Palmitoylation of Prolactin-Releasing Peptide Increased Affinity for and Activation of the GPR10, NPFF-R2 and NPFF-R1 Receptors: In Vitro Study

The anorexigenic neuropeptide prolactin-releasing peptide (PrRP) is involved in the regulation of food intake and energy expenditure. Lipidization of PrRP stabilizes the peptide, facilitates central effect after peripheral administration and increases its affinity for its receptor, GPR10, and for the neuropeptide FF (NPFF) receptor NPFF-R2. The two most potent palmitoylated analogs with anorectic effects in mice, palm11-PrRP31 and palm-PrRP31, were studied in vitro to determine their agonist/antagonist properties and mechanism of action on GPR10, NPFF-R2 and other potential off-target receptors related to energy homeostasis. Palmitoylation of both PrRP31 analogs increased the binding properties of PrRP31 to anorexigenic receptors GPR10 and NPFF-R2 and resulted in a high affinity for another NPFF receptor, NPFF-R1. Moreover, in CHO-K1 cells expressing GPR10, NPFF-R2 or NPFF-R1, palm11-PrRP and palm-PrRP significantly increased the phosphorylation of extracellular signal-regulated kinase (ERK), protein kinase B (Akt) and cAMP-responsive element-binding protein (CREB). Palm11-PrRP31, unlike palm-PrRP31, did not activate either c-Jun N-terminal kinase (JNK), p38, c-Jun, c-Fos or CREB pathways in cells expressing NPFF-1R. Palm-PrRP31 also has higher binding affinities for off-target receptors, namely, the ghrelin, opioid (KOR, MOR, DOR and OPR-L1) and neuropeptide Y (Y1, Y2 and Y5) receptors. Palm11-PrRP31 exhibited fewer off-target activities; therefore, it has a higher potential to be used as an anti-obesity drug with anorectic effects.


Introduction
Prolactin-releasing peptide (PrRP) was discovered as an endogenous ligand of the orphan G-protein coupled receptor GPR10 (also known as hGR3) in the hypothalamus and has been suggested to stimulate prolactin secretion [1,2]. However, soon after this finding, Lawrence et al. showed a reduction in food intake and body weight and an increase in energy expenditure after intracerebroventricular (ICV) PrRP injection in rats and questioned the role of PrRP in prolactin secretion [3,4]. The effects of PrRP, mostly mediated through the GPR10 receptor, which is widely expressed throughout the brain mainly in areas related to the regulation of food intake and energy homeostasis, confirm GPR10 knockout (KO) mouse studies showing an increase in body weight in KO mice [5][6][7].
PrRP occurs in two biologically active isoforms, PrRP31 and PrRP20. Our previous studies showed the induction of central c-Fos activation of regions related to food intake R1. GPR10 is a highly selective receptor for PrRP31 and analogs related to PrRP31. To control the selectivity of PrRP31 for GPR10s, we used NPFF and its stable analog 1DMe in this study. To determine whether the possible analgesic effect of PrRPs is caused by off-target activity, opioid receptors were investigated. Based on previously published data, affinity for the GPR10 and NPFF-R2 of PrRP31 and its analogs was studied [8,9]. PrRP31 and its two palmitoylated analogs of PrRP31 (see Table 1 for structures) have a high binding affinity for the GPR10 and NPFF-R2 receptors, and their K i values were in the nanomolar range (Table 2). Compared to natural PrRP31, palmitoylated analogs had a higher binding affinity for both of these receptors. Palm 11 -PrRP31 showed a higher affinity for the receptor GPR10 than for the receptor NPFF-R2. NPFF and its stable analog 1DMe displayed negligible affinity for the GPR10 receptor. The affinities of NPFF and 1DMe to NPFF-R2 were detected to be in the nanomolar range ( Table 2). -not determined; data presented as the means K i values ± SEM and analyzed in Graph-Pad Software were performed in 2-5 independent experiments in duplicates. K i was calculated using the Cheng-Prusoff equation [33].
To find another possible target of the two most potent palmitoylated analogs of PrRP31, binding to NPFF-R1 was tested. Membranes from CHO-K1 cells expressing the NPFF-R1 were isolated, and the K d was determined to be 0.94 ± 0.06 nM by saturation experiments using the radioligand [ 125 I]-1DMe. Although natural PrRP31 bound to NPFF-R1 with a lower affinity than to NPFF-R2, the binding affinity was still in the 10 −8 M range (Table 2). Palmitoylation increased the binding affinities of both analogs to NPFF-R1. Palm-PrRP31 showed binding affinities in the nanomolar range to both NPFF receptors compared to palm 11 -PrRP31 (Table 2) Several other potential off-target receptors of PrRP31 and its palmitoylated analogs were tested. The binding properties of PrRP31, palm 11 -PrRP and palm-PrRP31 to receptors Y 1 , Y 2 , and Y 5 , ghrelin receptor (also growth hormone secretagogue receptor-GHSR) and kappa-opioid receptor (KOR) were determined. The natural ligand PYY of Y receptors bound in the nanomolar range to the Y 1 , Y 2 , and Y 5 receptors (Table 2). From saturation binding experiments with [ 125 I]-PYY as a radioligand, the K d for each receptor was determined. The K d for Y 1 was 1.53 ± 0.08 nM, for Y 2 was 2.18 ± 0.85 nM and for Y 5 was 1.01 ± 0.27 nM. Natural PrRP31 had no affinity to the Y 1 and Y 2 receptors in the range of measured concentrations, but it showed a very low affinity to the Y 5 receptor. Compared to palm 11 -PrRP31, palm-PrRP31 exhibited a relatively high affinity for the Y 5 receptor. Both palmitoylated analogs bound to Y 1 and Y 2 with a negligible low affinity ( Table 2).
The K d determined by a saturation binding experiment with [ 125 I]-dynorphin as a radioligand was 2.38 nM. The agonist dynorphin showed a very high affinity for the KOR receptor, but no binding was observed with natural PrRP31 (Table 2). Palmitoylation enhanced binding to the KOR receptor. Palm-PrRP31 bound to KOR with a higher affinity than palm 11 -PrRP31, but both were in the 10 −7 -10 −6 M range.
Another tested potential off-target receptor was the ghrelin receptor GHSR. From saturation experiments using [ 125 I]-ghrelin as a radioligand, a K d of 0.44 ± 0.12 nM was determined. Natural PrRP31 showed no binding to GHSR in competitive binding experiments in the measured range, but palmitoylated analogs showed a low binding affinity for this receptor (Table 2). Palm-PrRP31 had a higher affinity for GHSR than palm 11 -PrRP31.

Palmitoylated PrRP31
Analogs Activate Different Intracellular Signaling Pathways in GPR10-, NPFF-R2-or NPFF-R1-Expressing Cells To determine the intracellular mechanism of action of PrRP31 and its palmitoylated analogs, several signaling pathways were tested in cells expressing GPR10, NPFF-R2 or NPFF-R1 receptors using immunoblotting (Figures 2-5; Supplementary Figure S1). No changes in total protein levels were observed (Supplementary Figure S1B); therefore, only activated/phosphorylated proteins were quantified and compared. NPFF and 1DMe were used as negative controls to validate GPR10 selective properties.
The activation of the cAMP-dependent protein kinase (PKA) was also studied (Supplementary Figure S1A). No significant changes were observed after treatment with PrRP31, palmitoylated PrRP31 analogs, NPFF or 1DMe in cells expressing GPR10, NPFF-R2 and NPFF-R1. Densitometric quantification was normalized to GAPDH, and the phosphorylation level in the untreated control was standardized as 100%. Data are presented as the mean ± SEM and analyzed by two-way ANOVA followed by Dunnett's post hoc test. Experiments were performed independently at least three times. Statistically significant differences from the control are indicated (* p < 0.05, ** p < 0.01, *** p < 0.001). Densitometric quantification was normalized to GAPDH, and the phosphorylation level in the untreated control was standardized as 100%. Data are presented as the mean ± SEM and analyzed by two-way ANOVA followed by Dunnett's post hoc test. Experiments were performed independently at least three times. Statistically significant differences from the control are indicated (* p < 0.05, ** p < 0.01, *** p < 0.001).  Densitometric quantification was normalized to GAPDH and the phosphorylation level in the untreated control was standardized as 100%. Data are presented as mean ± SEM and analyzed by two-way ANOVA followed by Dunnett's post hoc test. Experiments were performed independently at least three times. Statistically significant differences from the control are indicated (* p < 0.05, ** p < 0.01, *** p < 0.001).
To further characterize the signaling of receptors GPR10 and NPFF-R2, dose-response experiments were performed. The EC 50 of ERK activation in cells expressing GPR10 was in the nanomolar range after stimulation with PrRP31, palm 11 -PrRP and palm-PrRP31 ( Figure 4A). Cells expressing NPFF-R2 showed a strong response with EC 50 in nanomolar concentrations after stimulation with natural PrRP31, palmitoylated PrRP31 analogs, NPFF or 1DMe ( Figure 4B).
Finally, three DNA-binding proteins, cyclic AMP-responsive element-binding (CREB), c-Jun and c-Fos protein, which activate transcription factors, were tested ( Figure 5). Palm-PrRP31 significantly increased the activation of c-Jun ( Figure 5A) and c-Fos ( Figure 5B) and the phosphorylation of CREB ( Figure 5C) compared to the nontreated control in the CHO-K1 cells expressing GPR10, NPFF-R2 or NPFF-R1. Stimulation with palm 11 -PrRP31 significantly increased the activation of all three DNA-binding proteins ( Figure 5) in cells with GPR10 and NPFF-R2, but was ineffective in cells expressing the NPFF-R1 receptor. No activation in GPR10 after stimulation with NPFF and its stable analog 1DMe was observed, unlike in NPFF-R2 or NPFF-R1, where significantly increased activation was monitored.
The results showing signaling pathway activation determined using immunoblotting in CHO-K1 cells expressing GPR10, NPFF-R2 and NPFF-R1 incubated with peptides at final concentrations of 10 −6 M are summarized in Table 3. Table 3. Summary table of signaling pathways tested using immunoblot in cells expressing GPR10, NPFF-R2 and NPFF-R1.

Agonist and Antagonist Properties of PrRP31 and Its Palmitoylated Analogs at Other Potential Off-Target Receptors
Using the beta-lactamase reporter gene assay with a FRET substrate, receptor activation was studied to establish agonist and antagonist properties of natural PrRP31 and palmitoylated PrRP31 analogs.
Both tested palmitoylated PrRP analogs were strong agonists of the GPR10 receptor, and their EC 50 values were in the picomolar range (Table 4). Palm 11 -PrRP31 had stronger agonist activity on GPR10 than the analog palm-PrRP31. Table 4. Agonist properties on GPR10 and other potential off-target receptors determined using β-lactamase assay. Natural PrRP31 was not effective at any tested possible off-target receptor. Both palm 11 -PrRP31 and palm-PrRP31 did not show any agonist activity on the DOR, MOR and ORL-1 opioid receptors, but they did have very weak agonist activity on the KOR (Table 4). In addition, lipidized analogs exerted weak agonist effects on GHSR.

Discussion
Palmitoylated analogs of neuropeptide PrRP31 previously showed anorexigenic effects and central c-Fos activation after peripheral administration, as well as increased central insulin and leptin signaling, suggesting great potential for the treatment of not only obesity but also neurodegenerative disorders [9,34,35]. PrRP31 has a high affinity for its receptor GPR10, but it also binds to NPFF-R2 [13]. Based on the results of our previous studies the mechanism of action of the two most potent palmitoylated PrRP31 analogs, palm 11 -PrRP31 and palm-PrRP31 on the anorexigenic receptors GPR10 and NPFF-R2 was mapped.
Palmitoylation increased the binding properties of PrRP31 to both of these receptors. Palm 11 -PrRP31 had a higher affinity for the GPR10 receptor than palm-PrRP31, and both analogs displayed an affinity for the NPFF-R2 in the nanomolar range. In this study, several possible off-target receptors of PrR31 were tested. Both of the PrRP31 analogs showed a stronger affinity for the NPFF-R1 than natural PrRP31. Therefore, NPFF-R1 is now considered another relevant target of lipidized PrRP31 analogs.
The activation of intracellular Ca 2+ mobilization in the CHO-K1 AequoScreen cell line expressing NPFF-R2 showed the agonist properties of PrRP31 and its palmitoylated analogs. Palmitoylation increased the agonist properties of PrRP31 on the receptor NPFF-R2. However, NPFF and its stable analog 1DMe have much stronger agonist activity on its NPFF-R2 receptor than palm 11 -PrRP31 and palm-PrRP31. The activation of GPR10 was studied using the β-lactamase assay with a FRET substrate and a FLIPR calcium assay measuring intracellular Ca 2+ mobilization. The EC 50 values of palm 11 -PrRP31 and palm-PrRP31 were in the picomolar range, and the activation was increased three times after palmitoylation. Previous studies suggested that GPR10 is coupled with Gi/o proteins [30,32]. Other studies have shown the ability of PrRP to stimulate cAMP in rat PC12 cells [36] and CHO-K1 cells expressing GPR10 [37], which pointed to Gs protein coupling. However, Langmead et al. revealed the PrRP-induced mobilization of intracellular Ca 2+ after GPR10 activation, and the PrRP's inability to suppress cAMP levels after forskolin stimulation in HEK293 cells transfected with GPR10. These results suggested that GPR10 is coupled with the Gq protein [27]. We observed intracellular Ca 2+ mobilization after stimulation with PrRP31 and its palmitoylated analogs, which may suggest that GPR10 is coupled with either Gi or Gq. This also supported our finding that PKA was not activated after stimulation with PrRP31 and its palmitoylated analogs; thus, GPR10 was not coupled with Gs proteins.
In this study, the intracellular signaling pathways of PrRP31 and its palmitoylated analogs in CHO-K1 cells transfected with GPR10, NPFF-R2, or NPFF-R1 were explored using immunoblotting, and the possible signal transduction of GPR10 was suggested (Figure 7). Haykawa et al. previously showed the activation of Akt in rat pituitary GH3 cells after 5 min of stimulation with PrRP [32]. Our study found significant induction of Akt phosphorylation at T308 and S473 in CHO-K1 cells expressing GPR10, NPFF-R2 and NPFF-R1 after 5 min of stimulation with palm 11 -PrRP31 and palm-PrRP31, but no significantly increased phosphorylation was observed after stimulation with natural PrPR31 in cells with NPFF-R1. Palmitoylation helped stabilize PrRP31 and increased the induction activity of Akt through the receptors GPR10, NPFF-R2 and NPFF-R1. Previous studies demonstrated that PrRP activated the MAP kinases ERK and JNK in rat GH3 cells [30] and PC12 cells [38]. Our results showed significant activation of JNK, ERK and p38 MAPKs after PrRP31 incubation in CHO-K1 cells expressing GPR10, and of JNK and ERK in cells with NPFF-R2. Both palmitoylated PrRP31 analogs also significantly increased the phosphorylation of all three tested MAPKs in GPR10 and NPFF-R2-expressing cells. Dose-response experiments showed the ability of PrRP31, palm 11 -PrRP31 and palm-PrRP31 to activate ERK phosphorylation in cells with GPR10 and NPFF-R2 in the nanomolar range. JNK and ERK activation play important roles in cell proliferation, differentiation and apoptosis by promoting the formation of AP1 complexes, important transcription factors controlling the cell cycle, through the activation of c-Fos and c-Jun [39]. Similar to ERK and JNK, p38 is also connected with cell cycle regulation, regulation of stress responses, immune responses and cell differentiation [40]. Palm 11 -PrRP31 and palm-PrRP31 were found to significantly increase p38 phosphorylation in cells expressing GPR10 and NPFF-R2, and stimulation with palm-PrRP31 induced p38 phosphorylation in cells expressing NPFF-R1. Both inducible transcription factors, c-Fos and c-Jun, were significantly activated after stimulation with PrRP31 and its palmitoylated analogs in cells with GPR10 and NPFF-R2. Conversely, NPFF-R1 significantly activated c-Fos and c-Jun only after stimulation with palm-PrRP31. The transcription factor CREB is also important for the regulation of cell proliferation, cell survival and differentiation, for maintaining glucose homeostasis, and has an important role in activating immune responses [40,41]. Likewise, c-Fos and c-Jun activation and phosphorylation of the transcription factor CREB were significantly increased in cells expressing GPR10 and NPFF-R2 after stimulation with PrRP31 and its analogs. Compared to palm 11 -PrRP31, palm-PrRP31 showed a higher activity in cells transfected with NPFF-R1 in all tested signaling pathways. The results show that PrRP31, palm 11 -PrRP31 and palm-PrRP31 may play important roles in the regulation of cell proliferation and affect immune responses. These findings suggest that dysregulation of glucose homeostasis and inflammatory responses linked with obesity could be treated with PrRP31 analogs.
In this study, we tested potential off-target receptors of PrRP31, which are related to food intake and energy metabolism. Because PrRP and NPFF were found to have antinociceptive properties [16,17,22,23], their agonist and antagonist activities on opioid receptors were studied using a β-lactamase assay. In our study, palmitoylation increased the binding properties of natural PrRP31. Palm 11 -PrRP31 was found to have a lower affinity for KOR than palm-PrRP31, but they both had negligible ability to activate the KOR receptor in either agonist mode or antagonist mode. We did not observe any agonist or antagonist activity of either PrRP31 palmitoylated analog on the other opioid receptors MOR, DOR and ORL-1. The possible pain modulation properties of PrRP do not seem to be linked to opioid receptors, which supports the idea that GPR10 is involved in pain processing regulation. A study by Laurent et al. using GPR10 KO suggested that the central anti-opioid activity of NPFF in mice is regulated by GPR10. Moreover, they suggested that the dual coupling of GPR10 with Gq and Gi may be the reason for PrRP's involvement in different neuronal networks [23]. GPR10 could be involved either in pain modulation or food intake regulation, depending on the type of G protein coupled with GPR10. Figure 7. Scheme of mechanism of action of palmitoylated PrRP31 analogs at GPR10: ERK, extracellular signal-regulated kinase; JNK, c-Jun N-terminal kinase; CREB, cAMP-responsive element binding protein; PIP 2 , phosphatidylinositol 4,5-bisphosphate; IP 3 , inositol 1,4,5-triphosphate; PIP 3 , phosphatidylinositol (3,4,5)-trisphosphate; PDK, phosphoinositidedependent kinase 1; Akt, protein kinase B; mTORC2, mammalian target of rapamycin complex 2; GSK-3β, glycogen synthase kinase-3β.
NPY, together with PYY and pancreatic polypeptide (PP), controls energy homeostasis though NPY receptors. NPY receptors are expressed throughout the central nervous system but can also be found in the peripheral nervous system [42]. The affinity of PrRP31 and its palmitoylated analogs for the receptors Y 1 , Y 2 and Y 5 was tested. No binding affinity of PrRP31 and palmitoylated analogs for the Y 2 receptor was observed and a negligible affinity of palm-PrRP31 for Y 1 was detected. However, natural PrRP31, palm 11 -PrRP31 and palm-PrRP31 bound and activated the Y 5 receptor with a K i and an EC 50 in the micromolar range. PrRP31 affinity and agonist activity were increased with the attached palmitoyl group. Y 1 and Y 5 receptors are expressed in the same neurons, and they both have important regulatory functions in food intake and energy balance [42]. Although NPY is an orexigenic peptide, Y 1 and Y 5 receptor deletion leads to obesity and decreases food intake [43]. This study showed that palm 11 -PrRP31 and palm-PrRP31 had agonist activity ranging from 10 −7 to 10 −8 M on the Y 5 receptor, and palm 11 -PrRP31 was also shown to be a positive allosteric modulator, which suggests that PrRP31 analogs could mediate the in vivo ability to reduce food intake through Y 5 receptors.
Finally, the off-target properties of the palmitoylated PrRP31 analogs on the receptor of the orexigenic peptide ghrelin were studied. Palm-PrRP31 had a higher affinity for the GHSR receptor than palm 11-PrRP31, but both analogs had negligible activity on GHSR. Palm 11 -PrRP31 and palm-PrRP31 displayed higher affinity for GPR10 and NPFF-R2 receptors than natural PrRP31, and stimulation with PrRP31 analogs activated transcription factors c-Fos, c-Jun and CREB and also activated PKB/Akt, MAPK pathways in cells expressing these receptors. A new strong target of palmitoylated analogs was found to be NPFF-R1. Palm-PrRP31 induced activation of tested signaling pathways in cells expressing NPFF-R1. Both analogs revealed negligible affinity and ability to activate receptors Y, opioid receptors and GHSR, but palm-PrRP31 showed higher off-target binding affinity for these possible off-target receptors. Palm 11 -PrRP31 was a more selective agonist of anorexigenic receptors GPR10 and NPFF-R2, with less off-target activity; therefore, it has higher potential for the treatment of obesity and neurodegenerative diseases.

Material
Human PrRP31, palm 11 -PrRP31, palm-PrRP31, neuropeptide FF (NPFF), its stable analog 1DMe (see Table 1 for structures), and ghrelin (ghr) were synthetized and purified as described previously [9,31]. PrRP31 palmitoylation was performed on fully protected peptide on resin as a last step [44]. Peptide purification and identification were determined by analytical high-performance liquid chromatography and by using a Q-TOF micro MS technique (Waters, Milford, MA, USA). Purity of the synthesized peptides was greater than 95%. Human

Cell Membrane Isolation
Pellets of CHO-K1 cells containing NPFF-R2, NPFF-R1 and KOR receptors were homogenized in ice-cold homogenizing buffer (20 mM HEPES pH 7.1, 5 mM MgCl 2 , 0.7 mM bacitracin) with a DIAX 100 Homogenizer (Heidolph Instruments, Schwabach, Germany) and centrifuged in an ultracentrifuge (Beckman Coulter, Fullerton, CA, USA) at 26,000× g for 15 min at 4 • C. The pellets were homogenized in ice-cold homogenization buffer, and the previous steps were repeated 2 more times. After the third centrifugation, pellets were resuspended in ice-cold storage buffer (50 mM Tris-Cl pH 7.4, 0.5 mM EDTA, 10 mM MgCl 2 , 10% sucrose), and aliquots were stored at −80 • C. The concentration of isolated membrane proteins was determined by a PierceTM BCA Protein Assay Kit (Pierce, Rockford, IL, USA).

Competitive Binding Experiments
Competition binding experiments were performed according to [45]. [ 125 I]-PrRP31 was used to compete with human PrRP31, palmitoylated PrRP31 analogs, NPFF, and 1DMe in CHO-K1 cells expressing human GPR10 as described previously [31]. Binding experiments using U2OS cells were optimized and performed in assay buffer (50 mM Tris-Cl pH 7. Plasma membranes isolated from CHO-K1 cells containing receptors NPFF-R2, NPFF-R1 and KOR were used at a concentration of 5 µg of protein/tube, and binding experiments were performed in assay buffer (50 mM Tris-HCl pH 7.4 + 60 mM NaCl + 1 mM MgCl2 + 0.5% BSA). [ 125 I]-1DMe was used to compete with human PrRP31, palmitoylated PrRP31 analogs, NPFF or 1DMe in isolated membranes with NPFF-R2 or NPFF-R1, and [ 125 I]dynorphin A was used to compete with human PrRP31 and palmitoylated PrRP31 analogs in isolated membranes with KOR. The studied peptides and radioligands were incubated with plasma membranes for 60 min at RT and subsequently filtered in a Brandel cell harvester (Biochemical and Development Laboratories, Gaithersburg, MD, USA) using Whatman GF/B filters preincubated in 0.3% polyethylenimine. Filters were rinsed three times with 2 mL of wash buffer (50 mM Tris pH 7.4 + 60 mM NaCl).
Radioactivity was determined by a γ-counter Wizard 1470 Automatic Gamma Counter (Perkin Elmer). Experiments were carried out in duplicate at least three times, and K i was calculated using the Cheng-Prusoff equation.

Cell Signaling Detection by Immunoblotting
Activation of signaling pathways was studied in the CHO-K1 cell lines containing GPR10, NPFF-R2 and NPFF-R1. Cells were seeded in 24-well plates at 30,000 cells/well in assay medium (growth medium without selective antibiotics) and were grown for 2 days. The day before the experiment, the medium was changed to serum-free medium. On the day of the experiment, cells were incubated with PrRP31, lipidized PrRP31 analogs, NPFF or 1DMe at final concentrations from 10 −11 to 10 −5 M for 5 min or 60 min at 37 • C and then washed three times with ice-cold phosphate-buffered saline (PBS) pH 7.4. Cells were lysed with Laemmli sample buffer (62.5 mM Tris-HCl at pH 6.8, 2% SDS, 10% glycerol, 0.01% bromophenol blue, 5% β-mercaptoethanol, 50 mM NaF, and 1 mM Na 3 VO 4 ). Samples were stored at −20 • C. Electrophoresis and immunoblotting were performed as described previously [45]. For detection of signaling pathways, primary monoclonal antibodies (see Table 5 for the antibodies used) purchased from Cell Signaling Technology (Danvers, MA, USA) were used.

Calcium Mobilization Assays
Measuring the intracellular Ca 2+ level in CHO-K1 cells containing GRP10 was performed using the calcium-sensitive dye Fura-2 according to the manufacturer's protocol (Molecular Devices, Sunnyvale, CA, USA). The day before the experiment, cells were seeded at 40,000 cells/well in 96-well plates in growth media and kept at 37 • C in an incubator with 5% CO 2 overnight. Peptides were tested at concentrations from 10 −12 to 10 −5 M. Fura-2 fluorescent dye was detected using a FlexStation 3 fluorometric plate reader (Molecular Devices), and excitation was measured at 340 nm and 380 nm and emission at 510 nm.
The intracellular Ca 2+ level was measured using the AequoScreen stable CHO-K1 cell line containing NPFF-R2 purchased from Perkin Elmer according to the manufacturer's protocol. Cells at 80-90% confluence cultured in media without selective antibiotics were detached (PBS pH 7.4 + 0.5 mM EDTA) and centrifuged. Cells resuspended in phenol red-free DMEM with 0.1% protease-free BSA and 5 µM coelenterazine h (Thermo Fisher Scientific Inc. Brand) were seeded at 50,000 cells/well in 96-well plates and incubated in the dark at RT with gentle agitation for 4 h. Peptides were tested at concentrations from 10 −12 to 10 −5 M. Luminescent light emission was recorded using a FlexStation 3 plate reader.

Cell Signaling Determined Using Beta-Lactamase Reporter System
Cell lines containing beta-lactamase reporter genes with different receptors, GPR10, Y 5 , GHSR and opioid receptors, were used to study the agonist/antagonist properties of PrRP31 and lipidized PrRP31 analogs. Cells were seeded at 10,000 cells/well in a 384-well plate in assay medium, and the assay was performed according to Thermo Fisher's protocol and according to our previous study [10]. Receptor agonists were tested at concentrations from 10 −12 to 10 −5 M. The concentration of the agonist in antagonist assay mode ranged from 10 −12 to 10 −5 M, and the potential antagonists PrRP31 and palm 11 -PrRP31 were tested at concentrations from 10 −7 or 10 −6 to 10 −5 M. Fluorescence was detected at 409 nm excitation and 460 and 530 nm emissions using the FlexStation 3 fluorometric plate reader.

Statistical Analysis
Data were analyzed by GraphPad Software (San Diego, CA, USA) and are presented as the means ± SEM. The saturation and competitive binding experiments were analyzed according to [46] using the Cheng-Prusoff equation [33]. The competitive binding curves were plotted compared to the best fit for single-binding site models, and half maximal inhibitory concentration (IC 50 ) values were obtained from nonlinear regression analysis. From saturation binding experiments, the dissociation constant (K d ) and number of binding sites/cell (Bmax) were calculated. Inhibition constants (K i ) were calculated from IC 50 values, K d and the concentration of radioligands.
Experiments using immunoblotting were analyzed using one-way ANOVA followed by Dunnett's post hoc test; p < 0.05 was considered statistically significant. Dose-response curves were obtained from nonlinear regression.
The beta-lactamase assay results were analyzed by nonlinear regression as log agonist versus response, and EC 50 values were determined in agonist mode using GraphPad software. Data are representative of at least two experiments, each performed in duplicate.
Ca 2+ release assay data are shown as the percentage of maximal response, and the results were analyzed by nonlinear regression as log agonist versus response using GraphPad software. Data are representative of at least three experiments, each performed in duplicate.

Conclusions
Lipidized PrRP31 analogs have great potential for the treatment of obesity and neurodegenerative diseases. The in vitro properties of the two most potent palmitoylated analogs, palm-PrRP31 and palm 11 -PrRP31, were tested and compared. Palmitoylation of PrRP31 increased not only the activity and binding affinity to GPR10 and NPFF-R2, which are both connected with food intake regulation, but also the binding properties and activity to NPFF-R1. Therefore, NPFF-R1 is a new target of lipidized PrRP31 analogs. Both analogs activated the cellular signaling of the PKB/Akt and MAPK pathways and activated the transcription factors c-Fos, c-Jun and CREB in cells expressing GPR10 and NPFFR-2. Activation of all previously mentioned cellular pathways in cells expressing NPFF-R1 was observed only after incubation with palm-PrRP31. Palm-PrRP31 also showed higher off-target activity on GHSR receptors and Y receptors than palm 11 -PrRP31; therefore, the more selective palm 11 -PrRP31 has a better potential for obesity treatment. Our future studies will focus on further development of palmitoylated PrRP analogs with minimized off-target activity.