Receptor for the pain modulatory neuropeptides FF and AF is an orphan G protein-coupled receptor.

Opiate tolerance and dependence are major clinical and social problems. The anti-opiate neuropeptides FF and AF (NPFF and NPAF) have been implicated in pain modulation as well as in opioid tolerance and may play a critical role in this process, although their mechanism of action has remained unknown. Here we describe a cDNA encoding a novel neuropeptide Y-like human orphan G protein-coupled receptor (GPCR), referred to as HLWAR77 for which NPAF and NPFF have high affinity. Cells transiently or stably expressing HLWAR77 bind and respond in a concentration-dependent manner to NPAF and NPFF and are also weakly activated by FMRF-amide (Phe-Met-Arg-Phe-amide) and a variety of related peptides. The high affinity and potency of human NPFF and human NPAF for HLWAR77 strongly suggest that these are the cognate ligands for this receptor. Expression of HLWAR77 was demonstrated in brain regions associated with opiate activity, consistent with the pain-modulating activity of these peptides, whereas the expression in adipose tissue suggests other physiological and pathophysiological activities for FMRF-amide neuropeptides. The discovery that the anti-opiate neuropeptides are the endogenous ligands for HLWAR77 will aid in defining the physiological role(s) of these ligands and facilitate the identification of receptor agonists and antagonists.

Opiate tolerance and dependence are major clinical and social problems. The anti-opiate neuropeptides FF and AF (NPFF and NPAF) have been implicated in pain modulation as well as in opioid tolerance and may play a critical role in this process, although their mechanism of action has remained unknown. Here we describe a cDNA encoding a novel neuropeptide Y-like human orphan G protein-coupled receptor (GPCR), referred to as HLWAR77 for which NPAF and NPFF have high affinity. Cells transiently or stably expressing HLWAR77 bind and respond in a concentration-dependent manner to NPAF and NPFF and are also weakly activated by FMRF-amide (Phe-Met-Arg-Phe-amide) and a variety of related peptides. The high affinity and potency of human NPFF and human NPAF for HLWAR77 strongly suggest that these are the cognate ligands for this receptor. Expression of HLWAR77 was demonstrated in brain regions associated with opiate activity, consistent with the pain-modulating activity of these peptides, whereas the expression in adipose tissue suggests other physiological and pathophysiological activities for FMRF-amide neuropeptides. The discovery that the anti-opiate neuropeptides are the endogenous ligands for HL-WAR77 will aid in defining the physiological role(s) of these ligands and facilitate the identification of receptor agonists and antagonists.
Two highly studied mammalian neuropeptides, NPAF 1 (A-18-F-amide) and NPFF (F-8-F-amide), also known as mor-phine-modulating peptides, are members of a large family of neuropeptides related to the molluscan cardioexcitatory neuropeptide (FMRF-amide, Phe-Met-Arg-Phe-amide) (1). These mammalian neuropeptides have been implicated in pain modulation, and they also possess potent pro-and anti-opioid activity, although the effects observed are dependent on the route of administration. NPFF administered intracerebroventricularly demonstrated anti-opiate activity in rodents (2,3). Pretreatment with IgG from NPFF antiserum restored the analgesic effects of morphine in morphine-tolerant rats (4) and anti-NPFF-IgG-administered intracerebroventricularly potentiated opiate function (5). NPFF administered into the spinal cord, intrathecally, produced a long lasting, dose-dependent antinociception (6) and potentiated morphines antinociceptive effects (7). Additionally, NPFF and NPAF are present in high concentrations in dorsal spinal cord, periaqueductal gray, medulla pons, and the hypothalamus (8), and concentrations are elevated in the cerebrospinal fluid of morphine-dependent rats relative to naive rats (9). The mammalian peptides have also been implicated in cardiovascular regulation (10) and neuroendocrine function (11). Although the biologic activities of NPFF and NPAF and related FMRF-amides are well documented, their mechanism of action is largely unknown. Whereas an FMRF-amide peptide-gated, amiloride-sensitive sodium channel has been identified in snail (12) other studies indicate that the neuropeptides bind and signal through a specific GPCR, distinct from the known opiate receptors (13,14).
As part of an ongoing program to couple novel orphan GPCRs with their cognate ligands, we isolated a cDNA from a brain library that encoded a GPCR, referred to as HLWAR77, phylogenetically most similar to the orexin-A receptor, a recently identified neuropeptide receptor (15). In the present report we describe the identification, cloning, expression, and localization of HLWAR77. With expression of this cDNA in human embryonic kidney (HEK) 293 cells, we show with binding and functional assays that huNPFF and huNPAF are the cognate ligands for this receptor. The assays described will permit the rapid identification of selective agonists and antagonists of this NPFF/NPAF receptor that will be useful to elucidate the physiological role(s) of these ligands.

EXPERIMENTAL PROCEDURES
Receptor Cloning-Expressed sequences tag analysis (16) of cDNA clones derived from a human placenta cDNA library (oligo(dT)-primed and constructed in the ZAPII vector (Stratagene)) identified a 900-base * The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank TM /EBI Data Bank with accession number(s) AF257210.
§ To whom correspondence and requests for reprints should be addressed: Dept. of Molecular Biology, SmithKline Beecham Pharmaceuticals, 709 Swedeland Rd., King of Prussia, PA 19406-0939. Tel.: 610-270-7602; Fax: 610-270-5114; E-mail: bob_ames-1@sbphrd.com. 1 The abbreviations used are: NPAF, neuropeptide AF (A-18-F-amide); GPCR, G protein-coupled receptor; PCR, polymerase chain reaction; NPFF, neuropeptide FF (F-8-F-amide); hu, human; HEK, human embryonic kidney; GRK2, G protein-coupled receptor kinase 2; GFP, green fluorescent protein; FLIPR, fluorescence imaging plate reader; CRE, cAMP response element; MRE, multiple response element; PLC, phospholipase C; FMRF-amide, Phe-Met-Arg-Phe-amide. pair clone demonstrating low homology to the neuropeptide Y-like receptors. This cDNA clone encoded an incomplete GPCR. Nested primers (outside 5Ј-AAGACTGAAGCTGCGACAGATATTC-3Ј and inside 5Ј-CCTGGAACCAATCCACTGATCTTGC-3Ј) were designed at the 5Ј end of the gene and used to extend the 5Ј sequence using Marathon human brain, placenta, and fetal brain cDNA templates (CLONTECH). Primers were synthesized corresponding to the amino and the carboxyl termini of the predicted open reading frame and were used to obtain the full-length cDNA clone from human brain cDNA by PCR. The fulllength cloning process was confirmed twice using the human brain and fetal brain cDNA as templates. The cDNAs were completely sequenced on both strands using an ABI sequencer. The HLWAR77 cDNA was subcloned into the mammalian expression vector pCDN, and transfections were performed as described (17).
Ca 2ϩ Mobilization-Ca 2ϩ mobilization was performed as described previously (18) using the microtiter plate-based Ca 2ϩ mobilization FLIPR assay to identify ligands activating HEK 293 cells transiently or stably expressing HLWAR77 and G proteins.
Transient Transfection with GRK2 and ␤-Arrestin 2-GFP-HEK 293 cells stably expressing HLWAR77 receptor were grown to 40 -80% confluency in T80 flasks and transiently transfected with 1.5 g each of GRK2 and ␤-arrestin 2-GFP, using LipofectAMINE Plus as described by the manufacturers. Cells were seeded into chambered covered glass slides 24 h after transfection (Life Technologies, Inc., 1.8-cm 2 well), at a density of 100 -200,000 cells/well and incubated overnight at 37°C with 95:5% air/CO 2 before being analyzed by confocal microscopy.
Confocal Microscopy-For assay, growth medium was removed from covered glass-based chamber slides and replaced with 200 l of assay buffer (Hanks' balanced salt solution with 10 mM HEPES, 200 M CaCl 2 , 0.1% bovine serum albumin (pH 7.4)) at 37°C. Ligand (200 l of 1 M) was added to the cells. The response observed using a Bio-Rad MRC600 laser scanning confocal microscope interfaced to a Zeiss Axiovert inverted light microscope with a 63ϫ/1.4NA oil immersion objective. GFP was excited using the 488 nm line of a krypton/argon laser and the emitted fluorescence detected with a 515-540 nm band pass filter. Slides on the microscope were maintained at 37°C by use of a heated jacket.
FIG. 1. Nucleotide and the deduced peptide sequence of the HLWAR77 receptor. The seven hydrophobic domains are indicated in bold; two cysteine residues available for disulfide bridge formation are marked with asterisks, and four potential glycosylation sites are indicated in bold italics and underlined. The polyadenylation site (ATAAA) is underlined. This sequence has been submitted to GenBank TM , and the accession number is AF257210.

RESULTS
As part of an ongoing effort to identify ligands for novel orphan GPCRs, we isolated an 1888-base pair cDNA from a human brain cDNA library that encoded a 420-amino acid protein that structurally resembled members of the GPCR superfamily. As shown in Fig. 1, this orphan receptor, HL-WAR77, encodes the typical GPCR signature motif of 7 distinct hydrophobic domains of 16 -28 amino acids each linked by hydrophilic amino acid stretches of varying length. Two cysteines highly conserved in the GPCR family are present in the putative first and second extracellular loops. Among the GPCRs, HLWAR77 is most similar to the orexin-A receptor (15) (37% amino acid identity). Recently, an apparent splice variant of this cDNA (NPGPR), with an additional 102 residues at the amino terminus, was described (20). Repeated attempts were made to clone the NPGPR cDNA from different human cDNAs using primers designed at the 5Ј-and 3Ј-untranslated regions of the gene. The result was a gene product missing most of the 1st transmembrane domain; therefore, we concluded that this gene represents an aberrant variant.
Northern hybridization analysis of RNA isolated from various human tissues suggested that the HLWAR77 receptor is expressed at barely detectable levels, with a predominant 1.8kilobase pair mRNA transcript present in placenta (data not shown). TaqMan quantitative reverse transcriptase-polymerase chain reaction (PCR) analysis confirmed the Northern blot data demonstrating that HLWAR77 was present in placenta at relatively low levels of expression ( Fig. 2A). The sensitive Taq-Man methodology showed that low levels of receptor RNA expression were detectable in adipose tissue and many brain regions, most notably cingulate gyrus (Fig. 2, A and B). Taq-Man was also used to test for HLWAR77 expression in 40 cell lines. Significant expression was observed in only 8 cell lines with the highest expression obtained in neuroblastoma and astrocytoma cell lines.
By using the "reverse pharmacological approach" (21), HEK 293 cells transiently co-transfected with HLWAR77 and a "promiscuous" G protein ␣ subunit, G␣ 16 , which facilitates GPCR signaling through phospholipase C (22), were used in a microtiter plate-based (FLIPR) calcium mobilization assay to search for the ligand(s) activating this receptor (17). HLWAR77/G␣ 16 cells were challenged, as described previously, with a collection of Ͼ1,000 different ligands (17). Unexpectedly, and in contrast to our experience with other orphan GPCRs, numerous peptide ligands functioned as selective agonists, albeit with very different potencies, generating transient calcium mobilization responses in HLWAR77/G␣ 16 -expressing HEK 293 cells. The common feature of the diverse peptide ligands that functioned as HLWAR77/G␣ 16 agonists was the occurrence at the carboxyl terminus of the dipeptide, Arg-Phe-amide (Table I). The response to the RF-amide peptides of HEK 293 cells expressing HLWAR77 was dependent on the co-transfection of both HLWAR77 and G␣ 16   HLWAR77, or G␣ 16 alone, did not respond to the RF-amide peptides, nor did cells transfected with vector alone or vector encoding other orphan GPCRs (Fig. 3A). The 17 peptides identified in the initial screen that showed reproducible concentration-dependent calcium responses are presented in Table I. The most potent peptides were bovine NPFF (bNPFF) and bNPAF, exhibiting EC 50 values of 13 Ϯ 5 and 22 Ϯ 6 nM, respectively. Our initial activities were dependent on HL-WAR77 co-expression with G␣ 16 . Subsequently, we co-expressed HLWAR77 with a variety of other reported promiscuous G proteins and the "chimeric" G protein ␣ subunits (22)(23)(24), composed of ␣ q in which the five carboxyl-terminal residues are replaced with the carboxyl-terminal residues derived from ␣ i2 or ␣ o , referred to as G qi5 or G qo5 , respectively. Co-transfection of HLWAR77 with these chimeras resulted in a 3-4-fold enhancement of the calcium mobilization signal compared with the amplitude of the response obtained when the receptor was co-expressed with G␣ 16 (Fig. 3B). However, the potency of the response was not altered significantly, since all responding cells exhibited EC 50 values of 10 -30 nM (Fig. 3B).
In light of the responses obtained in HEK 293 cells transiently transfected with HLWAR77 and G qi5 , HEK 293 cell lines stably expressing both HLWAR77 and G qi5 were generated. Several clones were identified in the FLIPR calcium assay where huNPFF induced concentration-dependent calcium re-sponse with varying potencies and magnitude of responses (Fig. 3C). The most responsive clonal cell line, AA18, responded to huNPFF with an EC 50 of 0.39 Ϯ 0.08 nM, and the least potent cell line, AB5, produced a maximal response 25% that of AA18, and the EC 50 was 10-fold weaker at 5.2 nM (Fig. 3C). These data may reflect the critical importance of the ratio of receptor to G protein to obtain efficient coupling of the PLC pathway (25). Clone AA18 was used to assess the relative potency of RF-amide like peptides. As shown in Fig. 3D, bNPFF, huNPFF, bNPAF, and huNPAF were the most potent with EC 50 values of 2.4, 0.4, 1.56, and 1.26 nM, respectively. The next most potent agonists identified were synthetic peptides, Leu-enkephalin-RF-amide and Met-enkephalin-RF-amide, which exhibited EC 50 values of 125 and 165 nM, respectively. The other FMRFamide-like peptides in Table I had EC 50 values of 0.5-60 M.
The calcium response induced by huNPFF or huNPAF is rapid and sustained (Fig. 4A). Preliminary studies characterizing the calcium response in the AA18 cell line indicated that the large sustained calcium response was completely inhibited by removing extracellular calcium, resulting in a rapid transient response that returns to base line in less than 2 min (Fig. 4A). The huNPFF-induced calcium response was blocked by the PLC inhibitor, U 73122, with an IC 50 of 400 nM, and was not significantly affected by the inactive analog U 73343, at concentrations as high as 10 M. Additionally, nifedipine, the calcium channel blocker, did not significantly inhibit the response at concentrations up to 100 M (data not shown).
Since the calcium response induced by huNPAF and huN-PFF in HLWAR77 expressing cells was dependent on co-transfection with promiscuous G␣ protein subunits, we performed experiments to elucidate a natural second messenger pathway of HLWAR77 in HEK 293 cells. We hypothesized that HL-WAR77 would normally activate G i/o subunits of G proteins because G qi5 or G qo5 was able to elicit potent robust calcium mobilization responses from HLWAR77 in response to huN-PAF and huNPFF peptides. Activation of the G i/o pathway results in inhibition of forskolin-stimulated cAMP accumulation and subsequent decreases in transcription from promoters containing the cAMP response element (CRE). Therefore, a CRE/MRE-directed luciferase reporter assay (19) was used to assess the activation of this second messenger pathway. In cells co-transfected with HLWAR77 and reporter vector, bNPFF, bNPAF huNPFF, and huNPAF inhibited forskolin-stimulated luciferase activity in a concentration-dependent manner, exhibiting IC 50 values of 1.3 Ϯ 0.4, 0.41 Ϯ 0.10, 0.25 Ϯ 0.08, and 0.18 Ϯ 0.04 nM, respectively (Fig. 4B). Cells co-transfected with reporter vector alone showed no response to these ligands.
The amino-terminal residue of NPFF was replaced with tyrosine (Y-8-F-amide) to facilitate the radioiodination of the peptide for use in binding studies (14). A high affinity saturable binding site was obtained with membranes from the cells (AA18) stably expressing HLWAR77, with a K d ϭ 0.5 Ϯ 0.2 nM and a B max of 34 Ϯ 6 fmol/mg protein (Fig. 4C). In competition binding studies bNPFF, bNPAF, huNPFF, and huNPAF were evaluated for their ability to displace 125 I-Y-8-F-amide peptide from these membranes. The peptides competed with IC 50 values of 1.6 Ϯ 0.2, 2.5 Ϯ 0.8, 0.15 Ϯ 0.02, and 0.90 Ϯ 0.23 nM, respectively (Fig. 4D). There was no specific binding observed for HEK 293 cells stably expressing the pCDN vector or to the parental HEK 293 cells.
An alternate functional assay for GPCRs is to study receptormediated translocation of ␤-arrestin-GFP from the cytoplasm to the plasma membrane following receptor activation (26). Following transient expression of both GRK2 and ␤-arrestin 2-GFP in cells stably expressing HLWAR77, the cells were visualized by confocal microscopy. In the absence of receptor activation, fluorescence corresponding to ␤-arrestin 2-GFP was evenly distributed throughout the cytoplasm of the cells (Fig.  5A). Upon activation with 0.5 M NPFF, cytosolic ␤-arrestin 2-GFP translocated to the cell periphery within 4 -5 min, visualized as punctate foci at the plasma membrane (Fig. 5B), suggesting that it was interacting with the agonist-occupied receptor. Subsequent images showed that distribution of ␤-arrestin 2-GFP was not further altered in the presence of ligand for up to 60 min (Fig. 5C), and there was no evidence for internalization of ␤-arrestin 2-GFP from the plasma membrane over this time period. DISCUSSION NPFF and NPAF were originally isolated from bovine brain (27), and recently cDNAs encoding both human and bovine peptides were reported (28,29). Both peptides are encoded by the same cDNA in man and bovine and are flanked by consensus sequences for peptide processing (27)(28)(29). Human and bovine NPAF are both 18 amino acid peptides that differ in sequence by only 2 amino acid residues (Table I). However, the sequence of NPFF predicted from the conserved processing site in human cDNA (huNPFF predicted sequence, SQAFLFQPQRF-NH 2 ) is 3 residues longer than the sequence of bNPFF isolated from bovine brain or found in human serum (FLFQPQRF-NH 2 ) (29,30). There are conflicting reports on the relative potency of the short and long forms of huNPFF. It was originally reported that the extended, 11-residue human NPFF was inactive in rodent models in which the shorter, 8-residue NPFF showed activity. In addition it was suggested that the longer peptide required further processing to produce mature, active huNPFF (29). More recently it was shown that the long form was enzymatically converted to the short form in mouse brain and that both forms were equally active (30). This latter observation is consistent with the data obtained with HLWAR77 in the present study where both forms of huNPFF are active and show similar potency at human HLWAR77.
Agonist interaction with GPCRs reversibly activate heterotrimeric G proteins by catalyzing conversion of GDP to GTP bound to the ␣ subunit; this results in the ␣-GTP dissociating from the ␤␥ subunit dimer. Different receptors specifically activate G q , G i , or G s , whose ␣ subunits activate PLC, inhibit adenylate cyclase, or activate adenylate cyclase. Studies have shown that the carboxyl terminus of the G protein ␣ subunit is a critical determinant of receptor activation. Chimeric G proteins have been prepared by replacing the 5 terminal amino acids of G q ␣ with the corresponding amino acids of ␣ i2 , ␣ o , and ␣ s residues that have been shown to alter the signaling phenotype of receptors. We utilize the chimerics and the promiscuous G proteins, G␣ 15 and G␣ 16 , to induce the coupling of receptors to PLC that do not normally couple to PLC and calcium mobilization. Although there was no coupling of HLWAR77 to PLC in the absence of G protein, the co-transfection of HLWAR77 and G␣ 16 resulted in calcium responses for NPFF and NPAF.
The efficiency of the coupling appeared to improve when the G qi5 and HLWAR77 were stably co-transfected into HEK 293 cells. The data obtained for the stable clones are consistent with the importance of the ratio of GPCR to G protein for efficiency of coupling to the functional response. Similarly, other reports have demonstrated that some GPCRs, for example selected opioid receptors, couple more efficiently to chimeric G proteins than to G␣ 16 (24). The CRE reporter gene assay data presented confirm the high potency of NPAF and NPFF peptides for HLWAR77 and demonstrate receptor coupling to native G i/o ␣-G protein subunits.
In summary, we have identified a novel orphan GPCR that is activated by a number of naturally occurring and synthetic FMRF-amide-like ligands. The human neuropeptides huNPFF and huNPAF activated the receptor at concentrations consistent with that of a neuropeptide at its cognate receptor, suggesting that huNPAF and huNPFF are endogenous ligands for this receptor. The presence of HLWAR77 mRNA expression in brain is consistent with its potential role in the opiate modulatory activity induced by NPFF and NPAF. The current findings may lead to exciting new opportunities, including novel approaches for drug discovery in pain treatment. The assay systems described will be valuable in the identification of small molecule NPFF/NPAF agonists and antagonists which in turn will be useful tools to define the physiological role(s) of HL-WAR77 and NPFF/NPAF. In addition, since the receptor is expressed in other tissues, e.g. adipose, further investigation is required to determine other potential pathophysiological roles associated with this receptor.