µ-Opioid Receptor Phosphorylation Assays

The µ-opioid receptor (mu-opioid receptor, MOP) is the primary target for many potent analgesics used in the clinic including morphine, oxycodone, hydromorphone, fentanyl, sufentanil and methadone. The utility of µ-analgesics is limited by sever side effects such as respiratory depression, constipation, dependence and addiction. DAMGO ([D-Ala2,N-MePhe4, Gly-ol]-enkephalin) is an enkephalin derivative that is used as selective µ-agonist for in vitro studies. MOP desensitization, trafficking and tolerance are regulated by phosphorylation of carboxyl-terminal threonine370 (pT370-MOP), serine375 (pS375-MOP), threonine376 (pT376-MOP) and threonine379 (pT379-MOP) residues. This nomenclature refers to the mouse MOP. The human MOP sequence is highly conserved at this phosphorylation motif, however, individual sites correspond to T372, S377, T378 and T381 due to two additonal amino acid residues at the amino terminus in the human sequence. S375 is the primary site in a hierarchical phosphorylation cascade. High-efficacy agonists such as DAMGO, fentanyl, sufantanil and methadone induce phosphorylation of all four sites which is mediated by GRK2 and GRK3. Low-efficacy agonists such as morphine, oxycodone and hydromorphone induce primarily phosphorylation of S375 that is mediated in part by GRK5. T370 is subject to phosphorylation by PKC. Serine363 (pS363-MOP) is constitutively phosphorylated by PKC. Rapid MOP dephosphoryation is primarily mediated by protein phosphatase 1 gamma (PP1γ). The development of G protein-biased µ-agonists such as TRV130, PZM21 or SR-17018 has been proposed as a strategy for safer analgesics with fewer side effects. Recent work shows that phosphosite-specific antibodies provide more precise information about ligand bias then conventional ß-arrestin recruitment assays. For more information on µ-opioid receptor pharmacology please refer to the IUPHAR database. For further reading refer to:

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