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.