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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.

International audience; Given the importance of G-protein coupled receptors in the regulation of many physiological functions, deciphering the relationships between genotype and phenotype in past and present hominin GPCRs is of main interest to understand the evolutionary process that contributed to the present-day variability in human traits and health. Here, we carefully examined the publicly available genomic and protein sequence databases of the archaic hominins (Neanderthal and Denisova) to draw up the catalog of coding variations in GPCRs for peptide ligands, in comparison with living humans. We then searched in the literature the functional changes, phenotypes and risk of disease possibly associated with the detected variants. Our survey suggests that Neanderthal and Denisovan hominins were likely prone to lower risk of obesity, to enhanced platelet aggregation in response to thrombin, to better response to infection, to less anxiety and aggressiveness and to favorable sociability. While some archaic variants were likely advantageous in the past, they might be responsible for maladaptive disorders today in the context of modern life and/or specific regional distribution. For example, an archaic haplotype in the neuromedin receptor 2 is susceptible to confer risk of diabetic nephropathy in type 1 diabetes in present-day Europeans. Paying attention to the pharmacological properties of some of the archaic variants described in this study may be helpful to understand the variability of therapeutic efficacy between individuals or ethnic groups.

Neuropeptide FF (NPFF) has been shown to act as an endogenous anti-analgesic peptide. In this paper, several peptide analogs of the selective ligand dNP(NMe)AFLFQPQRF-NH(2) modified in the putative address segment, were designed to be selective NPFF(2) receptor probes, synthesized and assayed. One peptide dA(NMe)AAFLFQPQRF-NH(2) displays a very high affinity for NPFF(2) receptors transfected in CHO cells, and a high selectivity versus NPFF(1) receptors. The exact residues carried in the N-terminal part of the ligands are not decisive to obtain a high affinity only the length of the peptide in itself seems important to create selectivity.

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.

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.

Activation of the NPFF(2) receptor reduces the inhibitory effect of opioids on the N-type Ca(2+) channel. Although this anti-opioid effect is specific for opioid receptors in neurons and tissues, it also affects NPY Y2 and alpha(2)-adrenoreceptors in undifferentiated SH-SY5Y cells stably expressing the NPFF(2) receptor. To test whether this difference could be due to the immaturity of these cells, they were differentiated to a noradrenergic neuronal phenotype with staurosporine. The differentiated cells ceased to divide and grew long, thin neurites. The inhibition of the depolarization-triggered Ca(2+) transient by activation of G(i)-coupled receptors was either unaffected (micro-opioid), increased (NPY), reduced (NPFF(2)) or lost (alpha(2)-adrenoreceptors). Following a 20 min incubation with 1DMe, the effect of DAMGO was reduced, as in undifferentiated cells, but the effect of NPY was no longer affected. Staurosporine differentiation did not modify the coupling of the micro-opioid and NPFF(2) receptors to the G(i/o) proteins. We suggest that the specificity of the effect of NPFF may not reside in the molecular mechanism of its anti-opioid activity itself but in the organization of receptors within the membrane.

Neuropeptide FF (NPFF) modulates the opioid system by exerting functional anti-opioid activity on neurons, the mechanism of which is unknown. By using a model of SH-SY5Y cells, we recently postulated that anti-opioid activity likely takes place upstream from the signaling cascade, suggesting that NPFF receptors could block opioid receptors by physical interaction. In the present study, fluorescence techniques were used to monitor the physical association and the dynamic of NPFF2 and μ-opioid (MOP) receptors tagged with variants of the green fluorescent protein. Importantly, cyan fluorescent protein-tagged NPFF2 receptors retained their capacity to antagonize opioid receptors. Fluorescence resonance energy transfer (FRET) and coimmunoprecipitation studies indicate that NPFF and MOP receptors are close enough to generate a basal FRET signal. The opioid agonist Tyr-d-Ala-Gly-NMe-Phe-Gly-ol disrupts by 20-30% this FRET signal, mainly because it concomitantly induces 40% internalization of receptors. In contrast, the NPFF analog 1DMe significantly increases by 10-15% the basal FRET signal, suggesting an association between both receptors. In addition, 1DMe reduces, by half, MOP receptor internalization, indicating that, besides a functional blockade of opioid receptors, the NPFF analog also inhibits their internalization. Finally, as a first report showing the modulation of the mobility of a G-protein-coupled receptor by another one, fluorescence recovery after photobleaching analysis reveals that 1DMe modifies the lateral diffusion of MOP receptors in the cell membrane, changing them from a confined to a freely diffusing state. By promoting NPFF-MOP receptor heteromerization, 1DMe could disrupt the domain organization of MOP receptors in the membrane, resulting in a reduction of opioid response.

The selectivity of two new radioligands, [ 125 I]YVP ([ 125 I]YVPNLPQRF-NH 2 ) and [ 125 I]EYF ([ 125 I]EYWSLAAPQRF-NH 2 ), for neuropeptide FF (NPFF) receptor subtypes was determined using HEK293 cells expressing hNPFF 1 and CHO cells expressing hNPFF 2 receptors. Saturation binding and displacement experiments showed that [ 125 I]YVP and [ 125 I]EYF bound selectively with a very high affinity, K D =0.18 nM and 0.06 nM, to NPFF 1 and NPFF 2 receptors respectively. By using in vitro autoradiography with these radioligands and frog pancreatic polypeptide (PP) as selective unlabelled competitor of NPFF 2 binding sites, NPFF 1 and NPFF 2 receptor distribution was analyzed throughout the rat CNS. The highest densities of [ 125 I]EYF binding sites were seen in the most external layers of the dorsal horn of the spinal cord, the parafascicular thalamic nucleus, laterodorsal thalamic nucleus and presubiculum of hippocampus. All specific binding of this radioligand was inhibited by 200 nM frog PP. The density of 0.1 nM [ 125 I]YVP binding was much smaller in all brain areas and frog PP-insensitive binding sites (NPFF 1 receptor subtype) were detected in septal, thalamic and hypothalamic areas but were absent in the spinal cord. The restricted distribution of NPFF 1 receptors in the CNS supports its specific role in a limited number of neuronal functions. In contrast to the rat spinal cord where the NPFF 1 system is absent, there is no strict separation between NPFF 1 and NPFF 2 system at the supraspinal level.

A structure-activity study was carried out to determine the importance of the C-terminal amino acids of the octapeptide Neuropeptide FF (NPFF) in binding and agonistic activity. Affinities of NPFF analogues were tested toward NPFF receptors of the rat spinal cord and the human NPFF 2 receptors transfected in CHO cells. The activities of these analogues were evaluated by their ability to both inhibit adenylate cyclase in NPFF 2 receptor transfected CHO cells and to reverse the effect of nociceptin on acutely dissociated rat dorsal raphe neurons. The substitutions of Phenylalanine 8 by a tyrosine, phenylglycine or homophenylalanine were deleterious for high affinity. Similarly, the replacement of Arginine 7 by a lysine or D.Arginine induces a loss in affinity. The pharmacological characterization showed that the presence of the amidated Phe 8 and Arg 7 residues are also extremely critical for activation of anti-opioid effects on dorsal raphe neurons. The sequence of the C-terminal dipeptide seems also to be responsible for the high affinity and the activity on human NPFF 2 receptors. The results support the view that a code messaging the molecular interaction toward NPFF-receptors is expressed in the C-terminal region of these peptides but the N-terminal segment is important to gain very high affinity.

[ 125 I]EYF ([ 125 I]EYWSLAAPQRFamide), a new radioiodinated probe derived from a peptide present in the rat Neuropeptide FF precursor (EFWSLAAPQRFamide, EFW-NPSF) was synthesized and its binding characteristics investigated on sections of the rat spinal cord and on membranes of mouse olfactory bulb. In both tissues, [ 125 I]EYF binding was saturable and revealed a very high affinity interaction with a single class of binding sites in rat and mouse (K D = 0.041 and 0.019 nM, respectively). Competition studies showed that [ 125 I]EYF bound to one class of binding sites exhibiting a high affinity for all the different peptides the precursor could generate (NPA-NPFF, SPA-NPFF, NPFF, EFW-NPSF, QFW-NPSF) with the exception of NPSF which displayed a low affinity. Autoradiographic studies demonstrated that [ 125 I]EYF binding sites were fully inhibited by a synthetic Neuropeptide FF agonist (1DMe) in all areas of the rat brain. The density of [ 125 I]EYF binding sites was high in the intralaminar thalamic nuclei, the parafascicular thalamic nucleus and in the superficial layers of the dorsal horn. Non specific binding reached 5–10% of the total binding in all brain areas. Similarly, in mouse brain experiments, the non-specific binding was never superior to 10%. These findings demonstrate that putative neuropeptides generated by the Neuropeptide FF precursor and containing the NPFF or NPSF sequences should bind to the same receptor. Furthermore, these data indicate that [ 125 I]EYF is a useful radiolabeled probe to investigate the NPFF receptors; its major advantages being its high affinity and the very low non-specific binding it induces.

The distribution of prepronociceptin messenger RNA, the recently identified endogenous ligand of the ORL1 receptor (opioid receptor-like-1), has been studied in the adult mouse central nervous system using in situ hybridization. Prepronociceptin is a new peptide precursor that generates, upon maturation, at least three bioactive peptides: nociceptin, noc2 and the recently described nocistatin. Considering both the density of labeled neurons per region and their intensity of labeling, the distribution of prepronociceptin messenger RNA-containing neurons can be summarized as follows: the highest level of prepronociceptin messenger RNA expression was detected in the septohippocampal nucleus, bed nucleus of the stria terminalis, central amygdaloid nucleus, and in selective thalamic nuclei such as the parafascicular, reticular, ventral lateral geniculate and zona incerta. High to moderate levels of prepronociceptin messenger RNA expression were detected in the lateral, ventral and medial septum, and were evident in brainstem structures implicated in descending antinociceptive pathways (e.g., the gigantocellular nucleus, raphe magnus nucleus, periaqueductal gray matter), and also observed in association with auditory relay nuclei such as the inferior colliculi, lateral lemniscus nucleus, medioventral preolivary nucleus and lateral superior nucleus. A moderate level of prepronociceptin messenger RNA expression was observed in the medial preoptic nucleus, ventromedial preoptic nucleus, periventricular nucleus, pedonculopontine tegmental nucleus, solitary tract nucleus and spinal trigeminal nucleus. A weak level of prepronociceptin messenger RNA expression was present in some areas, such as the cerebral cortex, endopiriform cortex, hippocampal formation, medial amygdaloid nucleus, anterior hypothalamic area, medial mammillary hypothalamic nuclei, retrorubral field and substantia nigra pars compacta. No labeled cells could be found in the caudate-putamen, nucleus accumbens and ventral tegmental area. The present data confirm that nociceptin is expressed in a broad array of regions of the central nervous system. In good correlation with the presently known physiological actions of nociceptin, they include, amongst others, brain areas conveying/integrating pain and auditory sensory afferences.

Nociceptin, also known as orphanin FQ, was recently identified as the naturally occurring agonist of orphan opioid receptor-like ORL1 receptor (Meunier et al., 1995, Nature 377, 532; Reinscheid et al., 1995, Science 270, 792). Nociceptin is a heptadecapeptide which, although it resembles dynorphin A, the endogenous agonist of the kappa-opioid receptor, displays very low potency in competing with binding of [3H]diprenorphine to or inhibiting adenylate cyclase via mu-, delta- and kappa-opioid receptors. Tritium-labeled nociceptin ([3H]nociceptin) was used here to establish a pharmacological profile in vitro of the ORL 1 receptor. In membranes from recombinant Chinese hamster ovary (CHO) cells expressing the ORL 1 receptor, equilibrium binding of [3H]nociceptin is highly specific, saturable (Bmax in the range 1.3-1.8 pmol/mg protein) and of high affinity (Kd approximately equal to 0.1 nM). It is selectively decreased in the presence of Na+ ions and/or of the GTP analog 5'-guanylylimido-diphosphate, an allosteric regulation that is analogous to that of opiate binding to opioid receptors. A few opiates, namely lofentanil, a 4-anilinopiperidine derivative and etorphine, a 6,14-endo-ethenotetrahydrothebaine derivative, were found to be quite potent not only in competing with binding of [3H]nociceptin at the ORL 1 receptor but also in inhibiting forskolin-induced accumulation of cyclic AMP in intact recombinant CHO cells. In a preliminary attempt to delineate active parts of the neuropeptide, nociceptin analogs were also tested, including N- and C-terminal truncation products. Our results suggest that the highly basic, internal core of nociceptin might be essential in conferring on the peptide both affinity for and activity at the ORL 1 receptor. In this respect, the message and address division of dynorphin A, nociceptin's closest structural analog, do not seem to apply to nociceptin.

Digitonin treatment of frog brain membranes in 50 mM Tris-HCl yields a soluble extract that contains nearly equal amounts of free and G protein-bound opioid receptor molecules (Mollereau et al., 1988, J. Biol. Chem. 263, 18003). We report here that the balance of the two forms of the opioid receptor in digitonin solution is dependent on the environment of the membrane suspension at the time of solubilization with the detergent. Preincubating the membrane suspension with 50 microM GppNHp or with 120 mM NaCl results, in the two cases, in a digitonin extract that no longer displays the G protein-bound form of the receptor, i.e., the form of the receptor which exhibits high affinity for the opiate agonist etorphine in binding studies, as well as large apparent molecular size in sucrose gradients. Assuming that the situation in soluble extracts faithfully reflects the one in the membrane, these results would exclude the possibility that in a physiological environment the opioid receptor is in part precoupled with a G protein in the absence of an opioid agonist.

Functional and structural studies on G-protein-coupled receptors (GPCRs) at molecular levels require producing and purifying high levels of receptors, and recombinant mammalian cell expression systems constitute the best systems to obtain receptors resembling those expressed in natural environments. In the course of increasing GPCR expression in Chinese hamster ovary (CHO) cells, we have expressed mu (μ)- and kappa (κ)-opioid receptors and neuropeptide FF 1 and FF 2 receptors (NPFF 1 and NPFF 2 , respectively) in dimethyl sulfoxide. This treatment did not modify the affinity ( K d ) for any receptor, but a significant increase in functional expression levels was observed for all receptors with the noticeable exception of NPFF 1 .

[Phe1psi(CH2-NH)Gly2]nociceptin-(1-13)-NH2, a pseudopeptide analog of nociceptin, has been shown to be a selective 'antagonist' of the nociceptin receptor in the isolated guinea pig ileum and mouse vas deferens preparations (Guerrini et al., 1998. Br. J. Pharmacol. 123, 163-165). However, in recombinant chinese hamster ovary cells expressing the human nociceptin receptor, we find that the pseudopeptide is a potent (IC50 = 7.5 nM) and fully efficacious inhibitor of forskolin-induced accumulation of cAMP, thus behaving as a pure 'agonist' rather than an antagonist of the receptor. The contrary behaviour of the pseudopeptide in smooth muscle and transformed cells may suggest that different nociceptin receptor types are being addressed in the two systems.

When assayed for specific opiate binding in the presence of 120 mM NaCl, digitonin extracts from frog (Rana ridibunda) brain membranes were found to contain about the same quantity (0.5 pmol/mg of protein) of high (Kdh = 0.4 nM) and of lower (Kdl = 15-20 nM) affinity sites for the opiate agonist [3H]etorphine. The two classes of [3H]etorphine binding sites displayed equally high (Kd = 0.3 nM) affinity for the opiate antagonist [3H]diprenorphine. 5'-Guanylylimidodiphosphate (GppNHp) selectively and potently (IC50 = 0.1 microM) inhibited high affinity binding of the tritiated agonist, and this inhibition resulted from the GppNHp-induced conversion of the high into the lower affinity sites for [3H]etorphine. Following centrifugation of the digitonin extract in sucrose gradients, opioid binding activity was found to be associated with two clearly separated macromolecular components of apparent sedimentation coefficients 11.5 and 9.7 S, respectively. The two components bound [3H]diprenorphine equally well, whereas the fast sedimented component bound [3H]etorphine better than did the slower sedimented one. In addition, labeling of the component of bigger apparent size with [3H]etorphine was considerably reduced in the presence of 50 microM GppNHp. Finally, in soluble extracts which had been (i) preincubated with and (ii) centrifuged in the presence of GppNHp, the fast sedimented component was no longer observed while there was about twice as much of the component of smaller apparent size as in control (no GppNHp) extracts. Together, these results demonstrated the existence of an opioid receptor-G protein complex which, in digitonin solution, was still amenable to regulation (dissociation) by guanine nucleotides.