Prolactin Releasing Peptide Has High Affinity and Efficacy at Neuropeptide FF2 Receptors

Neuropeptide FF (NPFF) and prolactin-releasing peptide (PrRP) are two members of the RFamide peptide family. In this study we investigated whether these RFamide peptides, which have common structural features in their C-terminal RFamide motif and share several physiologically important functions, could exert their effects through the same set of receptors. The affinity and functional activity of several related RFamide peptides were determined at the human neuropeptide FF receptor subtype 2 (hNPFF2) and the human prolactin-releasing peptide (hPrRP) receptors. The full-length human prolactin releasing peptide 31 (hPrRP31) had significantly higher efficacy compared with NPFF and its stable analog, (1DMe)Y8Fa, at the hNPFF2 receptor. In contrast, NPFF and (1DMe)Y8Fa were not efficacious at the hPrRP receptor. Our study indicated a generally relatively low level of discrimination for RFamide peptides at the NPFF receptor, whereas the hPrRP receptor clearly preferred PrRP or very closely related peptides. The seemingly promiscuous binding of the RFamide peptides to the NPFF receptor was further confirmed by receptor autoradiography. PrRP may thus signal through the NPFF receptors in vivo. The RFamide peptide family is well represented in invertebrates, but so far, only four RFamide prepropeptide precursors have been described in mammals. These include neuropeptide FF (NPFF)/neuropeptide AF (Perry et al., 1997; Vilim et al., 1999), prolactin-releasing peptide (PrRP) (Hinuma et al., 1998), RFamide-related peptides (RFRPs) (Hinuma et al., 2000), and KiSS-1 (Ohtaki et al., 2001). NPFF, the first RFamide peptide identified in mammals, acts as a modulator of morphine analgesia, tolerance, and dependence, and influences many functions including pain mechanisms (Panula et al., 1996). Recently, two human receptors for NPFF have been identified: hNPFF1 and hNPFF2 (Bonini et al., 2000; Elshourbagy et al., 2000; Hinuma et al., 2000). These receptor subtypes have different tissue localizations in human and rat (Bonini et al., 2000). The recently cloned NPFF2 receptor (named HLWAR77 by Elshourbagy et al., 2000) has been described as Gi/o-coupled when stably expressed in HEK 293 cells (Elshourbagy et al., 2000) or CHO cells (Kotani et al., 2001). Among the receptors with the highest amino acid sequence homology to NPFF1 and NPFF2 are members of the orexin, human neuropeptide Y (NPY), and cholecystokinin family, which have been implicated in feeding (Bonini et al., 2000). BIBP3226, an anorexigenic Y1 receptor ligand, has been shown to bind to the NPFF1 receptor, thus further suggesting a potential role of the NPFF1 receptor in regulation of feeding (Bonini et al., 2000). PrRP, the second RFamide peptide, was identified as the endogenous ligand for the orphan G protein-coupled receptor (GPCR), hGR3 (Hinuma et al., 1998). The full physiological role of PrRP remains to be elucidated, but it has been envisioned to play a broader role in brain function than originally suggested, and the ability to stimulate prolactin release may not represent its primary biological function (Samson et al., 2000). The hGR3 receptor (named GPR10 by Marchese et al., 1995; and in this paper referred to as the hPrRP receptor) and its rat counterpart, UHR-1, have been shown to couple to at least Gq and Gi but not to Gs in CHO cells (Hinuma et al., 1998, 1999). Mammalian genes encoding a third class of RFamide peptides, namely hRFRP1 and hRFRP3 and their receptor OT7T022, have recently been reported (Hinuma et al., 2000). Financial support was received from the Technology Development Fund (TEKES) and the Academy of Finland. Article, publication date, and citation information can be found at http://jpet.aspetjournals.org. DOI: 10.1124/jpet.102.047118. ABBREVIATIONS: NPFF, neuropeptide FF; PrRP, prolactin-releasing peptide; RFRP, RFamide-related peptide; hNPFF2, human neuropeptide FF receptor subtype 2; CHO, Chinese hamster ovary; NPY, neuropeptide Y; GPCR, G protein-coupled receptor; [S]GTP S, [S]guanosine-5 -O(3-thio)triphosphate; (1DMe)Y8Fa, DYL(NMe)FQPQRF-NH2; rNPSF, rat neuropeptide SF; r, rat; b, bovine; h, human; HEK, human embryonic kidney cell; FMRFamide, phenylalanyl-methionyl-arginyl-phenylalaninamide; I-Y8Fa, I-YLFQPQRFamide; I-EYF, I-EYWSLAAPQRFNH2; RT, room temperature; BSA, bovine serum albumin; TB, total binding; NSB, nonspecific binding; SB, specific binding; PTX, pertussis toxin; VIP, vasoactive intestinal polypeptide. 0022-3565/03/3053-825–832$7.00 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 305, No. 3 Copyright © 2003 by The American Society for Pharmacology and Experimental Therapeutics 47118/1062949 JPET 305:825–832, 2003 Printed in U.S.A. 825 at A PE T Jornals on N ovem er 4, 2017 jpet.asjournals.org D ow nladed from OT7T022 is activated by NPFF and corresponds to the NPFF1 receptor characterized by Bonini et al. (2000). RFRPs (more specifically hRFRP1) have been shown to regulate prolactin secretion (Hinuma et al., 2000), but additional physiological functions cannot be ruled out. KiSS-1 is a metastasis suppressor gene that encodes the fourth class of RFamide peptides (Ohtaki et al., 2001). The gene product of KiSS-1, also known as “metastin,” was found to be the endogenous ligand of an orphan GPCR, hOT7T175 (Ohtaki et al., 2001). The cellular mechanisms by which NPFF, PrRP, and RFRP exert their functions in vivo are poorly understood. To clarify the cellular and pharmacological actions activated by these RFamide peptides, well characterized, functional in vitro assays on recombinant cell lines and/or cell lines expressing these receptors endogenously need to be developed. In the present report, we have used [S]guanosine-5 -O-(3thio)triphosphate ([S]GTP S) binding induced by RFamide peptides on the hNPFF2 and hPrRP receptors stably transfected in CHO cells (CHO-hNPFF2 and CHO-hPrRP-R, respectively) to determine agonist efficacies and rank orders of potency. The agonist potencies of RFamide peptides were compared with their affinities obtained in competition binding assays with cell membrane preparations. Receptor autoradiography was conducted to visualize and quantitate the NPFF binding sites in rat cervical spinal cord sections to investigate the binding properties of RFamide peptides at the NPFF receptor in a native environment. Materials and Methods Materials. The following natural peptides and analogs were purchased from Bachem (Bubendorf, Switzerland): (1DMe)Y8Fa [DYL(NMe)FQPQRF-NH2], hPrRP20 (TPDINPAWYASRGIRPVGRF-NH2), hPrRP31 (SRTHRHSMEIRTPDINPAWYASRGIRPVGRF-NH2), human neuropeptide Y (hNPY) (YPSKPDNPGEDAPAEDMARYYSALRHYINLITRQRY-NH2), hNPY18-36 (ARYYSALRHYINLITRQRYNH2), NPFF (FLFQPQRF-NH2), and BIBP3226. HPFRP-1 (MPHSFANLPLRF-NH2), and hPRFP-3 (VPNLPQRF-NH2) were purchased from Phoenix Pharmaceuticals Inc. (Belmont, CA). NPFF-OH (FLFQPQRF-OH), rat neuropeptide SF (rNPSF) (SLAAPQRF-NH2), FMRFamide (FMRF-NH2), hPrRP24–31, hRFRP(6G)-3 (VPNLPGRFamide), and bPrRP20 (TPDINPAWYAGRGIRPV-GRFamide) were custom synthesized by Peptide Technologies Corp. (Gaithersburg, MD). Substance P was purchased from Peninsula Laboratories (Belmont, CA). Iodinated (1DMe)Y8Fa was custom ordered from Amersham Biosciences UK, Ltd. (Little Chalfont, Buckinghamshire, UK). The specific activity of I-(1DMe)Y8Fa was 2000 Ci/mmol, as indicated by the product specification sheet. [S]GTP S was purchased from PerkinElmer Life Sciences (Boston, MA). The specific activity of [S]GTP S was 1250 Ci/mmol. I-(1DMe)Y8Fa Binding Assays on Rat Spinal Cord Membranes. Rat spinal cord membrane preparations and binding assays were performed using essentially the method for I-YLFQPQRFamide (I-Y8Fa) binding as described by Allard et al. (1989) and modified by Payza and Yang (1993). Briefly, membranes (100–160 g/sample) were incubated with 0.02 to 0.07 nM I-(1DMe)Y8Fa, and various concentrations of ligands in 50 mM Tris-HCl, pH 7.5 at RT, 60 mM NaCl, 1 mM MgCl2, 3 g/ml aprotinin, 7.5 g/l BSA, and 30 M bestatin. Total binding (TB) and nonspecific binding (NSB) were determined in the absence and presence, respectively, of 1 M (1DMe)Y8Fa. After 45 min at RT, incubations were terminated by rapid filtration (Tomtec Harvester96; Tomtec Inc., Hamden, CT) through GF/B glass fiber filter mats (presoaked at 4°C in 200 ml of 50 mM Tris-HCl, 60 mM NaCl, 1 mM MgCl2, 1 g/l BSA, 5 g/l polyethyleneimine, pH 7.4 at 4°C for 45 min). Filters were washed four times with 5 ml of ice-cold wash buffer (50 mM Tris-HCl, 60 mM NaCl, 1 mM MgCl2, pH 7.4 at 4°C). Specific binding (SB) was calculated as TB NSB. The SB in the presence of various concentrations of test compounds was expressed as percentage of control SB, which was obtained as the TB in the absence of any competing compound minus NSB. Analysis of competition binding experiments was carried out by nonlinear least squares curve fitting with Hill slopes (nH) being set to unity (Devillers et al., 1994). Affinity constants (Ki) were calculated from the IC50 values according to the Cheng-Prusoff equation (Cheng and Prusoff, 1973). The KD for this purpose was 0.19 nM, as determined in a pilot study. I-(1DMe)Y8Fa Binding Assay on CHO-hNPFF2 Membranes. Recombinant CHO cells expressing the hNPFF2 receptor (Euroscreen S.A., Brussels, Belgium) were grown in Ham’s F12 medium (Nutrient Mixture Ham’s F12; Invitrogen, Glasgow, UK) supplemented with 10% fetal bovine calf serum and 400 g/ml G418. Cells were harvested in phosphate-buffered saline and frozen at 70°C. To prepare membranes, thawed cell pellets were homogenized on ice in a Potter-Elvehjem homogenizer in 10 mM Tris-HCl, 0.1 mM EDTA, pH 7.5 at RT supplemented with 320 mM sucrose. The nuclear pellet obtained by centrifugation of the homogenate at 1,000g for 15 min at 4°C was discarded. From the supernatant, a membrane fraction was collected by centrifugation at 48,000gmax for 30 min at 4°C. The 48,000g pellet was re-suspended in 10 mM Tris-HCl, 0.1 mM EDTA, pH 7.5 at RT without sucrose and centrifuged again at 48,000gmax for 30 min. Binding of I-(1DMe)Y8Fa to membranes (2.5 g/sample) of hNPFF2 receptor expressing CHO cells and analysis of the binding experiments were carried out as described above for rat spinal cord membranes. The KD used in the analysis of the competition binding experiments was 0.10 nM, as determined in a pilot study. I-PrRP Binding Assay on CHO-hPrRP-R Membranes. Recombinant CHO cells expressing the human PrRP (hPrRP) receptor hGR3/GPR10 (Euroscreen S.A.) were grown, and membranes from these cells were prepared by the same method as described above for hNPFF2 expressing CHO cells. I-PrRP20 competition binding experiments were performed by incubating membranes (0.75 g/sample) with 0.02 to 0.05 nM iodinated peptide and various concentrations of ligands in 25 mM Hepes, pH 7.4 at RT, 1 mM CaCl2, 5 mM MgCl2, 3 g/ml aprotinin, 30 M bestatin, and 7.5 mg/ml BSA. NSB was determined in the presence of 1 M hPrRP20. Incubations were terminated after 45 min at RT as described above for rat spinal cord membranes except that 25 mM Hepes, 1 mM CaCl2, 5 mM MgCl2, and 0.5 M NaCl, pH 7.4, at 4°C was used as the washing buffer. The analysis of the binding data was also carried out as described above using a KD value of 0.3 nM, as determined in saturation binding experiments. [S]GTP S Binding Assay. The agonist activity of various ligands was determined by their ability to stimulate the receptormediated binding of [S]GTP S to G proteins in membranes of CHO-hNPFF2 or CHO-hPrRP-R cells. Membranes (2 and 10 g/ sample of CHO-hNPFF2 and CHO-hPrRP-R cells, respectively) were incubated in 50 mM Tris-HCl, 5 mM MgCl2, 1 mM dithiothreitol, 1 mM EDTA, 1 M GDP, 20 mM NaCl (CHO-hNPFF2 assay), or 100 mM NaCl (CHO-hPrRP-R assay), pH 7.4 at RT with 6 to 12 concentrations of test ligands and a tracer concentration of [S]GTP S (0.07–0.16 nM). After a 60-min incubation at RT (30-min preincubation without label followed by a 30-min stimulation after addition of label in CHO-hNPFF2 assay or 60 min stimulation in CHO-hPrRP-R assay without preincubation), the reaction was terminated by rapid vacuum filtration through glass fiber filters. Filters were washed four times with 5 ml of ice-cold wash buffer (20 mM Tris-HCl, 5 mM MgCl2, 1 mM EDTA, pH 7.4 at RT), dried, and counted for radioactivity in a scintillation counter. Experimental results were calculated with nonlinear least squares curve fitting. Agonist effects were normalized against the stimulation obtained with reference compounds (1DMe)Y8Fa, in the case of the hNPFF2 receptor, and hPrRP20, in 826 Engström et al. at A PE T Jornals on N ovem er 4, 2017 jpet.asjournals.org D ow nladed from the case of the hPrRP receptor. The response of the reference agonists was set as 100%. Experiments were repeated at least three times, unless indicated otherwise. Quantitative Receptor Autoradiography. Autoradiography was performed on rat spinal cord sections with I-(1DMe)Y8Fa and was carried out essentially as described earlier for I-Y8Fa (Allard et al., 1992). Coronal 20m cryosections at the cervical level of the rat spinal cord were collected onto poly-L-lysine-coated slides (Menzel-Gläser; Merck, Darmstadt, Germany) and stored dry at 70°C. The mounted sections were rehydrated in 50 mM Tris-HCl (pH 7.5), 140 mM NaCl and 0.5% BSA for 20 min at RT. The slides were washed twice with ice-cold 50 mM Tris-HCl (pH 7.5) for 2 min before the incubation with 0.05 nM I-(1DMe)Y8Fa in 50 mM Tris-HCl (pH 7.5), 120 mM NaCl, 0.5% BSA, 0.1 mM bestatin, 1 mM EDTA, and 3 M aprotinin. Displacement of I-(1DMe)Y8Fa was analyzed by including unlabeled competing agents at the following concentrations: rNPSF (500 nM, 5 nM), NPFF (20 nM, 0.2 nM), (1DMe)Y8Fa (20 nM, 0.2 nM), bPrRP20 (200 nM, 2 nM), hRFRP-1 (1 M, 10 nM), hRFRP(6G)-3 (1 M, 10 nM), BIBP3226 (1 M, 10 nM), and substance P (1 M). The concentrations of the ligands [except hRFRP(6G)-3 and BIBP3226, which were tested at 10 nM and 1 M] were chosen to represent approximately the Ki and the 100-fold Ki value (Table 1). After 60 min of incubation at RT, the slides were washed three times for 2 min in ice-cold Tris-HCl (pH 7.5) and were finally dipped in ice-cold distilled water, air-dried, and exposed on film (Biomax MR; Eastman Kodak, Rochester, NY). Autoradiographic films were analyzed using the MCID image analysis system (Imaging Research, St. Catherines, ON, Canada). The optical density of the area of the upper dorsal horn (laminae I–II) was measured and the integrated optical density values were determined based on a calibration curve derived from C standards (American Radiolabeled Chemicals, St. Louis, MO) exposed simultaneously with the samples to the films. The means S.E.M. of three animals and three sections in each area of every animal are given as the quantitative assessment of ligand binding activity. Statistical analysis was performed by one-way analysis of variance of grouped data (NewmanKeuls test; MicroComputer Specialists, Wynnewood, PA).