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Fluorescence-Based Measurements of Phosphatidylserine/Phosphatidylinositol 4-Phosphate Exchange Between Membranes
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Contribution of phospholipase C-beta3 phosphorylation to the rapid attenuation of opioid-activated phosphoinositide

D Strassheim1, P Y Law, H H Loh

  • 1Department of Pharmacology, University of Minnesota, Minneapolis, Minnesota 55455-0347, USA.

Molecular Pharmacology
|June 17, 1998
PubMed

Insights

Opioid receptor activation increases intracellular inositol-1,4,5-triphosphate (Ins(1,4,5)P3) but desensitizes via phospholipase C (PLC) phosphorylation. Protein kinase C (PKC) mediates this, facilitating opioid receptor desensitization.

Area of Science:

  • Neuropharmacology
  • Cell Signaling
  • Molecular Biology

Background:

  • Activation of delta-opioid receptors in NG108-15 cells transiently increases intracellular inositol-1,4,5-triphosphate (Ins(1,4,5)P3).
  • The time course of this Ins(1,4,5)P3 increase differs from opioid receptor desensitization, suggesting feedback regulation of phospholipase C (PLC) activity.

Purpose of the Study:

  • To investigate the mechanism of rapid loss in the Ins(1,4,5)P3 response following delta-opioid receptor activation.
  • To determine the role of protein phosphorylation, specifically protein kinase C (PKC), in regulating PLC activity and opioid receptor desensitization.

Main Methods:

  • Utilized NG108-15 neuroblastoma X glioma hybrid cells.
  • Employed delta-selective opioid agonist [D-Pen2, D-Pen5]enkephalin (DPDPE) and other signaling molecules like lysophosphatidic acid (LPA).
  • Investigated protein phosphorylation using immunoprecipitation of PLC-beta3 and assessed enzyme activity with inhibitors (calphostin C, U73122) and activators (phorbol-12-myristate-13-acetate).

Main Results:

  • DPDPE stimulation led to time- and concentration-dependent phosphorylation of PLC-beta3, which was naloxone-reversible.
  • PKC activation was responsible for DPDPE-mediated PLC-beta3 phosphorylation, as confirmed by calphostin C and phorbol ester treatments.
  • DPDPE- and LPA-induced PLC-beta3 phosphorylation were additive, suggesting distinct but converging signaling pathways, and were specific to PLC-beta3.

Conclusions:

  • PKC-mediated phosphorylation of PLC-beta3 plays a significant role in the desensitization of the opioid-activated phospholipase C response.
  • This phosphorylation facilitates the desensitization of the PLC pathway preceding the desensitization of adenylyl cyclase activity following opioid receptor activation.

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