Modulation by Neuropeptide FF of the interaction of Mu-opioid (MOP) receptor with G-proteins

The Neuropeptide FF (NPFF) system is known to modulate the effects of opioids in vivo and in vitro. In the present study, we have investigated the effect of NPFF agonists on the coupling of the Mu-opioid (MOP) receptor to G-proteins in a model of SH-SY5Y cells transfected with NPFF2 receptor, in which the neuronal anti-opioid activity of NPFF was previously reproduced. Activation of G-proteins was monitored by [35S]GTPγS binding assay and analysis of G-protein subunits associated with MOP receptors was performed by Western blotting after immunoprecipitation of the receptor. The results demonstrate that concentrations of NPFF agonists that produce a cellular anti-opioid effect, did not affect the ability of the opioid agonist DAMGO to activate G-proteins. However, at saturating concentration of agonist or when expression of receptor was high, opioid and NPFF agonists did not stimulate [35S]GTPγS binding in an additive manner, indicating that both receptors share a common fraction of a G-protein pool. In addition, stimulation of NPFF receptors in living cells modified the G-protein environment of MOP receptor by favoring its interaction with αs, αi2 and β subunits. This change in G-protein coupling to MOP receptor might participate in the mechanism by which NPFF agonists reduce the inhibitory activity of opioids.