Calmodulin regulation of basal and agonist-stimulated G protein coupling by the mu-opioid receptor (OP(3)) in morphine-pretreated cell

J Neurochem. 2000 Aug;75(2):763-71. doi: 10.1046/j.1471-4159.2000.0750763.x.

Abstract

Calmodulin (CaM) has been shown to suppress basal G protein coupling and attenuate 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 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 beta-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.

MeSH terms

  • Amino Acid Substitution
  • Animals
  • Calmodulin / physiology*
  • Cell Line
  • Cell Membrane / physiology
  • Chickens
  • Egtazic Acid / pharmacology
  • GTP-Binding Proteins / physiology*
  • Guanosine 5'-O-(3-Thiotriphosphate) / pharmacokinetics
  • Humans
  • Kidney
  • Kinetics
  • Morphine / pharmacology*
  • Mutagenesis, Site-Directed
  • Naloxone / pharmacokinetics
  • Naltrexone / analogs & derivatives
  • Naltrexone / pharmacology
  • Narcotic Antagonists / pharmacology
  • Receptors, Opioid, mu / chemistry
  • Receptors, Opioid, mu / genetics
  • Receptors, Opioid, mu / physiology*
  • Recombinant Proteins / metabolism
  • Transfection

Substances

  • Calmodulin
  • Narcotic Antagonists
  • Receptors, Opioid, mu
  • Recombinant Proteins
  • Naloxone
  • Guanosine 5'-O-(3-Thiotriphosphate)
  • Egtazic Acid
  • Naltrexone
  • chlornaltrexamine
  • Morphine
  • GTP-Binding Proteins