AGONIST-INDUCED CONSTITUTIVE ACTIVATION OF THE MU-OPIOID RECEPTOR BY PHOSPHORYLATION

We previously proposed that constitutive mu opioid receptor activation during prolonged agonist stimulation represents a key step in narcoti c tolerance and dependence. Morphine pretreatment of SH-SY5Y cells was shown to lead to a gradual conversion of mu receptors to a constituti vely active state, mu, detectable with an antagonist having negative intrinsic activity (naloxone) (1). mu formation was blocked and rever sed by the general protein kinase inhibitor H7, and further, H7 rapidl y reversed acute morphine induced tolerance and dependence in mice (1) . These results suggest that constitutive mu receptor activation occur s via phosphorylation. To test these hypothesis, we transfected the cl oned mu opioid receptor (2) into HEK 293 cells. Morphine pretreatment caused extensive formation of mu, as determined with cAMP measurement s using naloxone and the neutral antagonist CTAP. H7 inhibited mu for mation, whereas the phosphatase inhibitor calyculin A enhanced it. mu receptor phosphorylation, measured by immunoprecipitating an epitope t agged mu receptor following P-32 labeling, paralleled the activity sta te of the receptor. These combined results strongly support the hypoth esis that agonist induced phosphorylation of the mu receptor leads to its constitutive activation, rather than the commonly assumed desensit ization.