Calmodulin regulation of basal and agonist-stimulated G protein coupling by the mu-opioid receptor (OP3) in morphine-pretreated cells

Calmodulin (CaM) has been shown to suppress basal G protein coupling and at tenuate agonist-stimulated G protein coupling of the mu-opioid receptor (OP 3) through direct interaction with the third intracellular (i3) loop of the receptor. Here we have investigated the role of CaM in regulating changes in OP3-G protein coupling during morphine treatment, shown to result in CaM release from plasma membranes. Basal and agonist-stimulated G protein coup ling by OP3 was measured before and after morphine pretreatment by incorpor ation of guanosine 5'-O-(3-[S-35]thiotriphosphate) into membranes, obtained from HEK 293 cells transfected with human OP3 cDNA. The opioid antagonist beta-chlornaltrexamine fully suppressed basal G protein coupling of OP3, pr oviding a direct measure of basal signaling. Pretreatment of the cells with morphine enhanced basal G protein coupling (sensitization). In contrast, a gonist-stimulated coupling was diminished (desensitization), resulting in a substantially flattened morphine dose-response curve. To test whether CaM is involved in these changes, we constructed OP3-i3 loop mutants with reduc ed affinity for CaM (K273A, R275A, and K273A/R275A). Basal signaling of the se mutant OF, receptors was higher than that of the wild-type receptor and, moreover, unaffected by morphine pretreatment, whereas desensitization to agonist stimulation was only slightly attenuated. Therefore, CaM-OP3 intera ctions appear to play only a minor role in the desensitization of OP3. In c ontrast, release of CaM from the plasma membrane appears to enhance the inh erent basal G protein coupling of OP3, thereby resolving the paradox that O P3 displays both desensitization and sensitization during morphine treatmen t.