Premium
Calmodulin Regulation of Basal and Agonist‐Stimulated G Protein Coupling by the μ‐Opioid Receptor (OP 3 ) in Morphine‐Pretreated Cell
Author(s) -
Wang Danxin,
Surratt Christopher K,
Sadée Wolfgang
Publication year - 2000
Publication title -
journal of neurochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.75
H-Index - 229
eISSN - 1471-4159
pISSN - 0022-3042
DOI - 10.1046/j.1471-4159.2000.0750763.x
Subject(s) - agonist , g protein , medicine , g protein coupled receptor , μ opioid receptor , receptor , endocrinology , opioid receptor , chemistry , opioid , homologous desensitization , morphine , pharmacology , biophysics , biology , biochemistry
Calmodulin (CaM) has been shown to suppress basal G protein coupling and attenuate agonist‐stimulated G protein coupling of the μ‐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 OP 3 ‐G protein coupling during morphine treatment, shown to result in CaM release from plasma membranes. Basal and agonist‐stimulated G protein coupling by OP 3 was measured before and after morphine pretreatment by incorporation of guanosine 5′‐ O ‐(3‐[ 35 S]thiotriphosphate) into membranes, obtained from HEK 293 cells transfected with human OP 3 cDNA. The opioid antagonist β‐chlornaltrexamine fully suppressed basal G protein coupling of OP 3 , 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 OP 3 ‐i3 loop mutants with reduced affinity for CaM (K273A, R275A, and K273A/R275A). Basal signaling of these mutant OP 3 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‐OP 3 interactions appear to play only a minor role in the desensitization of OP 3 . In contrast, release of CaM from the plasma membrane appears to enhance the inherent basal G protein coupling of OP 3 , thereby resolving the paradox that OP 3 displays both desensitization and sensitization during morphine treatment.