Cholesterol-rich lipid rafts are involved in neuropeptide FF anti-nociceptin/orphanin FQ effect.

The participation of a signaling platform to the anti-nociceptin/orphanin FQ (N/OFQ) effect of neuropeptide FF (NPFF) receptors was investigated in both acutely dissociated neurons and SH-SY5Y human neuroblastoma cells. The NPFF anti-N/OFQ, not anti-µ opioid effect, on the Ca2+ transient triggered by depolarization was reversed by methyl-ß-cyclodextrin which depletes cholesterol from cell membranes. While the inactive α-cyclodextrin had no effect. By using [35 S]GTPγS binding assay, a significant 20% decrease in the activity of nociceptin/orphanin FQ peptide receptors induced by the NPFF analog 1DMe was observed in detergent-resistant membranes, but not in total membranes of SH-SY5Y cells. Moreover, siRNA knock-down of G-protein-coupled receptor kinase 2 indicated that G-protein-coupled receptor kinase 2, but not protein kinase C, acted as the mediator in the NPFF anti-N/OFQ process. These data indicate that cholesterol-rich lipid rafts play an important role in the anti-N/OFQ effect of NPFF receptors. We proposed the participation of a signaling platform to the anti-Nociceptin/Orphanin FQ (N/OFQ) effect of Neuropeptide FF (NPFF) receptors both in mouse neurons and SH-SY5Y cells, with GRK2 protein acting as the mediator in this process. These findings should provide a more precise way to understand the anti-opioid effect of NPFF. NOP, Nociceptin/Orphanin FQ peptide.

Optimized Proteomic Mass Spectrometry Characterization of Recombinant Human µ-Opioid Receptor Functionally Expressed in Pichia pastoris Cell Lines.

Human µ-opioid receptor (hMOR) is a class-A G-protein-coupled receptor (GPCR), a prime therapeutic target for the management of moderate and severe pain. A chimeric form of the receptor has been cocrystallized with an opioid antagonist and resolved by X-ray diffraction; however, further direct structural analysis is still required to identify the active form of the receptor to facilitate the rational design of hMOR-selective agonist and antagonists with therapeutic potential. Toward this goal and in spite of the intrinsic difficulties posed by the highly hydrophobic transmembrane motives of hMOR, we have comprehensively characterized by mass spectrometry (MS) analysis the primary sequence of the functional hMOR. Recombinant hMOR was overexpressed as a C-terminal c-myc and 6-his tagged protein using an optimized expression procedure in Pichia pastoris cells. After membrane solubilization and metal-affinity chromatography purification, a procedure was devised to enhance the concentration of the receptor. Subsequent combinations of in-solution and in-gel digestions using either trypsin, chymotrypsin, or proteinase K, followed by matrix-assisted laser desorption ionization time-of-flight MS or nanoliquid chromatography coupled with tandem MS analyses afforded an overall sequence coverage of up to >80%, a level of description first attained for an opioid receptor and one of the six such high-coverage MS-based analyses of any GPCR.

Identification and functional characterization of the phosphorylation sites of the neuropeptide FF2 receptor.

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.

A high-affinity, radioiodinatable neuropeptide FF analogue incorporating a photolabile p-(4-hydroxybenzoyl)phenylalanine.

A new radioiodinated photoaffinity compound, [(125)I]YE(Bpa)WSLAAPQRFNH2, derived from a peptide present in the rat neuropeptide FF (NPFF) precursor was synthesized, and its binding characteristics were investigated on a neuroblastoma clone, SH-SY5Y, stably expressing rat NPFF2 receptors tagged with the T7 epitope. The binding of the probe was saturable and revealed a high-affinity interaction (KD=0.24nM) with a single class of binding sites. It was also able to affinity label NPFF2 receptor in a specific and efficient manner given that 38% of the bound radioligand at saturating concentration formed a wash-resistant binding after ultraviolet (UV) irradiation. Photoaffinity labeling with [(125)I]YE(Bpa)WSLAAPQRFamide showed two molecular forms of NPFF2 receptor with apparent molecular weights of 140 and 95kDa in a 2:1 ratio. The comparison of the results between photoaffinity labeling and Western blot analysis suggests that all receptor forms bind the probe irreversibly with the same efficiency. On membranes of mouse olfactory bulb, only the high molecular weight form of NPFF2 receptor is observed. [(125)I]YE(Bpa)WSLAAPQRFamide is an excellent radioiodinated peptidic ligand for direct and selective labeling of NPFF2 receptors in vitro.

GRK2 protein-mediated transphosphorylation contributes to loss of function of µ-opioid receptors induced by neuropeptide FF (NPFF2) receptors.

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 ß-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.

Expression of opioid and anti-opioid receptors in Chinese hamster ovary cells after exposure to dimethyl sulfoxide.

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).

Solubilization and functional reconstitution of human neuropeptide FF2 receptors.

Neuropeptide FF (NPFF, FLFQPQRFamide) receptors modulate endogenous opioid functions. Here, we report the solubilization of the human NPFF(2) 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.

Modulation of basal and morphine-induced neuronal activity by a NPFF(2) selective agonist measured by c-Fos mapping of the mouse brain.

Neuropeptide FF (NPFF) is a neurotransmitter known to modulate opioid-induced analgesia, sensitization, and reward. The expression of the immediate early gene c-Fos was analyzed to map the distribution of neurons whose activity is regulated by central administration of the NPFF(2)-selective agonist dNPA in naive mice and in animals who had received a systemic injection of morphine. The number of c-Fos positive nuclei was quantified in 28 brain regions. Intracerebro-ventricular injection of 1 nmol dNPA alone produced an overall inhibition of basal c-Fos expression in the brain with a statistically significant decrease in the lateral ventral part of the bed nucleus of the stria terminalis, the medial preoptic area, and the medial parvicellular part of the paraventricular nucleus of the hypothalamus. In contrast, intraperitoneal injection of morphine 5 mg.kg(-1) induced a statistically significant increase in c-Fos expression in the prelimbic cortex, the nucleus accumbens core and shell, the ventral pallidum, the lateral hypothalamus, and the nucleus of the tractus solitarius. dNPA counteracted morphine effect only in the nucleus accumbens shell and the ventral pallidum. The inhibitory effects of a low dose of dNPA in the hypothalamus and its afferents suggest that NPFF(2) receptors negatively regulate the hypothalamic-pituitary-adrenal axis in mouse. Moreover, our study identified the nucleus accumbens shell and ventral pallidum as putative sites of interaction between NPFF and opioid systems in relation with the modulation of acute morphine rewarding and locomotor effects.

Agonist binding of human mu opioid receptors expressed in the yeast Pichia pastoris: Effect of cholesterol complementation.

This study compared pharmacological profiles between human mu opioid receptors (hMOR) overexpressed in the SH-SY5Y neuroblastoma cell line (SH-hMOR) and the methylotrophic yeast Pichia pastoris (Pp-hMOR). Affinity determinations were performed by direct binding with the tritiated agonist DAMGO and antagonist diprenorphine (DIP). Additionally, displacement of these drugs with agonists (morphine and DAMGO) and antagonists (ß-funaltrexamine, naloxone and diprenorphine) was examined. Tritiated DAMGO could bind to membranes prepared from Pp-hMOR, although the receptor was not coupled with G-proteins. The data obtained with this yeast strain suggested that only 7.5% of receptors were in a high-affinity-state conformation. This value was markedly less than that estimated in SH-hMOR membranes, which reached 50%. Finally, to understand the pharmacological discrepancies between Pp-hMOR and SH-hMOR, the role of sterols was evaluated. The major sterol in P. pastoris is ergosterol, while hMOR naturally functions in a cholesterol-containing membrane environment. Cell membranes were sterol-depleted or cholesterol-loaded with methyl-ß-cyclodextrine. The results indicated that cholesterol must be present to ensure Pp-hMOR function. The proportion of high-affinity-state conformation was reversibly increased by cholesterol complementation.

Pharmacological insight into the activation of the human neuropeptide FF2 receptor.

The neuropeptide FF2 (NPFF2) 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 NPFF2 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 NPFF2 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.

Development and characterization of sphingosine 1-phosphate receptor 1 monoclonal antibody suitable for cell imaging and biochemical studies of endogenous receptors.

Although sphingosine-1-phosphate receptor 1 (S1P1) has been shown to trigger several S1P targeted functions such as immune cell trafficking, cell proliferation, migration, or angiogenesis, tools that allow the accurate detection of endogenous S1P1 localization and trafficking remain to be obtained and validated. In this study, we developed and characterized a novel monoclonal S1P1 antibody. Mice were immunized with S1P1 produced in the yeast Pichia pastoris and nine hybridoma clones producing monoclonal antibodies were created. Using different technical approaches including Western blot, immunoprecipitation and immunocytochemistry, we show that a selected clone, hereinafter referred to as 2B9, recognizes human and mouse S1P1 in various cell lineages. The interaction between 2B9 and S1P1 is specific over receptor subtypes, as the antibody does not binds to S1P2 or S1P5 receptors. Using cell-imaging methods, we demonstrate that 2B9 binds to an epitope located at the intracellular domain of S1P1; reveals cytosolic and membrane localization of the endogenous S1P1; and receptor internalization upon S1P or FTY720-P stimulation. Finally, loss of 2B9 signal upon knockdown of endogenous S1P1 by specific small interference RNAs further confirms its specificity. 2B9 was also able to detect S1P1 in human kidney and spinal cord tissue by immunohistochemistry. Altogether, our results suggest that 2B9 could be a useful tool to detect, quantify or localize low amounts of endogenous S1P1 in various physiological and pathological processes.

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.

Staurosporine differentiation of NPFF2 receptor-transfected SH-SY5Y neuroblastoma cells induces selectivity of NPFF activity towards opioid receptors.

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.

Biochemical anti-opioid action of NPFF2 receptors in rat spinal cord.

Neuropeptide FF (NPFF) agonists counteract the cellular opioid actions. We demonstrate for the first time a biochemical anti-opioid effect of NPFF receptors in the rat spinal cord by using the [(35)S]GTPgammaS binding assay in autoradiography. The mu agonist DAMGO as well as the potent and selective NPFF(2) agonist dNPA, stimulated [(35)S]GTPgammaS binding at different optimal GDP concentrations. dNPA decreased the effects induced by DAMGO alone; the maximal of G-protein coupling was decreased but not the potency of opioid agonist. We conclude that NPFF(2) receptors are coupled to G-protein in the rat spinal cord and could exert a molecular anti-opioid effect.

Anti-opioid activities of NPFF1 receptors in a SH-SY5Y model.

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 mu- and delta-opioid receptors, with the human NPFF1 receptor. The SH1-C7 clone expresses high affinity NPFF1 receptors in the same range order of density as opioid receptors. Similarly to the opioids, acute stimulation with the NPFF1 agonist NPVF inhibits adenylyl cyclase activity and voltage-gated (N-type) Ca2+ currents and enhances the intracellular Ca2+ 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. SH1-C7 cells are therefore a suitable model to investigate the interactions between NPFF and opioid receptors.

A neuropeptide FF agonist blocks the acquisition of conditioned place preference to morphine in C57Bl/6J mice.

Neuropeptide FF behaves as an opioid-modulating peptide that seems to be involved in morphine tolerance and physical dependence. Nevertheless, the effects of neuropeptide FF agonists on the rewarding properties of morphine remain unknown. C57BL6 mice were conditioned in an unbiased balanced paradigm of conditioned place preference to study the effect of i.c.v. injections of 1DMe (D-Tyr1(NMe)Phe3]NPFF), a stable agonist of the neuropeptide FF system, on the acquisition of place conditioning by morphine or alcohol (ethanol). Morphine (10 mg/kg, i.p.) or ethanol (2 g/kg, i.p.) induced a significant place preference. Injection of 1DMe (1-20 nmol), given 10 min before the i.p. injection of the reinforcing drug during conditioning, inhibited the rewarding effect of morphine but had no effect on the rewarding effect of ethanol. However, a single injection of 1DMe given just before place preference testing was unable to inhibit the rewarding effects of morphine. By itself, 1DMe was inactive but an aversive effect of this agonist could be evidenced if the experimental procedure was biased. These results suggest that neuropeptide FF, injected during conditioning, should influence the development of rewarding effects of morphine and reinforce the hypothesis of strong inhibitory interactions between neuropeptide FF and opioids.

Opioid-modulating peptides: mechanisms of action.

Opioids are involved in the physiological control of numerous functions of the central nervous system, particularly nociception. It appears that some endogenous neuropeptides, called anti-opioids, participate in an homeostatic system tending to reduce the effects of opioids. Neuropeptide FF (NPFF) and cholecystokinin (CCK) possess these properties and, paradoxically, the opioid peptides nociceptin and dynorphin display some anti-opioid activity. All these peptides exhibit complex properties as they are able to both counteract and potentiate opioid activity, acting rather as modulators of opioid functions. The purpose of this review is to highlight that two different mechanisms are clearly involved in the control of opioid functions by opioid-modulating peptides: a circuitry-induced mechanism for nociceptin and dynorphin, and a cellular anti-opioid mechanism for NPFF and CCK. The knowledge of these mechanisms has potential therapeutic interest in the control of opioid functions, notably for alleviating pain and/or for the treatment of opioid abuse.

Comparison of pharmacological activities of Neuropeptide FF1 and Neuropeptide FF2 receptor agonists.

The pharmacological effects of Neuropeptide FF (NPFF) analogs exhibiting different selectivities towards Neuropeptide FF1 (NPFF1) and Neuropeptide FF2 (NPFF2) receptors were investigated after supraspinal administration in mice. Injected into the third ventricle, VPNLPQRF-NH2, which is selective for Neuropeptide FF1 receptor induced a hypothermia while EFWSLAAPQRF-NH2 and SPAFLFQPQRF-NH2 which are selective for Neuropeptide FF2 receptor, did not. Furthermore, EFWSLAAPQRF-NH2 significantly increased the body temperature when compared to saline treated mice, indicating that Neuropeptide FF1 receptor could be responsible for hypothermia while Neuropeptide FF2 mediated an hyperthermic effect. After administration into the lateral ventricle, 1DMe ([D.Tyr1,(N.Me)Phe3]NPFF), a weakly selective Neuropeptide FF2 receptor agonist, exerted a clear anti-opioid effect in the tail flick test. The selective Neuropeptide FF1 receptor agonist VPNLPQRF-NH2 did not induce significant anti-opioid actions but rather increased, dose-dependently, morphine analgesia while EFWSLAAPQRF-NH2, the highest selective Neuropeptide FF2 receptor analog, induced the same pharmacological effect as VPNLPQRF-NH2 at comparable doses. These features indicate that the pro- and anti-opioid actions are not strictly related to the selectivity towards Neuropeptide FF2 or Neuropeptide FF1 receptor. Our data demonstrate that Neuropeptide FF1 and Neuropeptide FF2 receptors differently modulate nervous system functions.

Identification of neuropeptide FF-related peptides in human cerebrospinal fluid by mass spectrometry.

Several neuropeptide FF (NPFF)-related peptides, known as modulators of the opioid system, have been previously characterized in bovine and rodent brain. Reverse-phase high pressure liquid chromatography (HPLC) fractions of a human with normal pressure hydrocephalus cerebrospinal fluid (CSF), co-migrating with NPFF-related synthetic peptides, were characterized by capillary HPLC coupled on-line to nanospray ion trap tandem mass spectrometry. Two peptides present in the pro-NPFF(A) precursor, NPAF (AGEGLNSQFWSLAAPQRF-NH2) and NPSF (SLAAPQRF-NH2), were identified. The monitoring of NPFF-related peptides in human CSF can be helpful to understand their roles in pain sensitivity.

Opposite alterations of NPFF1 and NPFF2 neuropeptide FF receptor density in the triple MOR/DOR/KOR-opioid receptor knockout mouse brains.

Mice lacking the mu-delta-kappa-opioid receptor (MOR/DOR/KOR) genes and their corresponding wild-type littermates have been used to quantify NPFF(1) and NPFF(2) (neuropeptide FF) receptors by in vitro autoradiography in the central nervous tissues. Adjacent coronal sections were labelled with [125I]YVP ([125I]YVPNLPQRF-NH(2)) and [125I]EYF ([125I]EYWSLAAPQRF-NH(2)) as specific radioligands for NPFF(1) and NPFF(2) receptors, respectively. NPFF(2) receptors are predominantly expressed in both genotypes, but their density increases significantly in non cortical regions of mutant mice: 64% in the amygdaloid area, 89, 308, 1214 and 49% in the nucleus of the vertical limb of the diagonal band, substantia nigra, the vestibular nucleus and the spinal cord, respectively. In contrast, the density of the NPFF(1) subtype is lower than NPFF(2) in both genotypes and significantly decreased in some brain areas of mutant mice: -99, -90 and -90% in the nucleus of the vertical limb of the diagonal band, substantia nigra and the spinal cord, respectively. This study shows that mice lacking opioid receptors have brain region-dependent increases (NPFF(2)) and decreases (NPFF(1)) in NPFF receptors densities and suggests a different functional participation of each NPFF receptor subtype in the actions of opioids.