GRK2 protein-mediated transphosphorylation contributes to loss of function of μ-opioid receptors induced by neuropeptide FF (NPFF2) receptors

J Biol Chem. 2012 Apr 13;287(16):12736-49. doi: 10.1074/jbc.M111.314617. Epub 2012 Feb 28.


Neuropeptide FF (NPFF) interacts with specific receptors to modulate opioid functions in the central nervous system. On dissociated neurons and neuroblastoma cells (SH-SY5Y) transfected with NPFF receptors, NPFF acts as a functional antagonist of μ-opioid (MOP) receptors by attenuating the opioid-induced inhibition of calcium conductance. In the SH-SY5Y model, MOP and NPFF(2) receptors have been shown to heteromerize. To understand the molecular mechanism involved in the anti-opioid activity of NPFF, we have investigated the phosphorylation status of the MOP receptor using phospho-specific antibody and mass spectrometry. Similarly to direct opioid receptor stimulation, activation of the NPFF(2) receptor by [D-Tyr-1-(NMe)Phe-3]NPFF (1DMe), an analog of NPFF, induced the phosphorylation of Ser-377 of the human MOP receptor. This heterologous phosphorylation was unaffected by inhibition of second messenger-dependent kinases and, contrarily to homologous phosphorylation, was prevented by inactivation of G(i/o) proteins by pertussis toxin. Using siRNA knockdown we could demonstrate that 1DMe-induced Ser-377 cross-phosphorylation and MOP receptor loss of function were mediated by the G protein receptor kinase GRK2. In addition, mass spectrometric analysis revealed that the phosphorylation pattern of MOP receptors was qualitatively similar after treatment with the MOP agonist Tyr-D-Ala-Gly (NMe)-Phe-Gly-ol (DAMGO) or after treatment with the NPFF agonist 1DMe, but the level of multiple phosphorylation was more intense after DAMGO. Finally, NPFF(2) receptor activation was sufficient to recruit β-arrestin2 to the MOP receptor but not to induce its internalization. These data show that NPFF-induced heterologous desensitization of MOP receptor signaling is mediated by GRK2 and could involve transphosphorylation within the heteromeric receptor complex.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Analgesics, Opioid / pharmacology
  • Arrestins / metabolism
  • Cell Line, Tumor
  • Enkephalin, Ala(2)-MePhe(4)-Gly(5)- / pharmacology
  • G-Protein-Coupled Receptor Kinase 2 / genetics
  • G-Protein-Coupled Receptor Kinase 2 / metabolism*
  • Gene Knockdown Techniques
  • Heterotrimeric GTP-Binding Proteins / metabolism
  • Humans
  • Molecular Sequence Data
  • Neuroblastoma
  • Phosphorylation / physiology
  • Receptors, G-Protein-Coupled / metabolism
  • Receptors, Neuropeptide / metabolism*
  • Receptors, Opioid, mu / agonists
  • Receptors, Opioid, mu / metabolism*
  • Second Messenger Systems / drug effects
  • Second Messenger Systems / physiology
  • Serine / metabolism
  • Signal Transduction / drug effects
  • Signal Transduction / physiology
  • beta-Arrestins


  • Analgesics, Opioid
  • Arrestins
  • Receptors, G-Protein-Coupled
  • Receptors, Neuropeptide
  • Receptors, Opioid, mu
  • beta-Arrestins
  • neuropeptide FF receptor
  • seven-transmembrane G-protein-coupled receptor
  • Enkephalin, Ala(2)-MePhe(4)-Gly(5)-
  • Serine
  • GRK2 protein, human
  • G-Protein-Coupled Receptor Kinase 2
  • Heterotrimeric GTP-Binding Proteins