MOR−/− mice restored somatic withdrawal jumping and wet dog shaking. The stereotaxic injection of the phosphorylation‐deficient mutant (Y336F) MOR, in which the mutation of Tyr336 to Phe blocks Src‐mediated phosphorylation, attenuated naloxone‐precipitated withdrawal.
Funding Information
  • National Institutes of Health (DA031442)
  • Ministry of Science and Technology of the People's Republic of China (2013CB835100)
  • Ministry of Science and Technology of the People's Republic of China (2015CB553502)
  • National Natural Science Foundation of China (81130087)
  • National Natural Science Foundation of China (81671322)
  • National Natural Science Foundation of China (81401107)
  • Chinese Academy of Sciences (2017334)
  • Chinese Academy of Sciences (2011T2S29)

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