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.
Highlights
Neuropeptide FF2 receptors interact with -opioid receptors and decrease their activity
As the antibody was not sensitive enough to detect the endogenous MOP receptor, which is expressed at low levels in SH-SY5Y cells [23], phosphorylation experiments were performed in the SH2D9 MOPYFP cell line that stably expresses the human NPFF2 receptor and the human MOP receptor fused to the yellow fluorescent protein (YFP) at its C terminus [32]
Treatment with 1 M 1DMe alone for 30 min induced a significant 2-fold increase in MOP receptor Ser-377 phosphorylation (Fig. 1B). This effect seems specific for the NPFF2 receptor because activation of another Gi/o-coupled receptor endogenously expressed in SH-SY5Y cells, the ␣2-adrenergic receptor, with
Summary
Neuropeptide FF2 receptors interact with -opioid receptors and decrease their activity. To direct opioid receptor stimulation, activation of the NPFF2 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 Gi/o proteins by pertussis toxin. Activation of NPFF2 receptors in the recombinant (SH2-D9) cell line inhibits the opioid modulation of Ca2ϩ channels, reproducing the cellular anti-opioid activity observed in isolated neurons [23] This anti-opioid effect is not prevented by inhibition of second messenger-dependent kinases PKA and PKC [23], suggesting that it does not involve typical pathways of heterologous desensitization [24]. Mass spectrometric analysis indicated that the phosphorylation pattern of immunopurified MOP receptor is similar after homologous and heterologous stimulation, but the phosphorylation level is more intense after DAMGO treatment
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