Molecular basis of the specificity of opioid signaling and the establishment of a cell culture model for opioid tolerance and dependence

Subtle differences in coupling to different G proteins exist among the three opioid receptors (δ, κ and μ). Recently, differential coupling of Gα16 to the three opioid receptors has been observed. To further examine the ability of these opioid receptors to couple to Gα16, four chimeric opioid receptors (M3D, D3M, M5D and D5M) were constructed. Like their parental μ- (MOR) and δ- (DOR) opioid receptors, these chimeras inhibited cAMP formation via pertussis toxin-sensitive Gi/Go protein. When coexpressed with Gα16, M3D chimera stimulated phospholipase C to a greater extent than that induced by MOR, but lower than those induced by DOR. D3M, M5D and D5M chimeras all behaved like MOR. These results indicated that all intracelluar domains of the receptor are involved in the coupling to Gα16, with some domains more crucial than others. Introduction of a point mutation in M3D further identified Leu80 in intracellular loop one of DOR can enhance the receptor's ability to interact with Gα16. To delineate the mechanisms underlying opioid tolerance and dependence, cultured cells stably expressing the MOR and κ- (KOR) receptor were subjected to long-term exposure to opioids. Such chronic opioid treatments led to compensatory increase in adenylyl cyclase activity upon removal of the agonists. The results showed that this response is mediated through both opioid receptors and pertussis toxin-sensitive G proteins, and did not involve receptor desensitization. This overshoot response can be induced via all three subtype of opioid receptors and did not show any differential pattern among the receptors