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International audience; The neuropeptide FF2 (NPFF 2) receptor, predominantly expressed in the central nervous system, plays an important role in the modulation of sensory input and opioid analgesia, as well as in locomotion, feeding, intestinal motility, reward, and the control of obesity. The NPFF 2 receptor belongs to the RFamide peptide receptor family and to the G protein coupled receptor (GPCR) super family, but contrary to many other class A GPCRs, no 3D structure has been solved. Thus, it is essential to perform mutagenesis to gain information on the fine functioning of the NPFF 2 receptor. In this study, we examined the role of aspartic acid (D) from the "D/ERY/F" motif found in the second intracellular loop (ICL2) and the role of the C-terminal end of the receptor in ligand binding and signal transduction. We found that mutation D3.49A does not impair binding capacities but inhibits G protein activation as well as adenylyl cyclase regulation. Truncation of the C terminal part of the receptor has different effects depending on the position of truncation. When truncation was realized downstream of the putative acylation site, ligand binding and signal transduction capabilities were not lost, contrary to total deletion of the C terminus, which totally impairs the activity of the receptor.

In the context of human evolution, the study of proteins may overcome the limitation of the high degradation of ancient DNA over time to provide biomolecular information useful for the phylogenetic reconstruction of hominid taxa. In this study, we used a shotgun proteomics approach to compare the tooth proteomes of extant human and non-human primates (gorilla, chimpanzee, orangutan and baboon) in order to search for a panel of peptides able to discriminate between taxa and further help reconstructing the evolutionary relationships of fossil primates. Among the 25 proteins shared by the five genera datasets, we found a combination of peptides with sequence variations allowing to differentiate the hominid taxa in the proteins AHSG, AMBN, APOA1, BGN, C9, COL11A2, COL22A1, COL3A1, DSPP, F2, LUM, OMD, PCOLCE and SERPINA1. The phylogenetic tree confirms the placement of the samples in the appropriate genus branches. Altogether, the results provide experimental evidence that a shotgun proteomics approach on dental tissue has the potential to detect taxonomic variation, which is promising for future investigations of uncharacterized and/or fossil hominid/hominin specimens. SIGNIFICANCE: A shotgun proteomics approach on human and non-human primate teeth allowed to identify peptides with taxonomic interest, highlighting the potential for future studies on hominid fossils.

International audience; The study demonstrates the high potential of MS-based proteomics coupled to an iterative database search strategy for the in-depth investigation of ancient proteomes. An efficient targeted PRM MS-based approach, although limited to the detection of a single pair of sex-specific amelogenin peptides, allowed confirming the sex of individuals in ancient dental remains, an essential information for paleoanthropologists facing the issue of sex determination and dimorphism.

Methionine-enkephalin-Arg-Phe is an endogenous amphiactive analgesic peptide. Neuropeptide FF, on the other hand, is reported for its role in opioid modulation and tolerance development. Based on these reports, in the present study we designed a chimeric peptide NPYFa (YGGFMKKKPQRFamide), having the Met-enkephalin (opioid) and PQRFamide sequence of neuropeptide FF, which can then target both the opioid and neuropeptide FF receptors. We hypothesized that the chimeric peptide so designed would have both analgesic properties and further aid in understanding of the role of neuropeptide FF in the development of opiate tolerance. Our studies indicated that NPYFa induced an early onset, potent, dose-dependent and prolonged antinociception. Additionally, antagonists (MOR, KOR, and DOR) pretreatment studies determined a KOR-mediated antinociception activity of the ligand. Further, in vitro binding studies using the Eu-GTP-γS binding assay on cell lines expressing opioid and NPFF receptors showed binding to both the opioid and neuropeptide FF receptors suggesting a multiple receptor binding character of NPYFa. Moreover, chronic (6 days) treatment with NPYFa exhibited an absence of tolerance development subsequent to its analgesia. The current study proposes NPYFa as a potent, long-acting antinociceptor lacking tolerance development as well as a probe to study opioid analgesia and the associated complex mechanisms of tolerance development.

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.

Derivatization of biologically active peptides by conjugation with fluorophores or radionuclide-bearing moieties is an effective and commonly used approach to prepare molecular tools and diagnostic agents. Whereas lysine, cysteine, and N-terminal amino acids have been mostly used for peptide conjugation, we describe a new, widely applicable approach to peptide conjugation based on the nonclassical bioisosteric replacement of the guanidine group in arginine by a functionalized carbamoylguanidine moiety. Four arginine-containing peptide receptor ligands (angiotensin II, neurotensin(8-13), an analogue of the C-terminal pentapeptide of neuropeptide Y, and a neuropeptide FF analogue) were subject of this proof-of-concept study. The N(ω)-carbamoylated arginines, bearing spacers with a terminal amino group, were incorporated into the peptides by standard Fmoc solid phase peptide synthesis. The synthesized chemically stable peptide derivatives showed high receptor affinities with Ki values in the low nanomolar range, even when bulky fluorophores had been attached. Two new tritiated tracers for angiotensin and neurotensin receptors are described.

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.

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.

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.

Through the development of a new class of unnatural ornithine derivatives as bioisosteres of arginine, we have designed an orally active peptidomimetic antagonist of neuropeptide FF receptors (NPFFR). Systemic low-dose administration of this compound to rats blocked opioid-induced hyperalgesia, without any apparent side-effects. Interestingly, we also observed that this compound potentiated opioid-induced analgesia. This unnatural ornithine derivative provides a novel therapeutic approach for both improving analgesia and reducing hyperalgesia induced by opioids in patients being treated for chronic pain.

The neuropeptide FF2 (NPFF2) receptor belongs to the rhodopsin family of G protein-coupled receptors and mediates the effects of several related RFamide neuropeptides. One of the main pharmacological interests of this system resides in its ability to regulate endogenous opioid systems, making it a potential target to reduce the negative effects of chronic opioid use. Phosphorylation of intracellular residues is the most extensively studied post-translational modification regulating G protein-coupled receptor activity. However, until now, no information concerning NPFF2 receptor phosphorylation is available. In this study, we combined mass spectrometric analysis and site-directed mutagenesis to analyze for the first time the phosphorylation pattern of the NPFF2 receptor and the role of the various phosphorylation sites in receptor signaling, desensitization, and trafficking in a SH-SY5Y model cell line. We identified the major, likely GRK-dependent, phosphorylation cluster responsible for acute desensitization, (412)TNST(415) at the end of the C terminus of the receptor, and additional sites involved in desensitization ((372)TS(373)) and internalization (Ser(395)). We thus demonstrate the key role played by phosphorylation in the regulation of NPFF2 receptor activity and trafficking. Our data also provide additional evidence supporting the concept that desensitization and internalization are partially independent processes relying on distinct phosphorylation patterns.

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.

The zwitterionic detergent CHAPS was used to solubilize the human mu-opioid receptor (hMOR) from SH-SY5Y neuroblastoma cells and recombinant hMOR-CHO (CHO-T7-hMOR) and hMOR-SH-SY5Y (SH-SY5Y-T7-hMOR) cell membranes. Agonist stimulation and G-protein activation by the mu-selective opioid agonist DAMGO ([D-Ala2, N-MePhe4, Gly-ol]-enkephalin) were recovered after removing of CHAPS after polyethylene glycol (PEG) precipitation. Binding assays show that hMOR solubilized and reconstituted this way was functional and able to interact with both agonist peptides and with G-protein. The effective solubilization and reconstitution of hMOR from mammalian cells, without truncation and extensive modification, represent an essential step toward the purification of a receptor bearing important post-translational modifications.

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.

1. Neuropeptides FF (NPFF) and AF (NPAF) are involved in pain modulation and opioid tolerance. These peptides were known to act through uncharacterized G protein-coupled receptors (GPCR). We describe here, using an aequorin-based assay as screening tool, that an orphan GPCR, previously designated HLWAR77, is a functional high affinity receptor for NPFF and related peptides. This receptor is further designated as NPFFR. 2. Binding experiments were performed with a new radioiodinated probe, [(125)I]-EYF, derived from the EFW-NPSF sequence of the rat NPFF precursor. Chinese hamster ovary (CHO) cell membranes expressing NPFFR bound [(125)I]-EYF with a K(d) of 0.06 nM. Various NPFF analogues and related peptides inhibited [(125)I]-EYF specific binding with the following rank order (K(i)): human NPAF (0.22 nM), SQA-NPFF (0.29 nM), NPFF (0.30 nM), 1DMe (0.31 nM), EYW-NPSF (0.32 nM), QFW-NPSF (0.35 nM), 3D (1.12 nM), Met-enk-RF-NH(2) (3.25 nM), FMRF-NH(2) (10.5 nM) and NPSF (12.1 nM). 3. The stimulatory activity of the same set of peptides was measured by a functional assay based on the co-expression of NPFFR, G(alpha 16) and apoaequorin. The rank order of potency was consistent with the results of the binding assay. 4. Membranes from NPFFR expressing CHO cells bound GTP gamma[(35)S] in the presence of SQA-NPFF. This functional response was prevented by pertussis toxin treatment, demonstrating the involvement of G(i) family members. 5. SQA-NPFF inhibited forskolin induced cyclic AMP accumulation in recombinant CHO cells in a dose dependent manner. This response was abolished as well by pertussis toxin pre-treatment. 6. RT -- PCR analysis of human tissues mRNA revealed that expression of NPFFR was mainly detected in placenta, thymus and at lower levels in pituitary gland, spleen and testis.

[ 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 cloning of several receptors activated by either CC or CXC chemokines and belonging to the G protein-coupled family of receptors has been reported recently. In the present work, we describe the cloning of a human gene, named ChemR13, encoding a new CC-chemokine receptor. The gene encodes a protein of 352 amino acids with a calculated molecular mass of 40 600 Da and displaying a single potential site for N-linked glycosylation. Using a set of overlapping lambda clones, the genomic organisation of the locus was investigated, demonstrating that the ChemR13 gene is physically linked, and in the same orientation, as the CC-CKR2 gene that encodes a receptor for the monocyte chemoattractant protein-1 (MCP-1). A distance of 17.5 kb separates the two coding regions, which share 75% identity in nucleic acid and amino acid sequences. Human ChemR13 was functionally expressed in a stably transfected CHO-K1 cell line. Physiological responses to chemokines were monitored using a microphysiometer. Macrophage inflammatory protein 1 alpha (MIP-1 alpha) was the most potent agonist. MIP-1 beta and RANTES were also active at physiological concentrations. The other CC-chemokines, MCP-1, MCP-2 and MCP-3, as well as CXC-chemokines (IL-8, GRO alpha) had no effect. ChemR13 receptor transcripts were detected by Northern blotting in the promyeloblastic cell line KG-1A, suggesting a potential role in the control of granulocytic lineage proliferation or differentiation. ChemR13 is thus a new member of the growing family of chemokine receptors that mediate the recruitment of cells involved in immune and inflammatory processes. Being the fifth functionally identified receptor in his class, this new CC-chemokine receptor (CC-CKR) is tentatively designated CC-CKR5.

Selective PCR amplification of human and mouse genomic DNAs with oligonucleotides encoding highly conserved regions of the delta-opioid and somatostatin receptors generated a human DNA probe (hOP01, 761 bp) and its murine counterpart (mOP86, 447 bp). hOP01 was used to screen a cDNA library from human brainstem. A clone (named hORL1) was isolated, sequenced and found to encode a protein of 370 amino acids whose primary structure displays the seven putative membrane-spanning domains of a G protein-coupled membrane receptor. The hORL1 receptor is most closely related to opioid receptors not only on structural (sequence) but also on functional grounds: hORL1 is 49-50% identical to the murine mu-, delta- and kappa-opioid receptors and, in CHO-K1 cells stably transfected with a pRc/CMV:hORL1 construct, ORL1 mediates inhibition of adenylyl cyclase by etorphine, a 'universal' (nonselective) opiate agonist. Yet, hORL1 appears not to be a typical opioid receptor. Neither is it a somatostatin or sigma (N-allylnormetazocine) receptor. mRNAs hybridizing with synthetic oligonucleotides complementary to mOP86 are present in many regions of the mouse brain and spinal cord, particularly in limbic (amygdala, hippocampus, septum, habenula, ...) and hypothalamic structures. We conclude that the hORL1 receptor is a new member of the opioid receptor family with a potential role in modulating a number of brain functions, including instinctive behaviours and emotions.

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.

Neuropeptide FF (NPFF, FLFQPQRFamide) receptors modulate endogenous opioid functions. Here, we report the solubilization of the human NPFF2 receptor expressed in Chinese hamster ovary (CHO) cells by the zwitterionic detergent Chaps. Chaps solubilization resulted in the abolishment of specific agonist binding activity, which was restored by a polyethylene glycol (PEG) precipitation method. Reincorporation after the precipitation step into liposomes made of endogenous lipids issued from CHO membranes or exogenous lipids significantly enhanced the specific agonist binding activity and G-protein coupling. This method of solubilization and lipid reconstitution could be useful for studies of NPFF receptors.

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.

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.

In order to elucidate the mechanisms of the neuronal anti-opioid activity of Neuropeptide FF, we have transfected the SH-SY5Y neuroblastoma cell line, which expresses μ- and δ-opioid receptors, with the human NPFF 1 receptor. The SH 1 -C7 clone expresses high affinity NPFF 1 receptors in the same range order of density as opioid receptors. Similarly to the opioids, acute stimulation with the NPFF 1 agonist NPVF inhibits adenylyl cyclase activity and voltage-gated (N-type) Ca 2+ currents and enhances the intracellular Ca 2+ release triggered by muscarinic receptors activation. In contrast, preincubation of cells with NPVF decreases the response to opioids on both calcium signaling, thus reproducing the cellular anti-opioid activity described in neurons. SH 1 -C7 cells are therefore a suitable model to investigate the interactions between NPFF and opioid receptors.

Neuropeptide FF, a member of the RFamide family of peptides, has demonstrated an interesting array of pharmacological effects. To date however, little information has been obtained as to the exact pharmacological roles of the individual NPFF 1 and NPFF 2 receptors. Through peptide analogs of NPFF and related peptides, the essential pharmacophore has emerged somewhat. Yet, the field is lacking small molecule ligands selective for each receptor. This review of the structure–activity relationships of the reported NPFF peptide analogs and some non-selective small molecule ligands highlights the current understanding of the pharmacophoric elements required for affinity and activity at the NPFF receptors. The lack of mutagenesis data on the receptor as well as a crystal structure has also hindered the understanding of ligand recognition at the receptor level. If the targets can be further investigated as to their requirements for ligand recognition, the successful development of highly selective ligands should follow.

NPFF agonists designed to be selective NPFF(2) receptor probes were synthesized. D.Asn-Pro-(N-Me)Ala-Phe-Leu-Phe-Gln-Pro-Gln-Arg-Phe-NH(2) (dNPA) displays a very high affinity (0.027nM) for NPFF(2) receptors transfected in CHO cells, and a very high selectivity with a discrimination ratio greater than 100 versus NPFF(1) receptors. dNPA acts as a potent and selective agonist in [(35)S]GTPgammaS binding experiments and inhibits intracellular cAMP production with the same efficacy as NPA-NPFF. In SH-SY5Y cells expressing NPFF(2) receptors dNPA, in the presence of carbachol, stimulates Ca(2+) release from the intracellular stores. In vivo, after intracerebroventricular injection dNPA increases body temperature in mice and reverses the morphine-induced analgesia. Also, dNPA displays anti-opioid activity after systemic administration. So far, dNPA exhibits the highest affinity and selectivity for NPFF(2) receptors and reveals that its behavioral anti-opioid activity depends on the degree of opioid-induced analgesia.

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 .

Neuropeptide FF (NPFF) belongs to an opioid-modulatory system including two precursors (pro-NPFF(A) and pro-NPFF(B)) and two G-protein coupled receptors (NPFF(1) and NPFF(2)). The pharmacological and functional profiles of human NPFF(1) and NPFF(2) receptors expressed in Chinese hamster ovary (CHO) cells were compared by determining the affinity of several peptides derived from both NPFF precursors and by measuring their abilities to inhibit forskolin-induced cAMP accumulation. Each NPFF receptor recognizes peptides from both precursors with nanomolar affinities, however, with a slight preference of pro-NPFF(A) peptides for NPFF(2) receptors and of pro-NPFF(B) peptides for NPFF(1) receptors. BIBP3226 ((R)-N(2)-(diphenylacetyl)-N-[(4-hydroxyphenyl)-methyl]-argininamide) and BIBO3304 ((R)-N(2)-(diphenylacetyl)-N-[4-(aminocarbonylaminomethyl)-benzyl]-argininamide trifluoroacetate), two selective neuropeptide Y (NPY) Y(1) receptor antagonists, display relative high affinities for NPFF receptors and exhibit antagonist properties towards hNPFF(1) receptors. The structural determinants responsible for binding of these molecules to NPFF receptors were investigated and led to the synthesis of hNPFF(1) receptor antagonists with affinities from 40 to 80 nM. Our results demonstrate differences in pharmacological characteristics between NPFF(1) and NPFF(2) receptors and the feasibility of subtype-selective antagonists.

The ORL1 receptor is a G protein-coupled receptor structurally related to the opioid receptors, whose endogenous ligand is the heptadecapeptide nociceptin/orphanin FQ. In this review, data which have contributed to the mapping of the anatomic distribution of the ORL1 receptor have been collated with an emphasis on their relation to physiological functions. The ORL1 receptor is widely expressed in the central nervous system, in particular in the forebrain (cortical areas, olfactory regions, limbic structures, thalamus), throughout the brainstem (central periaqueductal gray, substantia nigra, several sensory and motor nuclei), and in both the dorsal and ventral horns of the spinal cord. Regions almost devoid of ORL1 receptors are the caudate-putamen and the cerebellum. ORL1 mRNA and binding sites exhibit approximately the same distribution pattern, indicating that the ORL1 receptor is located on local neuronal circuits. The ORL1 receptor is also expressed at the periphery in smooth muscles, peripheral ganglia, and the immune system. The anatomic distribution of ORL1 receptor suggests a broad spectrum of action for the nociceptin/orphanin FQ system (sensory perception, memory process, emotional behavior, etc.).

The aim of the present study was to delineate the functional domains of nociceptin (noc), a neuropeptide which is structurally related to dynorphin A (dyn). The binding and biological potencies towards the nociceptin (ORL1) and dynorphin A (kappa-opioid) receptors of twenty dyn/noc and noc/dyn hybrid peptides were compared with those of the parent heptadecapeptides. Replacement of as many as eleven residues in the C-terminus of dynorphin by the corresponding nociceptin sequence has no significant effect on binding and biological activity towards the kappa-opioid receptor. In marked contrast, replacement of as few as six residues (RKLANQ) in the C-terminus of nociceptin by the corresponding dynorphin sequence (LKWDNQ) dramatically impairs both affinity and activity towards the ORL1 receptor. This clearly indicates that the two neuropeptides have different functional architectures, despite the dual structural homology of both ligands and receptors. Moreover, the recombinant peptide approach led us to identify hybrids whose sequences differ only at positions 5 and 6 and displaying opposite or no receptor selectivity. One contains the dynorphin Leu5-Arg6 sequence and prefers the kappa-opioid receptor, whereas the other comprises the nociceptin Thr5-Gly6 sequence and prefers the ORL1 receptor. A third, containing the mixed dynorphin/nociceptin Leu5-Gly6 sequence, does not discriminate between the two types of receptor.

G-protein coupled receptors (GPCRs) play a major role in a number of physiological and pathological processes. Thus, GPCRs have become the most frequent targets for development of new therapeutic drugs. In this context, the availability of highly specific antibodies may be decisive to obtain reliable findings on localization, function and medical relevance of GPCRs. However, the rapid and easy generation of highly selective anti-GPCR antibodies is still a challenge. Herein, we report that highly specific antibodies suitable for detection of GPCRs in native and unfolded forms can be elicited by immunizing animals against purified full length denatured recombinant GPCRs. Contrasting with the currently admitted postulate, our study shows that an active and well-folded GPCR is not required for the production of specific anti-GPCR antibodies. This new immunizing strategy validated with three different human GPCR (μ-opioid, κ-opioid, neuropeptide FF2 receptors) might be generalized to other members of the GPCR family.

A cDNA clone encoding a receptor protein which presents all the characteristics of a guanine-nucleotide-binding protein (G-protein)-coupled receptor was isolated from a human brain stem cDNA library. The probe used (HGMP08) was a 600 bp DNA fragment amplified by a low-stringency PCR, using human genomic DNA as template and degenerate oligonucleotide primers corresponding to conserved sequences amongst the known G-protein-coupled receptors. The deduced amino acid sequence encodes a protein of 472 residues which shares 97.3% identity with the rat cannabinoid receptor cloned recently [Matsuda, Lolait, Brownstein, Young & Bronner (1990) Nature (London) 346, 561-564]. Abundant transcripts were detected in the brain, as expected, but lower amounts were also found in the testis. The same probe was used to screen a human testis cDNA library. The cDNA clones obtained were partially sequenced, demonstrating the identity of the cannabinoid receptors expressed in both tissues. Specific binding of the synthetic cannabinoid ligand [3H]CP55940 was observed on membranes from Cos-7 cells transfected with the recombinant receptor clone. In stably transfected CHO-K1 cell lines, cannabinoid agonists mediated a dose-dependent and stereoselective inhibition of forskolin-induced cyclic AMP accumulation. The ability to express the human cannabinoid receptor in mammalian cells should help in developing more selective drugs, and should facilitate the search for the endogenous cannabinoid ligand(s).

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.

In order to gain further insight into the functional architecture of structurally related G protein-coupled receptors, the ORL1 (nociceptin) and opioid receptors, we have constructed chimeras of ORL1 and mu-, delta- and kappa-opioid receptors, and compared their binding and functional properties with those of the parent receptors. We find in particular that a ORL1-kappa-opioid (O-K) hybrid construct has retained high affinity for non-type-selective opiate ligands, and has acquired the ability to bind and respond to enkephalins and mu- and/or delta-opioid receptor-selective enkephalins analogs, thus behaving like a 'universal' opioid receptor. Most significantly however, whilst the ORL1 and kappa-opioid receptors display high binding preference (KD 0.1 vs. 100 nM) for their respective endogenous ligands, nociceptin and dynorphin A, the O-K chimeric receptor binds both nociceptin and dynorphin A, with high affinity (KD1 nM). Together, these data (i) add weight to the hypothesis that the extracellular loops of opioid receptors act as a filter for ligand selection, and (ii) demonstrate that different domains of the ORL1 and kappa-opioid receptors are involved in recognition of their endogenous peptide ligands.

Many in vitro data have shown that the efficacy of several opioid drugs is correlated with differential mu-opioid (MOP) receptor phosphorylation. Label-free semiquantitative on-line nanoflow liquid chromatography–tandem mass spectrometry (nanoLC–MS/MS) analyses were performed to compare the endogenous MOP receptor phosphorylation patterns of mice administered with morphine, etonitazene and fentanyl. The analysis identified S363, T370 and S375 as phosphorylated residues in the carboxy-terminus. Only T370 and S375 were regulated by agonists, with a higher propensity to promote double phosphorylation for high efficacy agonists. Our study provides confirmation that differential agonist-driven multi-site phosphorylation of MOP receptor occurs in vivo and validate the use of MS to study endogenous GPCR phosphorylation.

To understand how two structurally analogous ligand-receptor systems, the nociceptin/opioid receptor-like 1 (ORL1) and dynorphin A/kappa-opioid receptor 1 (KOR1) systems, achieve selectivity, receptor chimeras were generated and analyzed. Replacing discrete domains located between the N-terminus and top of the third transmembrane helix of the KOR1 by the homologous domains of the ORL1 receptor yields hybrid receptors, which, in comparison with the parent KOR1, display up to 300-fold increased affinity but low sensitivity toward nociceptin, and unaltered (high) affinity and sensitivity toward dynorphin A. These substitutions contribute elements for binding of nociceptin but do not suppress determinants necessary for binding and potency of dynorphin A. More importantly, further replacement in these chimeras of the second extracellular loop with that of the ORL1 receptor fully restores responsiveness to nociceptin without impairing responsiveness to dynorphin A. A bifunctional hybrid receptor has thus been identified that binds and responds to both nociceptin and dynorphin A as efficiently as the ORL1 receptor does to nociceptin and the KOR1 to dynorphin A. Together, these results suggest that distinct peptide activation mechanisms operate in the two receptor systems. In particular, the second extracellular receptor loop appears to be an absolute requirement for activation of the ORL1 receptor by nociceptin, but not for activation of the KOR1 by dynorphin A.