Deciphering µ-opioid receptor phosphorylation and dephosphorylation in HEK293 cells.
OBJECTIVE
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.


METHODS
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.


RESULTS
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.


CONCLUSIONS
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.
Institute of Pharmacology and Toxicology, Jena University Hospital - Friedrich Schiller University Jena, Jena, Germany.
Christian
C
Doll
Florian
F
Pöll
Kenneth
K
Peuker
Anastasia
A
Loktev
Laura
L
Glück
Stefan
S
Schulz
D000701:Analgesics, Opioid / Q000494:pharmacology; D000906:Antibodies / Q000494:pharmacology; D020875:Enkephalin, Ala(2)-MePhe(4)-Gly(5)- / Q000494:pharmacology; D054768:G-Protein-Coupled Receptor Kinases / Q000378:metabolism*; D057809:HEK293 Cells; D006801:Humans; D009020:Morphine / Q000494:pharmacology; D010766:Phosphorylation; D054645:Protein Phosphatase 1 / Q000378:metabolism*; D034741:RNA, Small Interfering / Q000235:genetics; D017450:Receptors, Opioid, mu / Q000378:metabolism*
D000701:Analgesics, Opioid; D000906:Antibodies; D034741:RNA, Small Interfering; D017450:Receptors, Opioid, mu; D020875:Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; D009020:Morphine; D054768:G-Protein-Coupled Receptor Kinases; D054645:Protein Phosphatase 1