ISSN:
1471-4159
Source:
Blackwell Publishing Journal Backfiles 1879-2005
Topics:
Medicine
Notes:
Calmodulin (CaM) has been shown to suppress basal G protein coupling and attenuate agonist-stimulated G protein coupling of the μ-opioid receptor (OP3) 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 coupling by OP3 was measured before and after morphine pretreatment by incorporation of guanosine 5′-O-(3-[35S]thiotriphosphate) into membranes, obtained from HEK 293 cells transfected with human OP3 cDNA. The opioid antagonist β-chlornaltrexamine fully suppressed basal G protein coupling of OP3, providing a direct measure of basal signaling. Pretreatment of the cells with morphine enhanced basal G protein coupling (sensitization). In contrast, agonist-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 reduced affinity for CaM (K273A, R275A, and K273A/R275A). Basal signaling of these mutant OP3 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 interactions appear to play only a minor role in the desensitization of OP3. In contrast, release of CaM from the plasma membrane appears to enhance the inherent basal G protein coupling of OP3, thereby resolving the paradox that OP3 displays both desensitization and sensitization during morphine treatment.
Type of Medium:
Electronic Resource
URL:
http://dx.doi.org/10.1046/j.1471-4159.2000.0750763.x
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