Heterologous activation of protein kinase C stimulates phosphorylation of delta-opioid receptor at serine 344, resulting in beta-arrestin- and clathrin-mediated receptor internalization.
The purpose of the current study is to investigate the effect of opioid-independent, heterologous activation of protein kinase C (PKC) on the responsiveness of opioid receptor and the underlying molecular mechanisms. Our result showed that removing the C terminus of delta opioid receptor (DOR) containing six Ser/Thr residues abolished both DPDPE- and phorbol 12-myristate 13-acetate (PMA)-induced DOR phosphorylation. The phosphorylation levels of DOR mutants T352A, T353A, and T358A/T361A/S363S were comparable to that of the wild-type DOR, whereas S344G substitution blocked PMA-induced receptor phosphorylation, indicating that PKC-mediated phosphorylation occurs at Ser-344. PKC-mediated Ser-344 phosphorylation was also induced by activation of G(q)-coupled alpha(1A)-adrenergic receptor or increase in intracellular Ca(2+) concentration. Activation of PKC by PMA, alpha(1A)-adrenergic receptor agonist, and ionomycin resulted in DOR internalization that required phosphorylation of Ser-344. Expression of dominant negative beta-arrestin and hypertonic sucrose treatment blocked PMA-induced DOR internalization, suggesting that PKC mediates DOR internalization via a beta-arrestin- and clathrin-dependent mechanism. Further study demonstrated that agonist-dependent G protein-coupled receptor kinase (GRK) phosphorylation sites in DOR are not targets of PKC. Agonist-dependent, GRK-mediated receptor phosphorylation and agonist-independent, PKC-mediated DOR phosphorylation were additive, but agonist-induced receptor phosphorylation could inhibit PKC-catalyzed heterologous DOR phosphorylation and subsequent internalization. These data demonstrate that the responsiveness of opioid receptor is regulated by both PKC and GRK through agonist-dependent and agonist-independent mechanisms and PKC-mediated receptor phosphorylation is an important molecular mechanism of heterologous regulation of opioid receptor functions.
Xiang
B
B
National Laboratory of Medical Neurobiology, Fudan University Medical Center, Shanghai 200032, People's Republic of China.
Yu
G H
GH
Guo
J
J
Chen
L
L
Hu
W
W
Pei
G
G
Ma
L
L
D019390:Arrestins / Q000235:genetics / Q000502:physiology*; D002118:Calcium / Q000378:metabolism; D002460:Cell Line; D002966:Clathrin / Q000502:physiology*; D015536:Down-Regulation; D004705:Endocytosis; D020881:Enkephalin, D-Penicillamine (2,5)- / Q000494:pharmacology; D006801:Humans; D009154:Mutation; D010766:Phosphorylation; D010768:Phosphoserine / Q000378:metabolism; D011493:Protein Kinase C / Q000378:metabolism*; D017465:Receptors, Opioid, delta / Q000235:genetics / Q000378:metabolism*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014162:Transfection; D000071557:beta-Arrestins
D019390:Arrestins; D002966:Clathrin; D017465:Receptors, Opioid, delta; D000071557:beta-Arrestins; D010768:Phosphoserine; D020881:Enkephalin, D-Penicillamine (2,5)-; D011493:Protein Kinase C; D013755:Tetradecanoylphorbol Acetate; D002118:Calcium