A New Peptidic Ligand and Its Receptor Regulating Adrenal Function in Rats* □ S

We searched for peptidic ligands for orphan G pro-tein-coupled receptors utilizing a human genome data base and identified a new gene encoding a preproprotein that could generate a peptide. This peptide con-sisted of 43 amino acid residues starting from N-terminal pyroglutamic acid and ending at C-terminal arginine-phenylalanine-amide. We therefore named it QRFP after pyroglutamylated arginine-phenylalanine-amide peptide. We subsequently searched for its receptor and found that Chinese hamster ovary cells expressing an orphan G protein-coupled receptor, AQ27, specifically responded to QRFP. We analyzed tissue dis-tributions of QRFP and its receptor mRNAs in rats utilizing quantitative reverse transcription-polymerase chain reaction and in situ hybridization. QRFP mRNA was highly expressed in the hypothalamus, whereas its receptor mRNA was highly expressed in the adrenal gland. The intravenous administration of QRFP caused the release of aldosterone, suggesting that QRFP and its receptor have a regulatory function in the rat adrenal gland.

We searched for peptidic ligands for orphan G protein-coupled receptors utilizing a human genome data base and identified a new gene encoding a preproprotein that could generate a peptide. This peptide consisted of 43 amino acid residues starting from Nterminal pyroglutamic acid and ending at C-terminal arginine-phenylalanine-amide. We therefore named it QRFP after pyroglutamylated arginine-phenylalanineamide peptide. We subsequently searched for its receptor and found that Chinese hamster ovary cells expressing an orphan G protein-coupled receptor, AQ27, specifically responded to QRFP. We analyzed tissue distributions of QRFP and its receptor mRNAs in rats utilizing quantitative reverse transcription-polymerase chain reaction and in situ hybridization. QRFP mRNA was highly expressed in the hypothalamus, whereas its receptor mRNA was highly expressed in the adrenal gland. The intravenous administration of QRFP caused the release of aldosterone, suggesting that QRFP and its receptor have a regulatory function in the rat adrenal gland.
In the last decade, advances in cDNA and genomic DNA sequencing have revealed the existence of hundreds of G protein-coupled receptor (GPCR) 1 genes in the human genome. Those for which the ligands have not yet been identified are referred to as orphan GPCRs. GPCRs play pivotal roles in cell-to-cell communication and in the regulation of cell functions. For this reason, the identification of endogenous ligands for orphan GPCRs will open the door for clarifying new regulatory mechanisms of the human body. Furthermore, as GPCRs are considered to be some of the most important drug target molecules, the identification of ligands of orphan GPCRs will provide opportunities for developing novel drugs (1). Orphan GPCR research is therefore important from the aspects of both basic and applied science. We have previously established a widely applicable method to identify ligands for orphan GPCRs through monitoring specific signal transductions in cells expressing orphan GPCRs (2,3). Through this method we succeeded in identifying various orphan GPCR ligands including peptides with arginine-phenylalanine-amide (RFamide) structure at their C termini (2,4).
The first report on a peptide with RFamide was the isolation of FMRFamide from bivalve mollusks. Since then, a number of bioactive peptides with the same structure have been found throughout the animal kingdom and are called RFamide peptides (5). It has been found that more than 20 RFamide peptide genes encode over 50 distinctive peptides in the nematode Caenorhabditis elegans (6). In mammals, four RFamide peptide genes have so far been identified, that is neuropeptide FF (7,8), prolactin-releasing peptide (2), RFamide-related peptide (RFRP) (4), and metastin (9). All of their receptors have been identified through orphan GPCR research. Based on the identification of prolactin-releasing peptide and RFRP, we have proposed that a variety of RFamide peptides exist and have physiological functions even in mammals and that RFamide peptides are evolutionally related to members of the neuropeptide Y family (8). Another group has recently reported the identification of a new RFamide peptide and its receptor (10). However, their precise molecular and functional characterizations remain to be elucidated. In our present research, we have independently found the same new RFamide peptide gene utilizing a human genome data base and subsequently identified its receptor among orphan GPCRs by applying our previously established method. Here we report the molecular characterization of this newly identified RFamide peptide and its receptor including their binding properties and precise tissue distribution determined through in situ hybridization. In addition, we demonstrate that they function to regulate hormone secretion from the adrenal gland in rats.

EXPERIMENTAL PROCEDURES
Cloning of QRFP cDNA-To isolate a human QRFP cDNA by reverse transcription (RT)-PCR, we designed primers (5Ј-ATGGTAAGGCCT-TACCCCCTGATCTAC-3Ј and 5Ј-CAAATCCTTCCAAGGCGTCCTG-GCCCT-3Ј) based on the Celera Discovery Systems and Celera Genomics-associated data base. PCR was performed in a reaction mixture (25 l in total) containing a 0.2 M concentration of the primers, a template cDNA synthesized from human brain poly(A) ϩ RNA (Clontech) using a Marathon cDNA amplification kit (Clontech), 0.1 mM dNTPs, 1.25 units of KlenTaq DNA polymerase (Clontech), and 2.5 l of a buffer provided by the manufacturer. This was conducted at 94°C for 2 min followed by 40 cycles at 98°C for 10 s, at 63°C for 20 s, and at 72°C for 60 s. We obtained a PCR product of about 300 bp containing a full coding region and determined its nucleotide sequence with an ABI Prism 377 DNA * 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  1 The abbreviations used are: GPCR, G protein-coupled receptor; QRFP, pyroglutamylated arginine-phenylalanine-amide peptide; RFamide, arginine-phenylalanine-amide; RFRP, RFamide-related peptide; RT, reverse transcription; CHO, Chinese hamster ovary; FLIPR, fluorometric imaging plate reader; HEK, human embryonic kidney; MALDI-TOF, matrix-assisted-laser desorption/ionization time-of-flight; ACTH, adrenocorticotropic hormone; TAMRA, 5-carboxytetramethylrhodamine; FAM, 5-carboxyfluorescein. sequencer using a dideoxyterminator cycle sequence kit (PE Biosystems, Foster City, CA). Various primers were synthesized on the basis of the human cDNA sequence thus obtained, and with these primers, a rat QRFP cDNA fragment was isolated by RT-PCR from rat brain poly(A) ϩ RNA. We subsequently synthesized primers based on this cDNA sequence, and then isolated rat QRFP cDNA with a full coding region by 5Ј-and 3Ј-rapid amplification of cDNA ends using a Marathon cDNA amplification kit (Clontech). In a manner similar to obtaining the rat cDNA fragment, we isolated mouse and bovine QRFP cDNA from poly(A) ϩ RNA fractions prepared from mouse brain and bovine hypothalamus, respectively.
Cloning of AQ27 cDNAs-We designed primers (5Ј-TGTCAGCATG-CAGGCGCTTAACATTACCCCGGAGCAG-3Ј and 5Ј-GACTAGTTTA-ATGCCCACTGTCTAAAGGAGAATTCTC-3Ј) to isolate a human AQ27 cDNA by RT-PCR. The PCR was performed in a reaction mixture (25 l in total) containing a 0.2 M concentration of the primers, a template cDNA synthesized from a human fetal brain cDNA (Clontech), 0.2 mM dNTPs, 1.25 units of Advantage 2 polymerase mixture (Clontech), and 2.5 l of a buffer provided by the manufacturer. The mixture was heated at 95°C for 1 min followed by 5 cycles at 95°C for 30 s and at 72°C for 4 min, 5 cycles at 95°C for 30 s and at 70°C for 4 min, 30 cycles of at 95°C for 30 s, at 68°C for 30 s, and at 66°C for 4 min, and finally an extension reaction at 68°C for 3 min. We obtained a product with about 1600 bp that contained a full coding region and determined its nucleotide sequence. Based on the human cDNA sequence thus obtained, we synthesized various primers and isolated rat and mouse AQ27 cDNA with full coding regions by PCR from rat and mouse brain cDNA, respectively.
Preparation of Chinese Hamster Ovary (CHO) Cells Expressing QRFP cDNA-The entire coding region of the human QRFP cDNA was cloned into the downstream region of an SR ␣ promoter in the expression vector pAKKO-111H (11). The resultant expression vector plasmid was transfected into dhfr Ϫ CHO cells, and then dhfr ϩ CHO cells were selected as described previously (11).
Reporter Gene Assays-An expression vector with the human AQ27 cDNA (pAKKO-hAQ27) was constructed by inserting the AQ27 coding region into pAKKO-111H (11). HEK293 cells were used as host cells. In transient expression assays, the AQ27 expression vector and a reporter gene (i.e. a fusion gene of cAMP-responsive element and luciferase) were co-transfected into the host cells with LipofectAMINE 2000 (Invitrogen). After culture overnight, the transfected cells were incubated with test samples for 4 h. After incubation, luciferase activity was measured with a PicaGene LT 2.0 kit (Toyo Ink). Agonistic activities of the samples were detected as the increase of luciferase activity in the presence of forskolin (2 M).
Structual Analysis of QRFP-Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry was performed using a Voyager-DE PRO (PE Applied Biosystems). Samples were mixed with a matrix of ␣-cyano-4-hydroxycinnamic acid on a MALDI target. External calibration was performed with ACTH (7-38) and insulin. Data were obtained in the linear and delayed extraction mode with positive polarity. According to the manufacturer, under these conditions mass accuracy at the 0.05% level can be achieved.
cAMP Assays-We transfected expression vectors with various GPCR cDNAs in pAKKO-111H into dhfr Ϫ CHO cells and established CHO cells expressing each GPCR. Synthetic QRFPs were prepared as described previously (2). The suppression of forskolin-induced cAMP production in CHO cells was determined as described previously (12).
Ca 2ϩ Mobilization Assays-Ca 2ϩ mobilization assays using a stable CHO cell line expressing AQ27 (CHO-hAQ27) cells and mock CHO cells were conducted as described elsewhere (13). Changes in intracellular Ca 2ϩ concentrations induced by QRFPs were measured with a fluorometric imaging plate reader (FLIPR; Amersham Biosciences).
Receptor Binding Assays-Receptor binding assays were conducted principally as described previously (8). In brief, a tyrosine residue of synthetic human QRFP (i.e. ϽEDEGSEATGFLPAAGEKTSGPLGN-LAEELNGYSRKKGGFSFRFamide, where ϽE indicates pyroglutamic acid) was radioiodinated with Na 125 I (IMS-30, Amersham Biosciences) by using lactoperoxidase. This labeling did not affect its cAMP production-inhibitory activity on CHO cells expressing AQ27 (data not shown). The membrane fractions of CHO-hAQ27 cells were mixed with [ 125 I-Tyr 32 ]QRFP and incubated at room temperature for 90 min. To determine the amount of nonspecific binding, 1 M unlabeled QRFP was added to the mixture. After filtration, the radioactivity of [ 125 I-Tyr 32 ]QRFP bound to the membrane fractions was determined.
Quantification of Rat QRFP and AQ27 mRNAs by RT-PCR-Poly(A) ϩ RNA fractions were prepared from rat tissues, and their expression levels of QRFP and AQ27 mRNAs were quantitatively ana-

FIG. 1. Amino acid sequences of human, bovine, rat, and mouse QRFP
preproproteins. The open arrowhead shows the predicted cleavage site of the N-terminal secretory signal peptide sequence. The motifs that could generate the C-terminal RFamide structure are underlined. The closed arrowhead indicates the N terminus of the fully active QRFP. Residues that are identical in at least two of the species are boxed. Amino acid numbers are shown on the right. Nucleotide and amino acid sequences for human, bovine, rat, and mouse cDNAs will appear in the DDBJ/EMBL/GenBank TM data bases with the accession numbers AB109625, AB109626, AB109627, and AB109628, respectively. lyzed by means of RT-PCR using an ABI Prism 7700 sequence detector as described elsewhere (14). The following primer sets and probes were used: 5Ј-AGCACACTGGCTTCCGTCTAG-3Ј, 5Ј-CGCTGGCCTTCTCT-GAGTCA-3Ј, and 5Ј(FAM)-AGGCAGGACAGTGGCAGTGAAGCC-(TA-MRA)3Ј for rat QRFP mRNA and 5Ј-CGGAAGCCTGGGAATTCTG-3Ј, 5Ј-ATGTGTCTCCTTTGGTTTCTTCCA-3Ј, and 5Ј(FAM)-AGCAAAGT-TATCTCGACCACAGCGTCCA-(TAMRA)3Ј for rat AQ27 mRNA. PCR was performed at 50°C for 10 min for the reaction of uracil-N-glycosylase to prevent the amplification of carried over PCR products, at 95°C for 2 min for the activation of AmplyTaq Gold DNA polymerase, and for 40 cycles at 95°C for 15 s and at 57°C for 80 s for the amplification.
In Situ Hybridization-Under pentobarbital anesthesia, male Wistar rats (8 -9 weeks old) were perfused with 4% paraformaldehyde via the left cardiac ventricle. Frozen sections prepared from the brains and adrenal glands were hybridized with digoxigenin-labeled antisense RNA probes synthesized from full-length rat QRFP and AQ27 template cDNAs according to the floating method described previously (15). Visualization of QRFP and AQ27 mRNAs was conducted with alkaline phosphatase-conjugated anti-digoxigenin antibody using 4-nitroblue tetrazolium chloride and 5-bromo-4-chloro-3-indolyl phosphate.
Assays for Plasma Hormone Concentrations-Male Wistar rats (8 -9 weeks old) were provided with food and water ad libitum and kept under controlled lighting (lights on 8:00 to 20:00) and temperature (25°C) until used for the experiments. A cannula was inserted into the right jugular vein of each rat under sodium pentobarbital anesthesia (50 mg/kg). The cannula-implanted rats were housed in individual cages where they were kept for 3 days before the experiments. The day after the implantation, 10 l of phosphate-buffered saline either with or without the peptide was injected into the right jugular vein via the inserted cannula. Before the intravenous injection of the peptide, 400 l of blood was withdrawn from each rat through the cannula, and 0.01 M EDTA with aprotinin (300 kallikrein-inactivating units/ml) was immediately added. This was performed between 13:00 and 17:00. Plasma aldosterone concentrations were determined by radioimmunoassay. To analyze other adrenal gland hormones (i.e. corticosterone and testosterone), other sets of rats were treated in the same manner, and their plasma concentrations were measured as described previously (8).

Identification of a Novel RFamide Peptide Gene-We
searched for unknown members of the RFamide peptide family in the human genome data base using queries to detect repetitive patterns generating RFamide peptide (i.e. RFGR or RFGK where RF is followed by G as an amide donor and by R or K as a proteolytic cleavage site) and a secretory signal peptide sequence upstream of the patterns as reported previously (8). Signal sequences were predicted with the SignalP Version 2.0 software program (16). By this search, we found a human genomic sequence that possibly encoded an RFamide peptide (i.e. QRFP). On the basis of the sequences detected, we isolated human, bovine, rat, and mouse cDNAs with full coding regions by RT-PCR. The isolated cDNAs encoded preproproteins with amino acid lengths of 124 -136 (Fig. 1). In each preproprotein sequence, 17 or 18 amino acid residues at the N-terminal were thought to comprise the secretory signal peptide. Two RFGR motifs were found in the human preproprotein. The motif at the C-terminal side was conserved among the different species, but that at the N-terminal was not. Based on these sequence analyses, we predicted that an RFamide peptide (QRFP) would be produced from the C-terminal motif in the human preproprotein. In addition, we noticed that the C-terminal amino acid sequence of the predicted RFamide peptide (i.e. GGFSFRFamide) quite resembled that of Met-enkephalinamide (i.e. YGGFMRFamide) (Fig. 2). However, QRFP did not show apparent homology to other known mammalian RFamide peptides except for its RFamide structure.
Identification of a Receptor for QRFP-As all of the RFamide peptides previously found in mammals act as ligands for certain GPCRs, we presumed that this newly identified RFamide  peptide would also function as a ligand for a GPCR. We have previously searched for ligands of various orphan GPCRs by exposing synthetic peptides to CHO cells expressing orphan GPCRs (2,3). We found that CHO cells expressing the orphan GPCR, AQ27, weakly but specifically responded to Met-enkephalinamide peptide (data not shown). AQ27 is a novel GPCR that we isolated from human brain poly(A) ϩ RNA based on public genome information (GenBank TM accession number AQ270411), and has been found to be identical to GPR103 as recently reported (17). We isolated its rat and mouse counterparts from their respective brain poly(A) ϩ RNA by RT-PCR. The amino acid sequences deduced from human, rat, and mouse cDNA are aligned in Supplemental Fig. 1. These had amino acid lengths of 431 or 433 and showed 84 -96% amino acid identity to each other. Among the GPCRs for which ligands are known, AQ27 showed homology with OT7T022 (the receptor for RFRP) and HLWAR77 (the receptor for neuropeptide FF), that is 30 and 32% amino acid identity, respectively, as determined by the Gapped Blast program. As a result of phylogenic analysis, GPR103 has been predicted to be an RFamide peptide receptor (18). We therefore inferred that QRFP might act as a ligand for AQ27. To examine this, we synthesized a short peptide with an amino acid length of 7 (GGFSFRFamide). We derived this from the C-terminal RFGR motif of the human QRFP preproprotein because, in RFamide peptides, C-terminal portions are essential sites to bind receptors, and even short C-terminal peptides frequently retain receptor binding activity (19). We subjected this short peptide to an assay with HEK293 cells transiently expressing AQ27 and a reporter gene (cAMP-responsive element-luciferase). This assay was modified from the method of Chen et al. (20,21). They demonstrated that the activation of GPCRs coupled with G s and/or G q causes an increase in the transcription of the cAMPresponsive element promoter. We modified their method for detecting G q signals so that, by our technique, G q signals were detected as the increase of luciferase activity in the presence of forskolin. As AQ27 coupled to G q , we detected the activation of AQ27 treated with the peptide as the increase of luciferase activity (data not shown). However, the agonistic activity of this peptide appeared to be very weak with its effective dose ranging from 10 Ϫ5 to 10 Ϫ6 M (data not shown).
Determination of a Fully Active Form of QRFP-As the agonistic activity of the 7-amino acid-long synthetic peptide was considerably weak, we thought that a longer form of the peptide would show full activity. To ascertain this, we expressed the human QRFP cDNA in CHO cells and examined whether more effective peptidic ligands were secreted in the culture supernatant. As we could detect specific and strong stimulatory activity on HEK293 cells expressing AQ27 in the culture supernatant, we decided to purify the ligands for AQ27 from the culture supernatant. To do so, we used affinity column chromatography using a monoclonal antibody (1F3) for RFRP-1 because 1F3 could cross-react with the short form of QRFP (GGFSFRFamide). The culture supernatant (2.4 L) was boiled and centrifuged, and the resulting supernatant was applied to the affinity chromatography. Elution was conducted with 0.2 M glycine-HCl (pH 2.2) containing 0.5 M NaCl. The eluate was subsequently fractionated through a Vydac C18 218TP5415 column with a 20 -35% linear gradient of CH 3 CN, and each fraction was examined in assays with HEK293 cells transiently expressing human AQ27 and a reporter gene. Finally active fractions in the assays were applied to RPC C2/C18 SC2.1/10 column chromatography. AQ27-stimulatory activities were detected in eluted fractions, which corresponded to at least two peaks (i.e. peaks 1 and 2 in Fig. 3A). Although peaks 1 and 2 matched absorbance peaks at 215 nm, the main absorbance peak at 280 nm matched only with peak 1. In the C-terminal region of QRFP there is a Tyr residue that should give absorbance at 280 nm. Since the absorbance of peak 2 was faint at 280 nm, we believe that it contained impurities. In the reporter gene assay, both peaks appeared to reach plateau levels of luciferase activity. Considering these results, we concluded that peak 1 was a major product derived from the QRFP cDNA-transfected CHO cells and that peak 2 was not a major peak, although it showed activity.
We determined the structure of the purified peptide in peak 1 as follows. Since the quantity of the peptide in peak 1 was calculated to be less than 1 pmol from its absorbance peak height, we analyzed it by MALDI-TOF mass spectrometry (Fig.  3B) and found that it produced a protonated molecular ion (m/z 4505.7). Considering this together with other evidence, we concluded the structure of the peptide to be ϽEDEGSEATGFL-PAAGEKTSGPLGNLAEELNGYSRKKGGFSFRFamide (ϽE indicates pyroglutamic acid). Although we could not determine from the mass spectrometric data whether or not the Cterminal residue was amidated, we determined the C-terminal RFamide structure of the peptide by the following reasoning. (i) The cDNA sequence of QRFP had motifs to generate RFamide peptides, and we knew that the synthetic 7-amino acid-long peptide with C-terminal RFamide showed weak but significant agonistic activity in the reporter assay (data not shown). (ii) It has been well established that RFamide structure is essential for RFamide peptides to interact with their receptors (23). (iii) We confirmed with synthetic peptides that the nonamidated form of this peptide showed drastically reduced agonistic activity (Table I). (iv) The M ϩ H ϩ calculated average masses of the amidated and nonamidated form were 4504.9 and 4505.9, respectively. Although the M ϩ H ϩ observed average mass (m/z 4505.7) seemed to be closer to the M ϩ H ϩ calculated average mass of the nonamidated peptide than that of the amidated form, both M ϩ H ϩ calculated average masses were within the allowable error limit (0.05%) of delayed extraction MALDI-TOF mass spectrometry in linear mode using external calibration. Therefore, the mass spectrometric data did not contradict the predicted RFamide structure. We determined the Nterminal structure as follows. (i) It is well known that an N-terminal glutamine or glutamic acid residue is easily circularized and converted to pyroglutamic acid (24). (ii) The calcu- lated molecular weight of the nonpyroglutamylated form was 4521.9, which differed from the mass spectrometric data (i.e. 4505.7) beyond the error limited. We therefore concluded that the purified peptide of peak 1 possessed the predicted structure. Our results indicated that this peptide was generated from the preproprotein encoded by the cDNA through processing. We therefore named it QRFP for pyroglutamylated RFamide peptide. In the reporter gene assays, the purified peptide was estimated to show agonistic activity even at 10 Ϫ10 -10 Ϫ9 M. Since this peptide apparently showed higher activity than the synthetic short peptide, we believe the purified QRFP with the 43-amino acid length to be a fully active form of QRFP.
Interaction between QRFP and AQ27-We next determined intracellular Ca 2ϩ and cAMP changes in CHO-hAQ27 after stimulation with QRFP. As shown in Fig. 4A, we detected a rapid rise of intracellular Ca 2ϩ concentrations in CHO-hAQ27 after the stimulation. We also detected the suppression of cAMP production in these cells (Fig. 4B and Table I). These results suggest that AQ27 couples to G proteins, that is G i/o and G q , in CHO cells. Because the purified QRFP had a long Nterminal region, we chemically synthesized various forms of QRFP and determined their cAMP production-inhibitory activities. QRFP inhibited the cAMP production of CHO-hAQ27 in a dose-dependent manner. The 43-amino acid-long QRFP, that is QRFP(43), was the most potent with a 50% inhibitory concentration (EC 50 ) of 2.7 nM (Table I). Serial deletion of the Nterminal region gradually attenuated the inhibitory activity of QRFP. However, the efficacy of the inhibitory activity of the peptides did not changed (Fig. 4B). A nonamidated form of the peptide, that is QRFP(26OH), and an N-terminal portion of QRFP, that is QRFP(28n), did not show the evident inhibitory activities, indicating that the C-terminal structure of QRFP is crucial in its interaction with the receptor.
As the human QRFP preproprotein had another N-terminal motif that might produce an RFamide peptide, we synthesized two different lengths of peptides, that is EHAGCRFamide and ASQPQALLVIARGLQTSGREHAGCRFamide, and then examined their stimulatory activities on CHO-hAQ27. However, their activities were considerably lower than QRFP(43) in Ca 2ϩ and cAMP assays (data not shown), indicating that they do not act as primary AQ27 ligands. Prolactin-releasing peptide and RFRPs also failed to inhibit cAMP production in CHO-hAQ27 (data not shown). These results indicate that QRFP functions specifically as a ligand for AQ27.
To examine the binding of QRFPs with AQ27, we labeled human QRFP(43) with 125 I. As shown in Fig. 4C, Scatchard plot analysis indicated that the membrane fractions of CHO-hAQ27 had a single class of high affinity binding sites for [ 125 I-Tyr 32 ]QRFP at the dissociation constant (K d ) of 5.3 ϫ 10 Ϫ11 M and maximal binding sites (B max ) of 0.51 pmol mg Ϫ1 of protein, indicating that QRFP binds with high affinity to AQ27 as a specific ligand. In competitive binding experiments, various lengths of QRFP inhibited the binding of [ 125 I-Tyr 32 ]QRFP with AQ27 (Table I). QRFP(43) proved the most potent in the competition with an IC 50 of 0.52 nM. The deletion of the Nterminal sequence in QRFP gradually diminished its binding affinity to AQ27 and was almost parallel to the decrease of cAMP production-inhibitory activities.
Tissue Distribution of AQ27 and QRFP mRNAs-We analyzed the tissue distribution of AQ27 and QRFP mRNAs in rats by quantitative RT-PCR. The highest levels of QRFP mRNA were detected in the hypothalamus and optic nerve, while moderate levels were found in the trachea and mammary gland (Fig. 5, upper panel). On the other hand, the highest level of AQ27 mRNA was detected in the adrenal gland, a high level was detected in the hypothalamus, and moderate levels were detected in the thalamus, midbrain, medulla oblongata, testis, and eye (Fig. 5, lower panel).
We subsequently performed in situ hybridization to determine the detailed localization of QRFP and AQ27 mRNAs in the brain and adrenal gland in rats. QRFP mRNA was detected in the retrochiasmatic area and posterior regions of the arcuate nucleus in the hypothalamus by antisense probe (Table II and Supplemental Fig. 2). The sense probe for QRFP mRNA did not give any significant signals. On the other hand, AQ27 mRNA was detected in various areas in the brain. Especially strong signals were detected in neurons within the piriform cortex, cortex-amygdala transition zone, ventral pallidum, lateral preoptic area, ventromedial hypothalamic nucleus, zona incerta, posterior hypothalamic area, marginal zone median geniculate, dorsal raphe nucleus, nucleus of the brachium inferior collicu-lus, intergeniculate leaf, locus caeruleus, and central gray ␣/␤ part. (Table II and Supplemental Fig. 3). The sense probe for AQ27 mRNA did not give any evident signals. In the adrenal gland, AQ27 mRNA was detected in the cortex. The cortex consists of three parts, that is the zona glomerulosa, zona fasciculate, and zona reticularis. A high level of AQ27 mRNA expression was detected in the zona glomerulosa with moderate levels found in the zona fasciculata and zona reticularis (Fig. 6).
Promotion of Aldosterone Secretion in Rats by QRFP-The zona glomerulosa in the adrenal gland is known to secrete mineral corticoids including aldosterone. We examined the effects of QRFP on aldosterone secretion by intravenous administration into rats. As shown in Fig. 7, human QRFP(43) at doses of 40 -400 nmol/kg of weight increased plasma aldosterone levels 5-15 min after administration. Five minutes after administration, plasma aldosterone concentrations in the treated rats reached about 5 times those in the untreated rats and then gradually declined reaching the basal level at 60 min. Although we examined other hormones (i.e. testosterone and corticosterone) secreted from the adrenal gland, we could not detect significant changes in their plasma levels (data not shown). Considered together with the expression of AQ27 mRNA in the adrenal gland, these results indicate that QRFP acts directly on the zona glomerulosa to induce aldosterone secretion in rats.

DISCUSSION
In this study, we have demonstrated that the novel RFamide peptide QRFP and its receptor exist and function in mammals. Although Jiang Z. et al. (10) have very recently reported the identification of the same peptide and its receptor independently of our research, they did not determine the form of this peptide possessing a full agonistic activity. We have shown here that a 43-amino acid-long sequence was necessary to exhibit full activity in QRFP on the basis of experiments ex-

FIG. 7. Effects of QRFP on plasma aldosterone levels in rats.
After the intravenous administration (indicated by the arrowhead) of human QRFP at 400 nmol/kg (E), 40 nmol/kg (OE), and 4 nmol/kg (छ) and phosphate-buffered saline (f) alone into rats, plasma samples were collected from the rats (n ϭ 3-5) at the indicated intervals. Each aldosterone concentration value represents the mean Ϯ S.E. Differences in aldosterone concentrations among the groups were analyzed by one-tailed Williams' test. *, p Յ 0.025 versus control.
pressing the QRFP gene in CHO cells and analyzing the interaction of the receptor with various lengths of synthetic peptides. Furthermore we unequivocally demonstrated that QRFP specifically bound with high affinity to the receptor, AQ27, on the basis of Scatchard plot analysis. The affinity of QRFP to AQ27 (i.e. K d of 5.3 ϫ 10 Ϫ11 M) was comparable in level to those of other known RFamide peptides to their receptors (2,8,9,19). Multiple binding sites have been reported in the case of recombinant GPCRs, and these are affected by guanine nucleotides (25). However, under our experimental conditions, we did not detect any low affinity binding site in the absence of additional guanine nucleotide.
Another RFamide motif was found in the N-terminal portion of the human preproprotein, that is 87 RFGR 90 . However, synthetic RFamide peptides deduced from this motif did not show high agonistic activity on HEK293 cells expressing human AQ27 (data not shown). In addition, alignment of the human, bovine, rat, and mouse preproprotein sequences revealed that this motif was not conserved in the other species (Fig. 1). Furthermore, in our experiments expressing the human QRFP cDNA in CHO cells, we could not detect agonistic peptides generated from this motif in the culture supernatant. In view of these results, we concluded that only one RFamide peptide (i.e. QRFP) would be produced from the C-terminal portion of the human preproprotein. QRFP is the fifth member of the RFamide peptide family to be found in mammals. Although we examined whether the fully active form of QRFP could crossreact with the receptors for the other RFamide peptides, such cross-reaction was not detected. However, when we tested the short 7-amino acid peptide, it showed very weak cross-reactions to OT7T022 (data not shown), suggesting that QRFP is closely related to RFRPs and neuropeptide FF not only in structure but also in functions. We concluded that the long N-terminal portion of QRFP is important in establishing its specificity and affinity for AQ27.
In our analyses for the tissue distribution of QRFP and AQ27 mRNAs in rats, we found that QRFP mRNA was highly expressed in the hypothalamus, optic nerve, trachea, and mammary gland. On the other hand, high levels of AQ27 mRNA were detected in the central nervous system, adrenal gland, and testis. These results suggest that QRFP and its receptor possess multiple functions in various tissues. The very high expression of AQ27 mRNA in the adrenal gland, especially in the zona glomerulosa of the cortex, suggested that QRFP affected the function of the adrenal glands in rats. Although the major hormones secreted from the adrenal cortex are corticosterone, testosterone, and aldosterone, the zona glomerulosa is the main site producing aldosterone, which controls electrolyte metabolism. We have shown here that the intravenous injection of QRFP resulted in the increase of plasma aldosterone in rats. Our results suggest that QRFP and its receptor play a regulatory role in aldosterone secretion from the adrenal cortex in rats. The release of aldosterone is mainly controlled by angiotensin II (26). Although the stimulatory activity of QRFP appeared to be weaker than that of angiotensin II (i.e. the efficacy of QRFP was one-tenth less than that of angiotensin II), QRFP might have regulatory functions similar to angiotensin II in aldosterone secretion in rats. As we noted, QRFP has a structure quite similar to Met-enkephalinamide. Enkephalins reportedly have regulatory functions in the release of catecholamine from the medulla of the adrenal gland. In addition, neuropeptide FF exists in the adrenal gland and inhibits aldosterone release (27). Considering these results, RFamide peptides might play roles in the regulation of adrenal functions. However, in comparison with rats, AQ27 mRNA expression levels in the adrenal gland are not so high in humans and mice (10). Therefore, we cannot rule out the possibility that in each species QRFP and its receptor show different expression and functions in respect to the adrenal gland.
A high level of AQ27 mRNA expression was detected in the hypothalamus in rats. It is noteworthy that high levels of AQ27 mRNA have been detected in the hypothalamus even in humans and mice (10,16). It is known that peptides found abundantly in the hypothalamus frequently influence the feeding behavior of animals. Since QRFP mRNA was detected in the retrochiasmatic nucleus and arcuate nucleus, we tested the effects of the intracerebroventricular injection of QRFP on food intake in rats. We could not, however, detect any evident effects of QRFP under our experimental conditions (data not shown). Using in situ hybridization, AQ27 mRNA was detected in the dorsal raphe nucleus, locus caeruleus, laterodorsal tegmental nucleus, and ventrolateral preoptic nucleus, which are known as regions related to sleep modulation (28), suggesting that QRFP has a regulating activity on sleep.
Based on sequence homology, AQ27 was found to correspond to NPR-1 in the nematode. Disruption of the NPR-1 gene reportedly affects the social behavior of the nematode (29). As AQ27 mRNA was detected widely in the brain including the cortex zones in rats (Table II), the suppression of AQ27 expression might influence behavior even in mammals. Future studies will be necessary to clarify the physiological significance of QRFP and its receptor.