title: GRK2 protein-mediated transphosphorylation contributes to loss of function of μ-opioid receptors induced by neuropeptide FF (NPFF2) receptors.
abstract: 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.
authors.affiliation: Institut de Pharmacologie et de Biologie Structurale, CNRS/Université de Toulouse, UMR 5089, 205 Route de Narbonne, 31077 Toulouse Cedex, France.
authors.forename: Lionel
authors.initials: L
authors.lastname: Moulédous
authors.forename: Carine
authors.initials: C
authors.lastname: Froment
authors.forename: Stéphanie
authors.initials: S
authors.lastname: Dauvillier
authors.forename: Odile
authors.initials: O
authors.lastname: Burlet-Schiltz
authors.forename: Jean-Marie
authors.initials: JM
authors.lastname: Zajac
authors.forename: Catherine
authors.initials: C
authors.lastname: Mollereau
mesh_terms: D000595:Amino Acid Sequence; D000701:Analgesics, Opioid / Q000494:pharmacology; D019390:Arrestins / Q000378:metabolism; D045744:Cell Line, Tumor; D020875:Enkephalin, Ala(2)-MePhe(4)-Gly(5)- / Q000494:pharmacology; D054769:G-Protein-Coupled Receptor Kinase 2 / Q000235:genetics / Q000378:metabolism*; D055785:Gene Knockdown Techniques; D020962:Heterotrimeric GTP-Binding Proteins / Q000378:metabolism; D006801:Humans; D008969:Molecular Sequence Data; D009447:Neuroblastoma; D010766:Phosphorylation / Q000502:physiology; D043562:Receptors, G-Protein-Coupled / Q000378:metabolism; D018013:Receptors, Neuropeptide / Q000378:metabolism*; D017450:Receptors, Opioid, mu / Q000819:agonists / Q000378:metabolism*; D015290:Second Messenger Systems / Q000187:drug effects / Q000502:physiology; D012694:Serine / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology; D000071557:beta-Arrestins
chemical_list: D000701:Analgesics, Opioid; D019390:Arrestins; D043562:Receptors, G-Protein-Coupled; D018013:Receptors, Neuropeptide; D017450:Receptors, Opioid, mu; D000071557:beta-Arrestins; C083929:neuropeptide FF receptor; C450520:seven-transmembrane G-protein-coupled receptor; D020875:Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; D012694:Serine; C496567:GRK2 protein, human; D054769:G-Protein-Coupled Receptor Kinase 2; D020962:Heterotrimeric GTP-Binding Proteins