Deciphering μ-opioid receptor phosphorylation and dephosphorylation in HEK293 cells

BACKGROUND AND PURPOSE The molecular basis of agonist-selective signalling at the mu-opioid receptor is poorly understood. We have recently shown that full agonists such as [D-Ala2-MePhe4-Gly-ol]enkephalin (DAMGO) stimulate the phosphorylation of a number of carboxyl-terminal phosphate acceptor sites including threonine 370 (Thr370) and serine 375 (Ser375), and that is followed by a robust receptor internalization. In contrast, morphine promotes a selective phosphorylation of Ser375 without causing rapid receptor internalization. EXPERIMENTAL APPROACH Here, we identify kinases and phosphatases that mediate agonist-dependent phosphorylation and dephosphorylation of the mu-opioid receptor using a combination of phosphosite-specific antibodies and siRNA knock-down screening in HEK293 cells. KEY RESULTS We found that DAMGO-driven phosphorylation of Thr370 and Ser375 was preferentially catalysed by G-protein-coupled receptor kinases (GRKs) 2 and 3, whereas morphine-driven Ser375 phosphorylation was preferentially catalysed by GRK5. On the functional level, inhibition of GRK expression resulted in enhanced mu-opioid receptor signalling and reduced receptor internalization. Analysis of GRK5-deficient mice revealed that GRK5 selectively contributes to morphine-induced Ser375 phosphorylation in brain tissue. We also identified protein phosphatase 1? as a mu-opioid receptor phosphatase that catalysed Thr370 and Ser375 dephosphorylation at or near the plasma membrane within minutes after agonist removal, which in turn facilitates receptor recycling. CONCLUSIONS AND IMPLICATIONS Together, the morphine-activated mu-opioid receptor is a good substrate for phosphorylation by GRK5 but a poor substrate for GRK2/3. GRK5 phosphorylates mu-opioid receptors selectively on Ser375, which is not sufficient to drive significant receptor internalization.