Deciphering µ-opioid receptor phosphorylation and dephosphorylation in HEK293 cells

The molecular basis of agonist-selective signalling at the µ-opioid receptor is poorly understood. We have recently shown that full agonists such as [D-Ala(2)-MePhe(4)-Gly-ol]enkephalin (DAMGO) stimulate the phosphorylation of a number of carboxyl-terminal phosphate acceptor sites including threonine 370 (Thr(370)) and serine 375 (Ser(375)), and that is followed by a robust receptor internalization. In contrast, morphine promotes a selective phosphorylation of Ser(375) without causing rapid receptor internalization.

Here, we identify kinases and phosphatases that mediate agonist-dependent phosphorylation and dephosphorylation of the µ-opioid receptor using a combination of phosphosite-specific antibodies and siRNA knock-down screening in HEK293 cells.

We found that DAMGO-driven phosphorylation of Thr(370) and Ser(375) was preferentially catalysed by G-protein-coupled receptor kinases (GRKs) 2 and 3, whereas morphine-driven Ser(375) phosphorylation was preferentially catalysed by GRK5. On the functional level, inhibition of GRK expression resulted in enhanced µ-opioid receptor signalling and reduced receptor internalization. Analysis of GRK5-deficient mice revealed that GRK5 selectively contributes to morphine-induced Ser(375) phosphorylation in brain tissue. We also identified protein phosphatase 1γ as a µ-opioid receptor phosphatase that catalysed Thr(370) and Ser(375) dephosphorylation at or near the plasma membrane within minutes after agonist removal, which in turn facilitates receptor recycling.

Together, the morphine-activated µ-opioid receptor is a good substrate for phosphorylation by GRK5 but a poor substrate for GRK2/3. GRK5 phosphorylates µ-opioid receptors selectively on Ser(375), which is not sufficient to drive significant receptor internalization.