Analytical Biochemistry 398 (2010) 225–229

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Analytical Biochemistry

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Solubilization and functional reconstitution of human neuropeptide FF2 receptors

Frank Talmont, Catherine Mollereau, Isabelle Muller, Jean-Marie Zajac *

Institut de Pharmacologie et de Biologie Structurale, CNRS/Université de Toulouse, UMR 5089, 205 route de Narbonne, 31077 Toulouse Cedex, France

a r t i c l e i n f o

Article history:Received 18 August 2009Received in revised form 27 November 2009Accepted 9 December 2009Available online 14 December 2009

Keywords:Neuropeptide FFReceptorSolubilizationReconstitutionBinding sites

0003-2697/$ - see front matter � 2009 Elsevier Inc. Adoi:10.1016/j.ab.2009.12.012

* Corresponding author.E-mail address: jean-marie.zajac@ipbs.fr (J.-M. Zaj

1 Abbreviations used: NPFF, neuropeptide FF; CHO, Chhuman embryonic kidney 293; MOP, l opioid;pyl)dimethylammonium]-1-propanesulfonate; PEG, pdodecyl-b-maltoside; EDTA, ethylenediaminetetraacealbumin; 1DMe, [D.Tyr1,(N-Me)Phe3]NPFF; NPA–NPFNPFF, SPAFLFQPQRF-NH2; SQA–NPFF, SQAFLFQPQRF-EYF, EYWSLAAPQRF-NH2; fPP, frog pancreatic polypept

a b s t r a c t

Neuropeptide FF (NPFF, FLFQPQRFamide) receptors modulate endogenous opioid functions. Here, wereport the solubilization of the human NPFF2 receptor expressed in Chinese hamster ovary (CHO) cellsby the zwitterionic detergent Chaps. Chaps solubilization resulted in the abolishment of specific agonistbinding activity, which was restored by a polyethylene glycol (PEG) precipitation method. Reincorpora-tion after the precipitation step into liposomes made of endogenous lipids issued from CHO membranesor 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.

� 2009 Elsevier Inc. All rights reserved.

Neuropeptide FF (NPFF, FLFQPQRF-NH2)1 is a neurotransmittersystem that modulates endogenous opioid functions [1–3]. NPFF-re-lated peptides issue from two precursors, NPFFA and NPFFB, that gen-erate peptides with a C-terminal PQRF-NH2 sequence [4]. The NPFFsystem involves two specific receptors named NPFF1 and NPFF2 thatare able to couple with Gi/o protein when expressed in Chinese ham-ster ovary (CHO) [5,6], human embryonic kidney 293 (HEK 293) [7],or human SH-SY5Y neuroblastoma [8] cells. In contrast, NPFF recep-tors appear to be G-protein coupled in mouse olfactory bulb [9], andcholera toxin blocks the effects of NPFF on opioid activity in the ratdorsal raphe [10]. NPFF anti-opioid activity has been studied in SH-SY5Y neuroblastoma cells that endogenously express opioid recep-tors and transfected with the human NPFF2 receptor. Results showthat NPFF analogs functionally antagonize the inhibition of N-typevoltage gated calcium channels by l and d opioid ligands. Moreover,fluorescence energy transfer studies have revealed that NPFF2 and lopioid (MOP) receptors may form heteromers [11].

Although NPFF analogs do not interact with opioid receptors[12], a close relationship between NPFF and opioid systems hasbeen clearly demonstrated, especially in pain perception [1,2].The distribution pattern of NPFF2 receptors [13,14] in the central

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ac).inese hamster ovary; HEK 293,Chaps, 3-[(3-cholamidopro-olyethylene glycol; DDM, n-tic acid; BSA, bovine serumF, NPAFLFQPQRF-NH2; SPA–NH2; NPVF, VPNLPQRF-NH2;ide.

nervous system is consistent with its potential role in the modula-tion of sensory input and opioid analgesia. In fact, supraspinalinjection of NPFF analogs, which has little or no effect in pain tests,decreases morphine-induced analgesia (anti-opioid activity) [1,2],whereas spinal administration induces a naloxone-sensitive anal-gesia and potentiates morphine-induced analgesia (pro-opioidactivity).

Molecular characterization of the receptor molecule and itsrelationships with G-protein requires its purification, the first stepof which is the successful solubilization of the receptor in an activeform. In this article, we describe the solubilization of NPFF2 recep-tors with a zwitterionic detergent, Chaps (3-[(3-cholamidopro-pyl)dimethylammonium]-1-propanesulfonate), with high sodiumconcentrations. The detergent inhibited the binding activity, butits elimination by a polyethylene glycol (PEG) precipitation meth-od that generates liposomes permitted the reconstitution of thefunctional receptor. Optimal conditions for restoration of bindingwere determined, and the reconstituted receptor was thencharacterized.

Materials and methods

Chemicals

Chaps, n-dodecyl-b-maltoside (DDM), ethylenediaminetetra-acetic acid (EDTA), MgCl2, NaCl, PEG, polyethylenimine, bovine ser-um albumin (BSA), and Tris were obtained from Sigma Chemical(USA).

Brain polar lipid extract (porcine) (phosphatidylethanolamine33.1 wt%, phosphatidylserine 18.5 wt%, phosphatidylcholine 12.6

226 Solubilization of human NPFF2 receptors / F. Talmont et al. / Anal. Biochem. 398 (2010) 225–229

wt%, phosphatidic acid 0.8 wt%, phosphatidylinositol 4.1 wt%) andsoybean lipid extract (phosphatidylethanolamine 18.06%, phos-phatidylinositol 11.5%, phosphatidylcholine 24.0%, phosphatidicacid 4.3%, lysophosphatidylcholine 4.6%) were purchased fromAvanti Polar Lipids (USA).

1DMe ([D.Tyr1,(N-Me)Phe3]NPFF), NPAFLFQPQRF-NH2 (NPA–NPFF), SPAFLFQPQRF-NH2 (SPA–NPFF), SQAFLFQPQRF-NH2 (SQA–NPFF), VPNLPQRF-NH2 (NPVF), EYWSLAAPQRF-NH2 (EYF), and frogpancreatic polypeptide (fPP) were synthesized using an automatedpeptide synthesizer (Applied Biosystems model 433A, USA) as de-scribed previously [15].

Iodinations of [125I]EYF ([125I]EYWSLAAPQRFamide) were per-formed as described previously [16]. The specific activity was as-sumed to be identical to that of Na125I (74 TBq/mmol).

[3H]EYF ([3H]EYWSLAAPQRFamide) (2.66 TBq/mmol) was syn-thesized as described previously [17].

Cell culture

Recombinant CHO cells expressing the human NPFF2 receptorwere grown in Ham-F12 medium supplemented with 7% fetal calfserum, 100 U/ml penicillin, 100 lg/ml streptomycin, 2 mM L-gluta-mine, 1 mM sodium pyruvate, and 400 lg/ml G418 (Gibco-BRL,France) in an atmosphere of 5% CO2 at 37 �C. Cells were passagedevery 2–3 days.

For membrane preparation, cells were harvested by centrifuga-tion at 1000g for 15 min at 4 �C in phosphate-buffered saline andfrozen at �80 �C for 1 h, and the pellet was homogenized in50 mM Tris–HCl (pH 7.4) in a Potter–Elvehjem tissue grinder. Aftercentrifugation at 1000g for 15 min at 4 �C, the nuclear pellet wasdiscarded and the membrane fraction was collected on centrifuga-tion of the supernatant at 100,000g for 30 min at 4 �C. The pelletwas resuspended in Tris–HCl (50 mM, pH 7.4) and homogenized,and aliquots (0.6–1.3 mg/ml protein) were stored at �80 �C. Pro-tein concentration was determined by the Bradford method withBSA as standard.

Solubilization of membranes

Membranes were incubated with 5 mM Chaps and 1 M NaCl inbuffer A (50 mM Tris, 1 mM EDTA, and 10 mM MgCl2, pH 7.4) at afinal protein concentration of 0.3 mg/ml for 30 min at 4 �C withoccasional shaking.

The preparation was centrifuged at 100,000g for 1 h at 4 �C, andthe clear supernatant was carefully removed from the pellet.

Liposome formation

PEG precipitation was carried out by diluting the extract withan equal volume of 40% (w/w) PEG 8000 in buffer A. Following vig-orous vortexing and incubation for 10 min on ice, the sample wascentrifuged at 15,000g for 10 min at 4 �C. The pellet was carefullyrinsed with buffer A, resuspended in the same buffer, and frozenat �80 �C.

In some experiments, the clear supernatant was incubated withvarious quantities of brain lipid extract or soybean lipid extract for15 min at 4 �C.

Binding assays

A classic soluble assay employing polyethylenimine-precoatedglass fiber filters was used to measure the binding of [125I]EYF or[3H]EYF to NPFF2 receptors by rapid filtration. Membranes or solu-ble proteins were incubated in polypropylene tubes in a final vol-ume of 500 ll containing 50 mM Tris–HCl (pH 7.4), 60 mM NaCl,0.1% BSA, and the radioligand. The nonspecific binding was deter-

mined in the presence of 1 lM EYWSLAAPQRFamide. Following 1 hof incubation at 25 �C, samples were rapidly filtered on WhatmanGF/B filters preincubated for at least 1 h in 50 mM Tris–HCl (pH7.4) and polyethylenimine (0.15%). Filters were rinsed three timeswith 3 ml of ice-cold Tris–HCl containing 0.1% BSA, and the boundradioactivity was quantified using a Packard gamma counter or aPackard liquid scintillation spectrophotometer.

[35S]GTPcS binding assay

The assay buffer consisted of 20 mM Hepes (pH 7.4), 100 mMNaCl, 3 mM MgCl2, 1 lM GDP, and 0.1% BSA. Aliquots of 50 ll wereincubated in polypropylene tubes at 30 �C for 60 min in 500 ll ofbuffer containing 0.05 nM [35S]GTPcS. The reaction was stoppedas described previously [18] or after diluting to half with a PEG(40%)–BSA (10%) solution, incubated for 10 min at 4 �C, and centri-fuged at 15,000g at 4 �C for 15 min. The radioactivity precipitatedwas determined in 0.1 ml of water by liquid scintillation spectro-photometry after overnight extraction in Ready Protein scintilla-tion fluid (Beckman, USA).

Liposome particle size was determined by dynamic light scat-tering using a DynaPro Protein Solutions (USA). Each liposomecomposition was diluted approximately 50-fold with Hepes bufferbefore the measure.

Results

Two radioligands were used in this study: [3H]EYF(KD = 0.54 nM on membrane receptors) [17] and [125I]EYF(KD = 0.05 nM) [19]. In the presence of very low density of sitesor low affinity, sufficient binding at a high ratio of specific to non-specific binding could be detected only with the radioiodinatedligand.

Optimization of conditions of solubilization of NPFF receptor

The mild, nondenaturing zwitterionic detergent Chaps is one ofthe most commonly used detergents in membrane biochemistry[20]. It effectively disrupts nonspecific protein interactions andhas been previously found to be an efficient detergent in functionalsolubilization of several receptors. NaCl was added to the mediumto lower the detergent concentration used; the critical micelle con-centration of Chaps is dependent on the salt concentration and de-creases from 6.41 mM in the absence of any salt to 4.34 mM in thepresence of 1 M NaCl [21]. A concentration of Chaps of 5 mM in thepresence of 1 M NaCl was used to solubilize the NPFF2 receptorfrom CHO membranes (4.8 pmol/mg) at a membrane protein con-centration of 0.45 mg/ml corresponding to a molar detergent/pro-tein ratio of 6.83:1.

As shown in Fig. 1, the binding activity of membrane NPFF2

receptors was sensitive to the detergent. A progressive decreaseof the binding, corresponding to both the solubilization and a di-rect inhibition of receptors, was noted.

However, the solubilized NPFF receptor was reconstituted byremoving the detergent by PEG precipitation. This procedure rap-idly removes the detergent along with salt, thereby overcomingthe problem of long dialysis periods. This method has been shownto restore functional interactions of membrane proteins by incor-porating them into vesicles [22,23].

Overall, as measured at 0.05 nM [125I]EYF, a large fraction of themembrane receptor binding (�30%) was solubilized (soluble after a100,000g centrifugation) and recovered following removal of thedetergent by precipitation with PEG. Chaps extract after precipita-tion was stable at �80 �C for at least 1 year.

Fig. 1. Effect of detergent concentration on binding activity of membrane andreconstituted NPFF2 receptor. Samples of CHO membrane (solid circles) or Chaps-solubilized and PEG-precipitated receptors (open squares) were used. [125I]EYFbinding was measured at 0.05 nM with 4 and 100 lg of protein for membrane andsoluble receptor, respectively, in the presence of the indicated detergent concen-tration. The specific counts per minute (cpm) correspond to the means (±standarddeviations) of three determinations.

Table 1Binding affinity and G-protein activity of various ligands on NPFF2 receptorsreconstituted with endogenous lipids.

SPA–NPFF 1DMe NPVF

Ki (nM), reconstituted NPFF2 0.61 ± 0.20 1.11 ± 0.52 18.3 ± 4.2Ki (nM), membranar NPFF2 [18] 0.047 ± 0.003 0.18 ± 0.04 17.4 ± 1.7Ratio reconstituted/membranar 12.97 6.16 1.05GTPcS binding stimulationEC50 (nM) 10.6 ± 5.0 35 ± 12 194 ± 25Emax (%) 156 ± 10 150 ± 13 140 ± 15

Note. Data are means ± standard errors of Ki or EC50 values (GTPcS stimulation) innanomolars (nM). The affinities were measured by competition experiments using0.05 nM [125I]EYF. The maximum response (Emax) is expressed as percentage ofbasal [35S]GTPcS binding in the absence of agonist.

Solubilization of human NPFF2 receptors / F. Talmont et al. / Anal. Biochem. 398 (2010) 225–229 227

On this latter preparation, low concentrations of Chaps induceda dose-dependent loss of the [125I]EYF binding (Fig. 1). Thus, thebinding was probably undetectable in crude Chaps extract because5 mM detergent caused a complete loss of binding.

Other detergents and methods were tested in attempts to im-prove receptor recovery. Higher concentrations of Chaps seriouslyimpaired the [125I]EYF binding activity. The removal of Chaps bydialysis in the presence or absence of glycerol did not lead to therecovery of much [125I]EYF binding activity. Glycerol alone inhib-ited the binding (27.4% of inhibition at a concentration of 10%).Similarly, the presence of an NPFF agonist during the solubilizationdid not improve the quantity of active receptor recovered. The useof a nonionic detergent such as digitonin at a concentration(8.1 mM) largely solubilizing or DDM (1% [w/v], 19.5 mM) in thepresence of cholesteryl hemisuccinate did not increase the recep-tor recovery.

Fig. 2. Critical lipid/protein ratio on the recovery of [3H]EYF binding solubilizedwith Chaps. Cell membranes solubilized with Chaps (5 mM) were incubated withvarious concentrations of brain lipid extract before PEG precipitation. The bindingwas measured with [3H]EYF (0.5 nM) and is expressed (means ± standard errors) aspercentages of maximal binding.

Binding properties of reconstituted NPFF receptor

Following PEG precipitation, the Chaps-extracted protein wasreconstituted into liposomes made of endogenous lipids issuedfrom CHO membrane. Measurement by dynamic light scatteringwas used to determine vesicle size [24]. After reconstitution in lip-osomes, 80% of the material exhibited a mean size of 71 nm.

The saturation isotherms of the specific binding of [125I]EYF toreceptors reconstituted by PEG precipitation were characterizedover the concentration range of 0.075–7.5 nM. [125I]EYF interactedwith a single class of binding sites, and the binding parameters cal-culated by nonlinear regression yielded a KD value of1.02 ± 0.35 nM (mean ± standard deviation). The nonspecific bind-ing was relatively high (�39%) for concentrations near the KD value(1.5 nM). The apparent affinity corresponds to a 20.4-fold decreasein the affinity given that the same radioligand binds receptors inCHO membrane with a KD value of 0.05 nM [19]. Thus, the fractionof NPFF receptor binding measured at one ligand concentration(equal to half saturation of membrane receptor, i.e., 0.05 nM) aftersolubilization with Chaps and reconstitution corresponded to

4.67% of the saturation, indicating that only 9.4% (4.7/50) of recep-tor had been reconstituted.

Interestingly, all NPFF agonists tested exhibited the same de-crease of affinity and the decrease was smaller when the affinityon membrane receptor was low (Table 1).

In the presence of 1 lM GDP, NPFF agonists stimulated[35S]GTPcS binding (Table 1). This result shows that Chaps wasable to extract the receptors coupled with G-proteins. The EC50 val-ues were between 6.3- and 17-fold higher than the Ki affinity val-ues determined in radioligand binding, similarly to that observedpreviously in membrane [18].

Optimization of NPFF2 receptor reconstitution

The addition of exogenous lipids during PEG precipitation in-creased the efficiency of receptor reconstitution. In fact, as seenin Fig. 2, the quantity of the specific [3H]EYF binding increasednearly 3-fold in the presence of brain polar lipid extract andreached a maximum for a lipid/protein ratio between 0.2 and 0.5.In the presence of a higher concentration of brain lipids, [3H]EYF

Fig. 3. Saturation binding isotherm of [3H]EYF to reconstituted NPFF2 receptorswith soybean lipid extract. Specific binding was measured at 25 �C after 60 minincubation of various concentrations of [3H]EYF. The nonspecific binding wasdetermined in the presence of 1 lM unlabeled EYF. Points represent themeans ± standard errors of triplicate determinations.

Table 2Binding affinity on [3H]EYF and activity of several ligands on NPFF2 receptors reconstituted in presence of brain lipid extract.

EYF SQA–NPFF NPA–NPFF fPP NPVF

Ki (nM), membranar NPFF2 [18] 0.24 ± 0.03 0.16 ± 0.02 0.033 ± 0.003 7 ± 2 17.4 ± 1.7Ki (nM), reconstituted NPFF2 2.33 ± 1.05 8.60 ± 1.42 6.8 ± 2.3 17.8 ± 8.2 190.4 ± 67.0Ratio reconstituted/membranar 9.70 53.75 206.06 2.50 10.94GTPcS binding stimulationEC50 (nM) 9.2 ± 1.6 4.7 ± 1.0 47.1 ± 6.1 1976 ± 1081Emax (%) 292 ± 13 263 ± 17 271 ± 13 216 ± 4

Note. Data were obtained for reconstitution with a lipid/protein ratio of 0.2. Data are means ± standard deviations of Ki or EC50 values (GTPcS) in nanomolars (nM). Themaximum response (Emax) is expressed as percentage of basal [35S]GTPcS binding in the absence of agonist.

228 Solubilization of human NPFF2 receptors / F. Talmont et al. / Anal. Biochem. 398 (2010) 225–229

binding decreased to a value inferior to control without the addi-tion of brain lipid.

NPFF2 receptors were insoluble after precipitation with PEG inthe presence of brain lipid extract; after centrifugation at100,000g for 1 h, 95% of the specific binding was recovered in thepellet (4132 ± 72 cpm specific binding vs. 4144 ± 162, n = 3, aftercentrifugation). Likewise, the addition of soybean lipid extract dur-ing the PEG precipitation step induced a similar effect, and themaximal increase of NPFF2 binding rose for a lipid/protein ratioof approximately 0.3.

The affinity of [3H]EYF to the material reconstituted with soy-bean lipid was 2.3 nM, that is, 4.95-fold lower than with mem-brane receptor (0.54 nM) (Fig. 3). Thus, the calculation of thetotal number of binding sites recovered, taking into account thisdifference in affinity, indicates that 35% of receptor had been solu-bilized and reconstituted. Because only 9% of receptors were recov-ered without exogenous lipids, this indicates that 90% of thereceptors solubilized by Chaps expressed a low affinity that couldbe converted in a higher affinity state by incorporation inliposomes.

Competition studies were undertaken to determine the changesin affinity of the reconstituted receptors (Table 2). In liposomes,NPFF2 receptor displayed a lower affinity compared with CHOmembranes (Table 2). The decrease depended on the ligand usedand was smaller when the membrane affinity was low. Similar rel-ative affinities were observed in the absence of exogenous lipidaddition.

NPFF2, agonists stimulated [35S]GTPcS binding with a high-effi-ciency indicating that receptor was reconstituted in a form coupled

with G-protein (Table 2). Interestingly, the maximum response ex-pressed as a percentage of basal binding was considerably largerthan with receptors precipitated without exogenous lipids. The ra-tio between EC50 values for membranar/reconstituted receptorsapproximates unity (2.77 for NPA–NPFF, 4.02 for fPP, and 0.8 forNPVF), suggesting that the receptor is recovered with an efficientcoupling to G-protein.

Measurement by dynamic light scattering showed that 85% ofmaterials exhibited a mean size of 69 nm after the addition ofexogenous lipids (as compared with 71 nm without lipids), sug-gesting that the efficient coupling of receptor to G-protein dependsmore on the lipid composition than on the size of liposomes.

Discussion

A common problem associated with most solubilization exper-iments is delipidation (i.e., loss of lipids). This often leads to re-duced activity of the solubilized protein or receptor becauselipid–protein interactions play a crucial role in maintaining thestructure and function of integral membrane proteins and recep-tors. By using highly selective radioligands, we have shown thathigh-affinity NPFF2 binding can be successfully solubilized fromCHO membranes with Chaps and reconstituted into a lipid envi-ronment that preserves binding properties and G-protein couplingactivity.

The NPFF2 receptor binding is sensitive to the detergent, and itwas necessary to eliminate Chaps to measure high-affinity bindingin a relatively high yield. The removal of Chaps by precipitationwith PEG led to the formation of liposomes, and the addition ofexogenous lipids increased the efficiency of reconstitution of NPFF2

binding in sedimentable materials (insoluble at 100,000g for 1 h).Because a high concentration of Chaps inhibits binding, it is possi-ble that a part of NPFF receptors was not recovered even after rein-corporation with lipids because of the presence of detergentmolecule in the receptor binding site or that detergent eliminatestoo large a proportion of lipids interacting with receptors.

Clearly, the binding properties of NPFF2 were modified duringthese steps in a different fashion depending on the ligand. Thus,it is difficult to determine the exact percentage of receptor recov-ery. Solubilization was at least 35% given that this is the percentageof receptor recovered after reconstitution. It is interesting thatliposome formation is accompanied by a highly efficient couplingof NPFF receptor with G-protein.

In conclusion, we have developed a protocol for producing high-affinity NPFF2 receptors that will be valuable for the detailedmolecular characterization of this receptor.

Acknowledgments

This research was supported by the French Agence Nationale dela Recherche, ‘‘Neurosciences, Neurologie et Psychiatrie” Program,Grant ANR-06-NEURO-041-04. The authors thank Neil Johnsonfor helpful comments on the paper.

Solubilization of human NPFF2 receptors / F. Talmont et al. / Anal. Biochem. 398 (2010) 225–229 229

References

[1] M. Roumy, J.M. Zajac, Neuropeptide FF, pain, and analgesia, Eur. J. Pharmacol.345 (1998) 1–11.

[2] P. Panula, E. Kalso, M. Nieminen, V.K. Kontinen, A. Brandt, A. Pertovaara,Neuropeptide FF and modulation of pain, Brain Res. 848 (1999) 191–196.

[3] C. Mollereau, M. Roumy, J.M. Zajac, Opioid-modulating peptides: mechanismsof action, Curr. Top. Med. Chem. 5 (2005) 341–355.

[4] J.M. Zajac, Neuropeptide FF: new molecular insights, Trends Pharmacol. Sci. 22(2001) 63.

[5] S. Hinuma, Y. Shintani, S. Fukusumi, N. Iijima, Y. Matsumoto, M. Hosoya, R.Fujii, T. Watanabe, K. Kikuchi, Y. Terao, T. Yano, T. Yamamoto, Y. Kawamata, Y.Habata, M. Asada, C. Kitada, T. Kurokawa, H. Onda, O. Nishimura, M. Tanaka, Y.Ibata, M. Fujino, New neuropeptides containing carboxy-terminal RFamide andtheir receptor in mammals, Nat. Cell Biol. 2 (2000) 703–708.

[6] M. Kotani, C. Mollereau, M. Detheux, E. Le Poul, S. Brezillon, J. Vakili, H.Mazarguil, G. Vassart, J.M. Zajac, M. Parmentier, Functional characterization ofa human receptor for neuropeptide FF and related peptides, Br. J. Pharmacol.133 (2001) 138–144.

[7] N.A. Elshourbagy, R.S. Ames, L.R. Fitzgerald, J.J. Foley, J.K. Chambers, P.G.Szekeres, N.A. Evans, D.B. Schmidt, P.T. Buckley, G.M. Dytko, P.R. Murdock, K.B.Tan, U. Shabon, P. Nuthulaganti, D.Y. Wang, S. Wilson, D.J. Bergsma, H.M. Sarau,Receptor for the pain modulatory neuropeptides NPFF and NPAF is an orphanG-protein-coupled receptor, J. Biol. Chem. 275 (2000) 25965–25971.

[8] C. Mollereau, H. Mazarguil, J.M. Zajac, M. Roumy, Neuropeptide FF (NPFF)analogs functionally antagonize opioid activities in NPFF2 receptor-transfectedSH-SY5Y neuroblastoma cells, Mol. Pharmacol. 67 (2005) 965–975.

[9] N. Gherardi, J.M. Zajac, Neuropeptide FF receptors of mouse olfactory bulb:binding properties and stimulation of adenylate cyclase activity, Peptides 18(1997) 577–583.

[10] M. Roumy, J.M. Zajac, Neuropeptide FF receptors couple to a cholera toxin-sensitive G-protein in rat dorsal raphe neurones, Eur. J. Pharmacol. 417 (2001)45–49.

[11] M. Roumy, C. Lorenzo, S. Mazères, S. Bouchet, J.-M. Zajac, C. Mollereau, Physicalassociation between neuropeptide FF (NPFF) and l-opioid (MOP) receptors asa molecular basis for anti-opioid activity, J. Biol. Chem. 282 (2007) 8332–8342.

[12] C. Gouarderes, J.A. Tafani, J.M. Zajac, Affinity of neuropeptide FF analogs toopioid receptors in the rat spinal cord, Peptides 19 (1998) 727–730.

[13] C. Gouarderes, I. Quelven, C. Mollereau, H. Mazarguil, S.Q. Rice, J.M. Zajac,Quantitative autoradiographic distribution of NPFF1 neuropeptide FF receptor

in the rat brain and comparison with NPFF2 receptor by using [125I]YVP and[125I]EYF as selective radioligands, Neuroscience 115 (2002) 349–361.

[14] C. Gouardères, A. Puget, J.M. Zajac, Detailed distribution of neuropeptide FFreceptors (NPFF1 and NPFF2) in the rat, mouse, octodon, rabbit, guinea pig, andmarmoset monkey brains: a comparative autoradiographic study, Synapse 51(2004) 249–269.

[15] H. Mazarguil, C. Gouarderes, J.A. Tafani, D. Marcus, M. Kotani, C. Mollereau, M.Roumy, J.M. Zajac, Structure-activity relationships of neuropeptide FF: role ofC-terminal regions, Peptides 22 (2001) 1471–1478.

[16] C. Gouarderes, C. Mollereau, J.A. Tafani, H. Mazarguil, J.M. Zajac, [125I]EYF: anew high affinity radioligand to neuropeptide FF receptors, Peptides 22 (2001)623–629.

[17] F. Talmont, L. Piedra Garcia, H. Mazarguil, J-M. Zajac, C. Mollereau,Characterization of two novel tritiated radioligands for labellingneuropeptide FF (NPFF1 and NPFF2) receptors, Neurochem. Intl. 55 (2009)815–819.

[18] C. Gouarderes, H. Mazarguil, C. Mollereau, N. Chartrel, J. Leprince, H. Vaudry,J.M. Zajac, Functional differences between NPFF1 and NPFF2 receptor coupling:high intrinsic activities of RFamide-related peptides on stimulation of[35S]GTPcS binding, Neuropharmacology 52 (2007) 376–386.

[19] C. Mollereau, H. Mazarguil, D. Marcus, I. Quelven, M. Kotani, V. Lannoy, Y.Dumont, R. Quirion, M. Detheux, M. Parmentier, J.M. Zajac, Pharmacologicalcharacterization of human NPFF1 and NPFF2 receptors expressed in CHO cellsby using NPY Y1 receptor antagonists, Eur. J. Pharmacol. 451 (2002) 245–256.

[20] L.M. Hjelmeland, A nondenaturing zwitterionic detergent for membranebiochemistry: design and synthesis, Proc. Natl. Acad. Sci. USA 77 (1980)6368–6370.

[21] A. Chattopadhyay, K.G. Harikumar, Dependence of critical micelleconcentration of a zwitterionic detergent on ionic strength: implications inreceptor solubilization, FEBS Lett. 391 (1996) 199–202.

[22] D. Ofri, A.M. Ritter, Y.F. Liu, T.L. Gioannini, J.M. Hiller, E.J. Simon,Characterization of solubilized opioid receptors: reconstitution anduncoupling of guanine nucleotide-sensitive agonist binding, J. Neurochem.58 (1992) 628–635.

[23] A. Pellegrino de Iraldi, J. Santiago Aguilar, E.L. Ochoa, Ultrastructure ofreconstituted membranes containing the muscarinic receptor, Neurochem.Res. 11 (1986) 983–996.

[24] M. Kinuta, K. Takei, Utilization of liposomes in vesicular transport studies, CellStruct. Funct. 27 (2002) 63–69.