Catherine Mollereau

Involvement of neuropeptide FF receptors in neuroadaptive responses to acute and chronic opiate treatments.

BACKGROUND AND PURPOSE Opiates remain the most effective compounds for alleviating severe pain across a wide range of conditions. However, their use is associated with significant side effects. Neuropeptide FF (NPFF) receptors have been implicated in several opiate‐induced neuroadaptive changes including the development of tolerance. In this study, we investigated the consequences of NPFF receptor blockade on acute and chronic stimulation of opioid receptors in mice by using RF9, a potent and selective antagonist of NPFF receptors that can be administered systemically.

Opioid-modulating properties of the neuropeptide FF system

Opioid receptors are involved in the control of pain perception in the central nervous system together with endogenous neuropeptides, termed opioid‐modulating peptides, participating in a homeostatic system. Neuropeptide FF (NPFF) and related peptides possess anti‐opioid properties, the cellular mechanisms of which are still unclear. The purpose of this review is to detail the phenomenon of cross‐talk taking place between opioid and NPFF systems at the in vivo pharmacological level and to propose cellular and molecular models of functioning. A better knowledge of the mechanisms underlying opioid‐modulating properties of NPFF has potential therapeutic interest for the control of opioid functions, notably for alleviating pain and/or for the treatment of opioid abuse.

Loss of Morphine Reward and Dependence in Mice Lacking G Protein–Coupled Receptor Kinase 5

BACKGROUND The clinical benefits of opioid drugs are counteracted by the development of tolerance and addiction. We provide in vivo evidence for the involvement of G protein-coupled receptor kinases (GRKs) in opioid dependence in addition to their roles in agonist-selective mu-opioid receptor (MOR) phosphorylation. METHODS In vivo MOR phosphorylation was examined by immunoprecipitation and nanoflow liquid chromatography-tandem mass spectrometry analysis. Using the hot-plate and conditioned place preference test, we investigated opioid-related antinociception and reward effects in mice lacking GRK3 or GRK5. RESULTS Etonitazene and fentanyl stimulated the in vivo phosphorylation of multiple carboxyl-terminal phosphate acceptor sites, including threonine 370, serine 375, and threonine 379, which was predominantly mediated by GRK3. By contrast, morphine promoted a selective phosphorylation of serine 375 that was predominantly mediated by GRK5. In contrast to GRK3 knockout mice, GRK5 knockout mice exhibited reduced antinociceptive responses after morphine administration and developed morphine tolerance similar to wild-type mice but fewer signs of physical dependence. Also, morphine was ineffective in inducing conditioned place preference in GRK5 knockout mice, whereas cocaine conditioned place preference was retained. However, the reward properties of morphine were evident in knock-in mice expressing a phosphorylation-deficient S375A mutation of the MOR. CONCLUSIONS These findings show for the first time that MOR phosphorylation is regulated by agonist-selective recruitment of distinct GRK isoforms that influence different opioid-related behaviors. Modulation of GRK5 function could serve as a new approach for preventing addiction to opioids, while maintaining the analgesic properties of opioid drugs at an effective level.

Characterization of two novel tritiated radioligands for labelling Neuropeptide FF (NPFF1 and NPFF2) receptors

The binding characteristics of [(3)H]-NPVF and [(3)H]-EYF, the two first tritiated probes for the respective labelling of NPFF(1) and NPFF(2) receptors, are presented. In membranes from CHO cells transfected with the human NPFF(1) receptor, [(3)H]-NPVF labelled one class of binding sites with a high affinity (Bmax=4pmol/mg protein, Kd=2.65nM). In membranes from CHO cells transfected with the human NPFF(2) receptor, [(3)H]-EYF labelled one class of binding sites with a high affinity (Bmax=16pmol/mg protein, Kd=0.54nM). Both radioligands exhibited time-dependent binding, low (10-20%) non-specific binding and poor cross-reactivity towards the related receptor subtype. The potency of different NPFF ligands to displace [(3)H]-NPVF and [(3)H]-EYF binding profiles was in good agreement with the profile previously measured by using (125)I-probes (NPFF(1) receptor: NPVF> or =1DMe=SPA-NPFF>NPFF=SQA-NPFF=QFW-NPSF>NPSF>RF9; NPFF(2) receptor: SPA-NPFF>>SQA-NPFF=QFW-NPSF=1DMe=NPFF>>NPSF=NPVF>RF9). Therefore, [(3)H]-NPVF and [(3)H]-EYF are new valuable tools for performing binding on NPFF receptors.

Neuropeptide FF-sensitive confinement of mu opioid receptor does not involve lipid rafts in SH-SY5Y cells.

Mu opioid (MOP) receptor activation can be functionally modulated by stimulation of Neuropeptide FF 2 (NPFF(2)) G protein-coupled receptors. Fluorescence recovery after photobleaching experiments have shown that activation of the NPFF(2) receptor dramatically reduces the fraction of MOP receptors confined in microdomains of the plasma membrane of SH-SY5Y neuroblastoma cells. The aim of the present work was to assess if the direct observation of receptor compartmentation by fluorescence techniques in living cells could be related to indirect estimation of receptor partitioning in lipid rafts after biochemical fractionation of the cell. Our results show that MOP receptor distribution in lipid rafts is highly dependent upon the method of purification, questioning the interpretation of previous data regarding MOP receptor compartmentation. Moreover, the NPFF analogue 1DMe does not modify the distribution profile of MOP receptors, clearly demonstrating that membrane fractionation data do not correlate with direct measurement of receptor compartmentation in living cells.

Heterologous regulation of Mu-opioid (MOP) receptor mobility in the membrane of SH-SY5Y cells.

Background: MOP receptor function is presumably linked to a specific dynamic organization in the membrane. Results: Inhibition of MOP receptor signaling by NPFF2 and α2 receptors is accompanied by diffusion changes, with a particular behavior for heterodimers. Conclusion: MOP receptor function, diffusion, and confinement are subject to specific heterologous regulation by other GPCRs. Significance: Specific GPCR regulation is associated with particular dynamic organization in the membrane. The dynamic organization of G protein-coupled receptors in the plasma membrane is suspected of playing a role in their function. The regulation of the diffusion mode of the mu-opioid (MOP) receptor was previously shown to be agonist-specific. Here we investigate the regulation of MOP receptor diffusion by heterologous activation of other G protein-coupled receptors and characterize the dynamic properties of the MOP receptor within the heterodimer MOP/neuropeptide FF (NPFF2) receptor. The data show that the dynamics and signaling of the MOP receptor in SH-SY5Y cells are modified by the activation of α2-adrenergic and NPFF2 receptors, but not by the activation of receptors not described to interact with the opioid receptor. By combining, for the first time, fluorescence recovery after photobleaching at variable radius experiments with bimolecular fluorescence complementation, we show that the MOP/NPFF2 heterodimer adopts a specific diffusion behavior that corresponds to a mix of the dynamic properties of both MOP and NPFF2 receptors. Altogether, the data suggest that heterologous regulation is accompanied by a specific organization of receptors in the membrane.

Pharmacological characterization of the mouse NPFF2 receptor

This study presents the binding and functional properties of the mouse NPFF(2) (mNPFF(2)) receptor, in comparison with its human counterpart (hNPFF(2)). Binding experiments were performed by using the NPFF(2) selective radioligand [(3)H]-EYF in membranes from CHO cells transfected with mouse and human NPFF(2) receptors and compared to membranes from mouse olfactory bulb, the brain region expressing the highest density of NPFF(2) receptors in mouse. mNPFF(2) receptors exhibited a high affinity (Kd=0.2-0.4 nM) for [(3)H]-EYF, comparable to that of hNPFF(2) receptors. Also, the binding selectivity profile of mNPFF(2) receptors was comparable to that of hNPFF(2) receptors, except for three ligands (NPSF, NPVF, RF9) that were about tenfold more potent and active on mouse receptors than on human receptors. In particular, compared to hNPFF(2) receptors, mNPFF(2) receptors were less discriminative towards the proNPFF(B)-derived peptide. This suggests some species-related differences in the binding properties of NPFF(2) receptors that could have repercussion when evaluating the pharmacological properties of drugs in vivo.

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 NPFF(2) receptor, in which the neuronal anti-opioid activity of NPFF was previously reproduced. Activation of G-proteins was monitored by [(35)S]GTPgammaS 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 [(35)S]GTPgammaS 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 alpha(s), alpha(i2) and beta 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.

Development of sub-nanomolar dipeptidic ligands of neuropeptide FF receptors

Based on our earlier reported neuropeptide FF receptors antagonist (RF9), we carried out an extensive structural exploration of the N-terminus part of the amidated dipeptide Arg-Phe-NH(2) in order to establish a structure-activity relationships (SAR) study towards both NPFF receptor subtypes. This SAR led to the discovery of dipeptides (12, 35) with subnanomolar affinities towards NPFF1 receptor subtype, similar to endogenous ligand NPVF. More particularly, compound 12 exhibited a potent in vivo preventive effect on opioid-induced hyperalgesia at low dose. The significant selectivity of 12 toward NPFF1-R indicates that this receptor subtype may play a critical role in the anti-opioid activity of NPFF-like peptides.

Neanderthal and Denisova tooth protein variants in present-day humans

Environment parameters, diet and genetic factors interact to shape tooth morphostructure. In the human lineage, archaic and modern hominins show differences in dental traits, including enamel thickness, but variability also exists among living populations. Several polymorphisms, in particular in the non-collagenous extracellular matrix proteins of the tooth hard tissues, like enamelin, are involved in dental structure variation and defects and may be associated with dental disorders or susceptibility to caries. To gain insights into the relationships between tooth protein polymorphisms and dental structural morphology and defects, we searched for non-synonymous polymorphisms in tooth proteins from Neanderthal and Denisova hominins. The objective was to identify archaic-specific missense variants that may explain the dental morphostructural variability between extinct and modern humans, and to explore their putative impact on present-day dental phenotypes. Thirteen non-collagenous extracellular matrix proteins specific to hard dental tissues have been selected, searched in the publicly available sequence databases of Neanderthal and Denisova individuals and compared with modern human genome data. A total of 16 non-synonymous polymorphisms were identified in 6 proteins (ameloblastin, amelotin, cementum protein 1, dentin matrix acidic phosphoprotein 1, enamelin and matrix Gla protein). Most of them are encoded by dentin and enamel genes located on chromosome 4, previously reported to show signs of archaic introgression within Africa. Among the variants shared with modern humans, two are ancestral (common with apes) and one is the derived enamelin major variant, T648I (rs7671281), associated with a thinner enamel and specific to the Homo lineage. All the others are specific to Neanderthals and Denisova, and are found at a very low frequency in modern Africans or East and South Asians, suggesting that they may be related to particular dental traits or disease susceptibility in these populations. This modern regional distribution of archaic dental polymorphisms may reflect persistence of archaic variants in some populations and may contribute in part to the geographic dental variations described in modern humans.