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CELLULAR AND MOLECULAR

Pharmacological Characterization of Human and Murine Neuropeptide S Receptor Variants

Dpartments of Pharmacology (R.K.R., Y.-L.X., N.O., J.Z., S.C., Z.W., O.C.) and Developmental and Cell Biology (O.C.), University of California Irvine, Irvine, California; and Department of Gastroenterology, Veterans Affairs Medical Center, Long Beach, California (R.P.)

Received July 28, 2005; accepted September 2, 2005.

	Abstract

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We have recently shown that Neuropeptide S (NPS) can promote arousal and induce anxiolytic-like effects after central administration in rodents. Another study reported a number of natural polymorphisms in the human NPS receptor gene. Some of these polymorphisms were associated with increased risk of asthma and possibly other forms of atopic diseases, but the physiological consequences of the mutations remain unclear. One of the polymorphisms produces an Asn-Ile exchange in the first extracellular loop of the receptor protein, and a C-terminal splice variant of the NPS receptor was found overexpressed in human asthmatic airway tissue. We sought to study the pharmacology of the human receptor variants in comparison with the murine receptor protein. Here, we report that the N107I polymorphism in the human NPS receptor results in a gain-of-function characterized by an increase in agonist potency without changing binding affinity in NPSR Ile¹⁰⁷. In contrast, the C-terminal splice variant of the human NPS receptor shows a pharmacological profile similar to NPSR Asn¹⁰⁷. The mouse NPS receptor, which also carries an Ile residue at position 107, displays an intermediate pharmacological profile. Structure-activity relationship studies show that the amino terminus of NPS is critical for receptor activation. The altered pharmacology of the Ile¹⁰⁷ isoform of the human NPS receptor implies a mechanism of enhanced NPS signaling that might have physiological significance for brain function as well as peripheral tissues that express NPS receptors.

Central administration of NPS promotes behavioral arousal and suppresses all stages of sleep in rodents. Furthermore, NPS was found to produce anxiolytic-like effects in a battery of four different tests that measure behavioral responses of rodents to novelty or stress. NPS was shown to induce transient increases of intracellular Ca²⁺, indicating that it might have excitatory effects at the cellular level (Xu et al., 2004).

Recently, a number of polymorphisms in the human NPS receptor gene were identified and a specific set of these polymorphisms was linked to an increased susceptibility for asthma and potentially other forms of allergy that are characterized by high IgE serum levels in Finnish and Canadian asthma patients (Laitinen et al., 2004). The study described a number of risk haplotypes and originally termed the receptor "GPRA isoform A" (for G protein-coupled receptor associated with asthma; GenBank accession number NP_997055 [GenBank] ; the protein has also been termed GPR 154). This receptor protein is identical to the NPS receptor that we have studied extensively for its pharmacology, distribution, and function in brain (Xu et al., 2004). One of the single-nucleotide polymorphisms changes the primary structure of the NPS receptor protein to code for an Asn-Ile exchange at position 107 of the mature protein (SNP591694 A>T; refSNP ID: rs324981; Fig. 1). The study also found that a C-terminal splice variant of the receptor, originally termed "GPRA isoform B" (GenBank accession number NP_997056 [GenBank] ; Fig. 1), was overexpressed in human asthmatic airway tissue and that the orthologous murine receptor mRNA was up-regulated in a mouse model of chronic airway inflammation. Immunostaining showed that human bronchial smooth muscle cells express the receptor protein, indicating a potential role in bronchial constriction. These data suggest a possible involvement of the C-terminal splice variant of the NPS receptor in the pathophysiology of asthma, but the report did not describe a functional role for the receptor or its isoforms due to the lack of a pharmacologically useable agonist (Laitinen et al., 2004). Because of technical constraints, the study also did not distinguish between the Asn¹⁰⁷ and Ile¹⁰⁷ isoforms.

View larger version (32K): [in this window] [in a new window]   Fig. 1. Schematic diagram ("snake plot") of the human NPS receptor protein showing the presumed location of the N107I polymorphism and the sequence of the alternatively spliced carboxyl-terminal tail in NPSR C-alt. Putative transmembrane domains are boxed and were predicted with TMPred (www.ch.embnet.org/software/TMPRED_form.html; Hofmann and Stoffel, 1993).

	Materials and Methods

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Cloning and Functional Expression of NPSR Isoforms. Human NPSR Asn¹⁰⁷ cDNA was cloned as described previously (Xu et al., 2004). The Ile¹⁰⁷ isoform of NPSR was generated by PCR using synthetic oligonucleotides and the QuikChange site-directed mutagenesis kit from Stratagene (La Jolla, CA). The C-terminal splice variant of NPSR (NPSR C-alt) was generated by recombinant PCR. First, the alternatively spliced exon was cloned by PCR from human genomic DNA using primers NPSRB5 (5'-ATCTCTTTCCCCTGCAGGGTCATCCGTCTCC-3') and NPSRB3-XbaI (5'-TTTCTAGAGAGCTGTCACCTTGGAA-3', XbaI site underlined). Recombinant PCR was carried out with the cloned exon and human NPSR Asn¹⁰⁷ cDNA as templates using primers NPSRA5-XhoI (5'-ATACTCGAGCCATGCCAGCCAACTTCACAGAGGGCA-3', XhoI site underlined) and NPSRB3-XbaI. The products were digested, gel-purified, and cloned into pcDNA3.1-hygromycin (Invitrogen, Carlsbad, CA). Mouse NPSR was cloned by nested PCR from mouse total brain cDNA (BD Biosciences Clontech, Palo Alto, CA). First-round primers were 5'-GCTGCAGGTGCAGAGACAGTGAG-3' and 5'-GAGAGCTGACTAAGTTCAGCC-3'. PCR products were further amplified using primers 5'-TTGGATCCCTGCCTGAGCCATGCCA-3' (BamHI site underlined) and 5'-TTTCTAGATCAGGGTTTAGATGAATTC-3' (XbaI site underlined). The products were digested with the respective restriction enzymes and cloned into pcDNA 3.1-hygromycin. Transfection of CHO and HEK293 cells was carried out using Lipofectamine (Invitrogen) as described previously (Xu et al., 2004). Selection of stable clones was achieved by culturing transfected cells in medium containing 400 mg/l hygromycin (Omega Scientific, Tarzana, CA). For transient expression, transfected cells were cultured for 72 h without antibiotic selection.

Detection of Endogenous NPSR Expression in Cell Lines. Total RNA from a number of human cell lines (HEK293, U373 MG, 1321N, Caco-2, LoVo, DLD-1, Colo205, HCT116, HT-29, SW480, and SW1116) was extracted and converted into single-strand cDNA using reverse transcriptase (New England Biolabs, Beverly, MA) and oligo(dT) primers. Quantitative real-time PCR was carried out as described using primers specific for human NPSR (Xu et al., 2004).

Measurement of Intracellular Ca²⁺ Mobilization. Changes of agonist-induced intracellular Ca²⁺ were measured using the fluorimetric imaging plate reader technology as described previously (Reinscheid et al., 2003; Xu et al., 2004). NPS and truncated NPS peptides were a gift from Phoenix Pharmaceuticals (Belmont, CA). Dose-response curves were calculated using Prism (GraphPad Software Inc., San Diego, CA). Mean EC₅₀ values of populations of stable clones expressing individual NPSR variants were compared using unpaired Student's t test, and p < 0.05 was considered significant.

Measurement of cAMP Accumulation. cAMP was measured as described previously (Reinscheid et al., 1996). In brief, stably transfected cells were seeded into 24-well plates and cultured for 24 h. Culture medium was aspirated and exchanged for 200 µl of Opti-MEM (Invitrogen) containing 100 µM 3-isobutyl-1-methylxanthine and agonists at various concentrations. After incubation for 15 min at 37°C, cells were lysed by adding ethanol to a final concentration of 66%. Aliquots of the lysate were lyophilized, and cAMP content was measured in a radioimmunoassay [either Flashplate; PerkinElmer Life and Analytical Sciences (Boston, MA), or SPA Biotrak; GE Healthcare, Little Chalfont, Buckinghamshire, UK]. Dose response curves were calculated using Prism.

CRE-Luciferase Reporter Gene Assays. HEK293 cells were stably transfected with a reporter gene containing six copies of a cAMP-response element (CRE; sequence CCAAT) in front of a luciferase reporter gene (Promega, Madison, WI) cloned into pcDNA3.1-neomycin (Himmler et al., 1993). One stable clone showing robust induction of reporter gene expression after forskolin challenge was chosen for further experiments. The different NPSR isoform cDNAs (cloned into pcDNA3.1-hygromycin) were transfected into these cells using Lipofectamine, and stable clones were selected. The same cells and procedures were also used for transient transfection. For induction of reporter gene expression, cells were plated in 96-well plates and incubated with agonist for 5 h in serum-free medium followed by aspiration of the medium and cell lysis with 25 mM Tris phosphate, pH 7.8, 2 mM dithiothreitol, 2 mM 1,2-diaminocyclohexane-N,N,N',N'-tetraacetic acid, 10% glycerol, and 1% Triton X-100. After one freeze-thaw cycle, aliquots of supernatant were transferred to white clear-bottom 96-well plates (Greiner Bio-One, Longwood, FL), and luciferase content was quantified by bioluminescence (Luc-Screen; Applied Biosystems, Foster City, CA). Plates were counted in a scintillation counter using bioluminescence settings (MicroBeta; PerkinElmer Wallac, Gaithersburg, MD).

Cell Proliferation Assays. Cells were seeded in 24-well plates and grown overnight to 60 to 70% confluency. After 24 h of serum starvation, 2 µCi of [methyl-³H]thymidine was added together with increasing concentrations of NPS. 500 nM prostaglandin E₂ (PGE₂) served as a positive control. After a 3-h incubation, cells were washed three times with phosphate-buffered saline and then lysed with 0.5 N NaOH. The lysate was neutralized with 0.5 N HCl, and aliquots were counted in a liquid scintillation counter for incorporated radioactivity.

MAPK Phosphorylation. Agonist-induced phosphorylation of p42/p44 mitogen-activated protein kinase (MAPK) was determined as described previously (Saito et al., 2001). In brief, cells were cultured in 24-well plates in serum-free cell culture medium for 24 h. After stimulation for 5 min at 37°C with increasing concentrations of NPS, cells were washed with phosphate-buffered saline and lysed with 10 mM Tris-HCl, pH 7.5, 150 mM NaCl, 5 mM EDTA, 0.1% SDS, 1.5% Nonidet P-40, 0.5% sodium deoxycholate, 1 mM sodium orthovanadate, 1 mM sodium fluoride, 0.5 mM phenylmethylsulfonyl fluoride, 1 µg/ml aprotinin, 0.5 µg/ml leupeptin, 0.7 µg/ml pepstatin, and 100 µg/ml bacitracin. Lysates were centrifuged, and aliquots of supernatant were electrophoresed on 4 to 10% SDS-polyacrylamide gels. Phosphorylated p42/p44 MAPK was assayed by Western blot. After transfer to Hybond C membranes (GE Healthcare), blots were incubated with anti-phospho-p42/p44 MAPK antibody (dilution 1:1500; Cell Signaling Technology Inc., Beverly, MA) in Tris-buffered saline, 1% nonfat dried milk, 0.2% Tween 20 at 4°C overnight. Horseradish peroxidase-conjugated goat anti-rabbit IgG (dilution 1:2000; Jackson ImmunoResearch Laboratories Inc., West Grove, PA) was used as a secondary antibody. Immunoblots were developed using an enhanced chemiluminescence detection kit (GE Healthcare), and films were scanned by densitometry (UN-SCAN-IT; Silk Scientific Inc., Orem, UT) for quantitative analysis.

Radioligand Binding. Saturation binding experiments in intact cells were carried out exactly as described previously (Xu et al., 2004). K_d and B_max values of [¹²⁵I]Tyr¹⁰-NPS binding were calculated using Prism. [¹²⁵I]Tyr¹⁰-NPS was a gift from PerkinElmer Life and Analytical Sciences. Nonspecific binding was determined in the presence of 1 µM NPS. Competition binding was measured in HEK cells stably expressing human or mouse NPSR isoforms, respectively, with 0.3 nM [¹²⁵I]Tyr¹⁰-NPS as tracer.

	Results

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We have previously described that NPS produces an increase in intracellular Ca²⁺ in cells stably expressing NPSR Asn¹⁰⁷ with an EC₅₀ of approximately 5 to 10 nM (Xu et al., 2004). Cells transiently transfected with NPSR Asn¹⁰⁷, NPSR Ile¹⁰⁷, or NPSR C-alt cDNAs did not show agonist-induced changes in Ca²⁺ (data not shown). We also could not detect significant receptor binding in transiently transfected cells with any of the constructs, indicating that the proteins might be either difficult to express or expressed at low levels. Therefore, we established stable cells lines expressing NPSR Asn¹⁰⁷, NPSR Ile¹⁰⁷, or NPSR C-alt in HEK293 cells.

Three individual cells lines expressing similar levels (5 fmol/10⁵ cells) of human NPSR Asn¹⁰⁷, NPSR Ile¹⁰⁷, or NPSR C-alt were chosen for detailed analyses of pharmacological parameters. As shown in Fig. 2A, NPS induced a dose-dependent increase in intracellular free Ca²⁺ in all cell lines. Cells expressing NPSR Asn¹⁰⁷ displayed an EC₅₀ of 12.4 ± 1.24 nM, whereas the clone expressing NPSR Ile¹⁰⁷ responded to agonist stimulation with an EC₅₀ of 1.4 ± 1.17 nM. Cells stably expressing NPSR C-alt displayed an EC₅₀ of 21.7 ± 1.4 nM. As shown in Fig. 2B, NPS also induced cAMP accumulation in HEK cells stably expressing NPSR Asn¹⁰⁷ with an EC₅₀ of 31.9 ± 1.17 nM. In HEK cells expressing NPSR Ile¹⁰⁷, NPS stimulated cAMP formation at 10-fold lower agonist concentrations with an EC₅₀ of 3.45 ± 1.26 nM. As mentioned before, we could not observe second messenger signaling in transiently transfected cells measuring either mobilization of Ca²⁺ or formation of cAMP. However, because of the high rate of signal amplification of the luciferase reporter gene assay, it was possible to establish agonist dose-response curves with this assay in transiently transfected cells. As shown in Fig. 2C, NPSR Asn¹⁰⁷ displayed only a weak induction of luciferase activity with an EC₅₀ of 76.6 ± 21.5 nM. In contrast, NPSR Ile¹⁰⁷ produced a robust increase in reporter gene expression with an EC₅₀ of 17.3 ± 1.31 nM. The NPSR variant containing the alternatively spliced C terminus (NPSR C-alt) displayed an EC₅₀ of 146.5 ± 15.1 nM, similar to the NPSR Asn¹⁰⁷ variant. The maximum of reporter gene expression in HEK cells transiently transfected with NPSR Ile¹⁰⁷ was 2-fold higher than in cells expressing NPSR Asn¹⁰⁷ or NPSR C-alt, suggesting an increase in agonist efficacy. The magnitude of reporter gene induction was 20-fold lower in transiently transfected cells compared with stable clones (see below).

View larger version (13K): [in this window] [in a new window]   Fig. 2. Pharmacological activity of NPS at NPSR Asn107, NPSR Ile107, and NPS C-alt. A, dose-response curves of NPS to elicit mobilization of intracellular Ca2+ in HEK293 cells stably expressing NPSR Asn107, NPSR Ile107, or NPS C-alt. NPS displays greater agonist potency at NPSR Ile107. Incubations were performed in triplicate and repeated three times. Data are shown as means ± S.E.M. B, dose-response curve of NPS to stimulate cAMP formation in two individual HEK293 clones expressing either NPSR Asn107 or NPSR Ile107. NPS displays greater agonist potency at NPSR Ile107. Incubations were performed in triplicate and repeated three times. Data are shown as means ± S.E.M. C, dose-response curves of NPS stimulating CRE-mediated luciferase reporter gene expression. HEK293 cells were transiently transfected with plasmids containing NPSR Asn107, NPSR Ile107, or NPSR C-alt cDNA constructs. Bioluminescence was determined from triplicate incubations repeated at least twice, and data are presented as means ± S.E.M.

To investigate whether this increase in agonist potency at NPSR Ile¹⁰⁷ was a general property of the receptor protein, we compared a large number of stable clones expressing NPSR Asn¹⁰⁷, NPSR Ile¹⁰⁷, or NPSR C-alt by establishing dose-response curves for each construct and calculating mean EC₅₀ values for the populations. As shown in Fig. 3, the mean EC₅₀ for NPS-induced mobilization of intracellular Ca²⁺ at NPSR Asn¹⁰⁷ was found to be 13.02 ± 1.18 nM (n = 16 individual stable clones). In contrast, NPSR Ile¹⁰⁷ displayed a more than 10-fold lower mean EC₅₀ of 1.01 ± 0.13 nM (n = 21). In the clones expressing NPSR C-alt, the peptide showed a mean EC₅₀ of 32.5 ± 1.15 nM (n = 14). These data demonstrate that NPSR Ile¹⁰⁷ is activated by significantly lower concentrations of agonist than NPSR Asn¹⁰⁷ or NPSR C-alt (F_2,51 = 79.65; p < 0.0001).

View larger version (14K): [in this window] [in a new window]   Fig. 3. Scatter-plot of logarithmic EC50 values of individual HEK293 clones expressing human NPSR Asn107 (Asn107), NPSR Ile107 (Ile107), or NPSR C-alt (C-alt) and mouse NPSR (mNPSR). Dose-response curves for each individual clone were established measuring either mobilization of intracellular Ca2+ (left) or induction of luciferase reporter gene transcription (right). Horizontal bars indicate mean EC50 values.

Using the same approach as described before, we compared the EC₅₀ values of a large number of stable clones established in HEK cells that coexpress the CRE-luciferase reporter gene. As shown in Fig. 3, HEK cells stably expressing both NPSR Asn¹⁰⁷ and the reporter gene displayed a mean EC₅₀ of 45.08 ± 1.19 nM for NPS-induced reporter gene expression (n = 7 individual clones). In contrast, cells coexpressing NPSR Ile¹⁰⁷ and the reporter gene showed a mean EC₅₀ of 0.63 ± 0.43 nM for NPS (n = 21). Cells expressing NPSR C-alt displayed a mean EC₅₀ of 28.8 ± 1.42 nM in the reporter gene assay (n = 12). Again, these data indicate that NPSR Ile¹⁰⁷ is activated at significantly lower agonist concentrations than NPSR Asn¹⁰⁷ or the alternatively spliced NPS receptor (F_2,37 = 67.31; p = 0.0001). Taken together, these data indicate that the Ile¹⁰⁷ mutation produces a gain-of-function in the NPS receptor protein, whereas the alternatively spliced C terminus does not appear to affect agonist potency.

Many GPCRs have been shown to affect cell growth that might have significance in malignant or other pathological processes. To investigate whether NPSR can affect cell proliferation at natural levels of receptor expression, we screened a number of cancer cell lines for endogenous NPSR mRNA expression by reverse transcription-PCR. The human colon cancer line Colo205 was found to express NPSR transcripts but did not display agonist-induced second messenger responses (data not shown). However, NPS produced a dose-dependent stimulation of thymidine incorporation in Colo205 cells, indicating that the peptide can stimulate cell proliferation and mitogenic signals in these cells (Fig. 4A). Doses of 0.1 to 10 nM NPS produced maximal thymidine incorporation, exceeding the effect of the well characterized mitogen PGE₂ on these cells. The human colon cancer cell line DLD-1, which does not express NPSR transcripts, served as a negative control and showed no NPS-dependent thymidine incorporation (data not shown). We next examined potential intracellular mediators of the proliferative response elicited by NPS. The p42/p44 MAPK is a well known integrator of mitogenic signals, and many GPCRs have been shown to increase phosphorylation of MAPK upon agonist stimulation. We found that NPS can stimulate MAPK phosphorylation in a dose-dependent manner in HEK cells stably expressing NPSR Asn¹⁰⁷ or NPSR Ile¹⁰⁷ isoforms. As observed before, NPS was more potent to induce MAPK phosphorylation in cells expressing NPSR Ile¹⁰⁷ (EC₅₀ 0.32 ± 0.25 nM) than in cells expressing NPSR Asn¹⁰⁷ (EC₅₀ 1.23 ± 0.38 nM) (Fig. 4B).

View larger version (13K): [in this window] [in a new window]   Fig. 4. NPS effect on cell proliferation and MAPK phosphorylation. A, NPS-induced stimulation of [3H]thymidine incorporation in Colo205 human colon cancer cells. NPS produces a dose-dependent stimulation of cell proliferation. 500 nM PGE2 was used as a positive control. All incubations were performed in triplicates, and experiments were repeated twice. Data are shown as means ± S.E.M. **, p < 0.01 versus buffer control. B, stimulation of MAPK phosphorylation by increasing concentrations of NPS. Values were normalized to levels of phospho-MAPK produced by incubation with 1 µM NPS (= 100%). Phospho-MAPK was quantified by densitometric scanning of Western blots as described under "Materials and Methods." Assays were performed in duplicate and repeated twice. Data are shown as means ± S.E.M.

Two individual clones, expressing either NPSR Asn¹⁰⁷ or NPSR Ile¹⁰⁷ with EC₅₀ values close to the average EC₅₀ in mobilizing intracellular Ca²⁺, were chosen to examine the structure-activity relationships of various NPS fragments. These NPS fragments represent potential processing products that could result from proteolytic cleavage of human NPS 1–20. In addition, we also tested the effect of rat and mouse NPS 1–20 on these cells. As shown in Fig. 5, A and B, and Table 1, most carboxyl-terminally truncated fragments of NPS retained full agonist potency at both NPSR isoforms. Rat and mouse NPS 1–20 seem to be slightly more potent agonists at both NPSR isoforms compared with the human peptide. Interestingly, NPS 1–12 shows greatly decreased agonist activity at NPSR Asn¹⁰⁷ while still behaving as a full but weakly potent agonist at NPSR Ile¹⁰⁷. Further deletion of the two lysine residues at positions 11 and 12 produced NPS 1–10. NPS 1–10 displayed full agonist activity at both NPSR isoforms, albeit with significantly higher potency at NPSR Ile¹⁰⁷. Deletion of the three amino-terminal amino acids (NPS 4–20) completely abolished agonist activity. These data indicate that the N terminus of NPS contains the pharmacophore. The two lysine residues at positions 11 and 12 seem to attenuate activation of the receptor when exposed at the C terminus, because further C-terminal deletion to NPS 1–10 restored agonist activity. Because of the peculiar pharmacology of NPS 1–12, this fragment was also tested for possible antagonist activity but failed to block activation of the two human receptor isoforms by NPS 1–20 (data not shown). Overall, the various NPS fragments displayed a 5 to 10-fold higher potency at NPSR Ile¹⁰⁷ compared with NPSR Asn¹⁰⁷ (Table 1).

View larger version (12K): [in this window] [in a new window]   Fig. 5. Structure-activity relationships of NPS peptides and truncated NPS fragments at human or mouse NPSR variants. Ca2+ mobilization elicited by human (h), mouse (m), or rat (r) NPS and various NPS fragments was determined in individual HEK293 clones expressing NPSR Asn107 (A), NPSR Ile107 (B), or mouse NPSR (C). Dose-response curves were calculated from triplicate incubations, and all assays were repeated at least twice. See Table 1 for comparison of EC50 values and peptide sequences. Data from triplicate experiments are shown as means ± S.E.M.

Some of the previous observations could be explained by an increased affinity or higher receptor expression of NPSR Ile¹⁰⁷ versus NPSR Asn¹⁰⁷. Therefore, we determined receptor binding parameters in a number of stable clones for both variants. Surprisingly, both NPSR Asn¹⁰⁷ and NPSR Ile¹⁰⁷ bind the radioligand with very similar affinities (K_d range of NPSR Asn¹⁰⁷ clones: 0.2–0.45 nM, n = 4; average K_d: 347.1 ± 44 pM; K_d range of NPSR Ile¹⁰⁷ clones: 0.17–0.5 nM, n = 4; average K_d: 402.5 ± 49 pM). However, stable NPSR Ile¹⁰⁷ clones tended to express more functional receptor protein per cell than NPSR Asn¹⁰⁷ clones (average B_max of NPSR Ile¹⁰⁷ clones: 12.5 ± 3.5 fmol/10⁵ cells; average B_max of NPSR Asn¹⁰⁷ clones: 3.9 ± 1.5 fmol/10⁵ cells; n = 4 for each receptor variant). Overall, the levels of NPS receptor expression are low compared with other GPCRs expressed in the same cellular environment. Dose-response relationship experiments for Ca²⁺ mobilization, cAMP formation, and MAPK phosphorylation were performed with individual clones that display very similar receptor levels to correct for possible confounds caused by varying numbers of functional receptors (Figs. 2A, 2B, and 4B). Taken together, our data indicate that the increased potency of NPS at NPSR Ile¹⁰⁷ is not caused by a change in receptor affinity but might reflect an increased intrinsic efficacy of the receptor protein to couple to G proteins and thus to the various second messenger pathways, as described above.

We also investigated the pharmacological profile of the mouse NPS receptor since much in vivo studies on the physiological functions of NPS have been performed in this species (Xu et al., 2004) and increased expression of NPSR has been reported in a murine model of allergic airway inflammation (Laitinen et al., 2004). The mouse NPSR carries an Ile residue at amino acid position 107, and currently available genetic data do not indicate polymorphic variants at this site in the mouse genome. As shown in Fig. 5C and Table 1, HEK cells stably expressing mouse NPSR responded to stimulation with various NPS peptides by increasing intracellular free Ca²⁺. The EC₅₀ values and rank order of potency of the various NPS peptides for mobilizing Ca²⁺ were found to lie in between those obtained with cells expressing human NPSR Asn¹⁰⁷ and NPSR Ile¹⁰⁷, respectively. Mouse and rat NPS appear to be slightly more potent at mouse NPSR. In contrast to the human NPSR isoforms, the rank order of potency for NPS 1–12 and NPS 1–10 was reversed, indicating that the two carboxyl-terminal lysine residues in NPS 1–12 might interact differently with mouse NPSR than with the human receptors. When analyzing a population of individual HEK clones stably expressing mouse NPSR, we obtained a mean EC₅₀ value of 4.58 ± 1.28 nM (n = 15 individual clones; Fig. 3). Statistical analysis indicated that the mean EC₅₀ of mouse NPSR was significantly different from both human NPSR Asn¹⁰⁷ (p < 0.05) and human NPSR Ile¹⁰⁷ (p < 0.05). Radioligand binding experiments showed that mouse NPSR binds NPS with approximately the same affinity compared with the human NPS receptor variants (Fig. 6). Note that the IC₅₀ values for half-maximal displacement of the radioligand in stable HEK cells are 10-fold higher than in CHO cells (IC₅₀ [CHO] = 0.4 nM; IC₅₀ [HEK] = 4 nM) that we reported previously (Xu et al., 2004).

View larger version (16K): [in this window] [in a new window]   Fig. 6. Radioligand binding. Displacement of 0.3 nM [125I]Tyr10-NPS by increasing concentrations of unlabeled human NPS in HEK cells stably expressing human NPSR Asn107, NPSR Ile107, or mouse NPSR. Data from triplicate experiments were normalized to correct for different levels of receptor expression and are shown as means ± S.E.M.

	Discussion

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Our data provide evidence for an increased agonist potency at human NPSR Ile¹⁰⁷. Surprisingly, the N107I point mutation does not affect ligand binding affinity, even though this amino acid is expected to be close to the ligand binding pocket of the receptor protein (Fig. 1). The endogenous agonist NPS displays approximately a 10-fold higher potency at NPSR Ile¹⁰⁷ compared with NPSR Asn¹⁰⁷ in mobilizing intracellular Ca²⁺, stimulating cAMP formation or inducing MAPK phosphorylation. The Ile¹⁰⁷ mutation does not produce increased constitutive activity, as judged from the cAMP accumulation and reporter gene assays. A plausible explanation for our observations could be that the N107I polymorphism is producing a conformational change of the receptor protein that facilitates agonist-induced G protein interaction and thus increases agonist potency in NPSR Ile¹⁰⁷. However, the Ile residue at position 107 is not solely sufficient to convey increased agonist potency because mouse NPSR, which also carries an Ile residue at this position, displays intermediate agonist potencies when compared with human NPSR Asn¹⁰⁷ and NPSR Ile¹⁰⁷, respectively. We also observed a trend to higher levels of receptor protein expression in stable clones expressing human NPSR Ile¹⁰⁷. It is currently not known whether NPSR Ile¹⁰⁷ expression is also facilitated in vivo or whether our observation is due to the overexpression system used in our studies. Together, our data indicate that the N107I polymorphism in human NPSR produces a gain-of-function that could have significant functional consequences in physiological processes that involve NPS signaling.

The C-terminal splice variant of NPSR (NPSR C-alt, GPRA isoform B) was described to be significantly overexpressed in airway smooth muscle cells from asthmatic patients compared with healthy controls when studied by immunohistochemical staining (Laitinen et al., 2004). Our data provide no evidence for an altered second messenger response elicited by NPSR C-alt compared with NPSR Asn¹⁰⁷. Both receptor variants contain an Asn residue at position 107. It remains to be determined whether the alternative C-terminal tail of the receptor protein can affect other signaling pathways or receptor desensitization and thus have functional significance. Interestingly, the alternative exon giving rise to NPSR C-alt in human is absent from the mouse and rat genome.

Our data show that NPSR can couple to intracellular Ca²⁺ as well as cAMP pathways, indicating interaction with both G_q and G_s types of G proteins. The pharmacophore of NPS is contained within the N-terminal part of the peptide, and we describe NPS 1–10 as a minimally active structure. All NPS fragments used in our studies could be produced by proteolytic processing involving trypsin-like cleavage at basic amino acid residues. Some of these fragments retain potent agonist activity. Therefore, it will be important to determine the enzymatic steps involved in the inactivation of this neuropeptide in vivo. Apparently, the NPSR protein cannot be studied easily in transient transfection systems that made the pharmacological analysis of the receptor variants more tedious. One common problem of using stable clones for second messenger assays is caused by the fact that each clone displays an individual pharmacology, and a comparison of too few stable clones can lead to inaccurate assumptions regarding general pharmacological properties. Therefore, we chose to analyze a large population of stable clones and determined mean EC₅₀ values followed by statistical analysis. This procedure allowed us to detect significant differences in agonist-induced second messenger coupling among the NPSR Asn¹⁰⁷, NPSR Ile¹⁰⁷, and NPSR C-alt isoforms, respectively.

At present, the functional involvement of NPSR in airway smooth muscle contraction still remains to be verified. According to the report by Laitinen et al. (2004), the receptor protein seems to be expressed in airway smooth muscle cells that might contribute to bronchial constriction. Our data indicate that activation of NPSR produces an increase in intracellular free Ca²⁺ and that the NPSR Ile¹⁰⁷ variant, if expressed in airway smooth muscle cells, could thus transmit an enhanced contractile response requiring lower agonist concentrations. Because increased bronchial constriction is one of the physiological hallmarks of asthma (Bousquet et al., 2000), the gain-of-function mutation in NPSR Ile¹⁰⁷ could therefore be associated with this phenotype. Also, our studies provide evidence for a proliferative effect of NPS using a cellular model of endogenous NPSR expression and demonstrating enhanced phosphorylation of MAPK. Tissue remodeling in asthmatic airways involves proliferation of smooth muscle cells and thickening of basal membranes (Bousquet et al., 2000). It remains to be determined whether these pathological changes are influenced by NPS receptors endogenously expressed in airway smooth muscle cells. Therefore, further investigations into the functional role of NPSR in airway tissue will be necessary to determine whether the gain-of-function polymorphism in NPSR Ile¹⁰⁷ that we describe in this paper might be involved in any of the pathological events underlying asthma. Ultimately, the development of NPS antagonists will be a critical step to fully understand the physiological functions of NPS signaling.

Naturally occurring polymorphisms that affect receptor function have been identified in numerous GPCRs. Not surprisingly, most of these mutations lead to inactive receptor proteins. The few examples of gain-of-function mutations can be divided into two classes based on their pharmacological phenotype. One group of mutations produces constitutively active receptors that promote second messenger signaling in the absence of endogenous agonist. This type of activating mutations has been found in the glycoprotein-hormone receptor subfamily (luteinizing hormone and thyroid-stimulating hormone receptor), parathyroid and parathyroid-related peptide receptor, and rhodopsin (Spiegel and Weinstein, 2004). The other type of activating polymorphisms increases ligand affinity or agonist efficacy in a way similar to the NPSR Ile¹⁰⁷ variant. Such mutations have been found in the Ca²⁺-sensing receptor and result in hypocalcemia and hypercalciuria (Pollak et al., 1994).

Genetic variations in several GPCRs have also been associated with asthma susceptibility or effectiveness of asthma pharmacotherapy. A coding polymorphism in the cysteinyl-leukotriene receptor type 2 was found to reduce the affinity of the receptor to one of its major endogenous ligands, leukotriene D₄. Because leukotriene D₄ is an important mediator of inflammatory responses in asthma, this polymorphism in cysteinyl-leukotriene receptor type 2 provides an asthma-protective effect (Pillai et al., 2004). Similarly, particular haplotypes in the promotor region of the prostanoid DP receptor (PTGDR) were found underrepresented in asthmatic patients. These polymorphisms lead to a reduced transcription of PTGDR mRNA and thus lower levels of receptor protein. Prostaglandin D2 (the endogenous ligand of PTGDR) is an important mediator of asthma, and PTGDR was found to be required for the development of airway sensitization in a mouse model of asthma. This might explain why reduced levels of PTGDR expression lead to an overall asthma-protective effect (Oguma et al., 2004). Coding polymorphisms in the ₂-adrenoreceptor that influence receptor down-regulation in response to adrenergic agonists were found to be associated with the therapeutic benefit of ₂-agonists to treat symptoms of asthma (Israel et al., 2004). Although ₂-adrenoreceptors are not causally involved in the pathophysiology of asthma, they are important therapeutic targets for acute and intermittent treatment of asthma symptoms. These examples illustrate the important contribution of specific GPCR genotypes for asthma susceptibility or therapy.

The observation of enhanced NPS-induced second messenger responses at NPSR Ile¹⁰⁷ could also have important consequences for brain function, because the predominant sites of NPSR expression are found in the central nervous system (Xu et al., 2004). It might be possible that the Ile¹⁰⁷ isoform of NPSR is associated with changes in behavior or neuronal processing. In summary, we provide evidence that a naturally occurring polymorphism in the NPS receptor is producing a gain-of-function, resulting in enhanced second messenger signaling. Future research will have to demonstrate whether this polymorphism has functional significance in asthma or other physiological processes involving NPS signaling.

	Acknowledgements

 
We thank Phoenix Pharmaceuticals and PerkinElmer Life and Analytical Sciences for generous supply of research materials.

	Footnotes

 
This study was supported in part by grants from National Institute of Mental Health (to R.K.R. and O.C.), National Alliance for Research on Schizophrenia and Depression (to R.K.R.), and the Stanley Medical Research Institute (to O.C.).

doi:10.1124/jpet.105.093427.

ABBREVIATIONS: NPS, neuropeptide S; NPSR, neuropeptide S receptor; GPCR, G protein-coupled receptor; GPRA, G protein-coupled receptor associated with asthma; SNP, single-nucleotide polymorphism; PCR, polymerase chain reaction; CHO, Chinese hamster ovary; MAPK, mitogen-activated protein kinase; HEK, human embryonic kidney; CRE, cAMP-response element; PGE₂, prostaglandin E₂; PTGDR, prostanoid DP receptor.

Address correspondence to: Dr. Rainer K. Reinscheid, Department of Pharmacology, University of California at Irvine, 360 Med Surge II, Irvine, CA 92697-4625. E-mail: rreinsch{at}uci.edu

	References

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