Agonist induced constitutive activation of the μ opioid receptor by phosphorylation

Abstract We previously proposed that constitutive μ opioid receptor activation during prolonged agonist stimulation represents a key step in narcotic tolerance and dependence. Morphine pretreatment of SH-SY5Y cells was shown to lead to a gradual conversion of μ receptors to a constitutively active state, μ ∗ , detectable with an antagonist having negative intrinsic activity (naloxone) (1). μ ∗ formation was blocked and reversed by the general protein kinase inhibitor H7, and further, H7 rapidly reversed acute morphine induced tolerance and dependence in mice (1). These results suggest that constitutive μ receptor activation occurs via phosphorylation. To test these hypothesis, we transfected the cloned μ opioid receptor (2) into HEK 293 cells. Morphine pretreatment caused extensive formation of μ ∗ , as determined with cAMP measurements using naloxone and the neutral antagonist CTAP. H7 inhibited μ ∗ formation, whereas the phosphatase inhibitor calyculin A enhanced it. μ receptor phosphorylation, measured by immunoprecipitating an epitope tagged μ receptor following 32 P labeling, paralleled the activity state of the receptor. These combined results strongly support the hypothesis that agonist induced phosphorylation of the μ receptor leads to its constitutive activation, rather than the commonly assumed desensitization.