A New Peptidic Ligand and Its Receptor Regulating Adrenal Function in Rats
2003; Elsevier BV; Volume: 278; Issue: 47 Linguagem: Inglês
10.1074/jbc.m305270200
ISSN1083-351X
AutoresShoji Fukusumi, Hiromi Yoshida, Ryo Fujii, Minoru Maruyama, Hidetoshi Komatsu, Yugo Habata, Yasushi Shintani, Shuji Hinuma, Masahiko Fujino,
Tópico(s)Regulation of Appetite and Obesity
ResumoWe 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 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 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. 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 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 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) 1The abbreviations used are: GPCRG protein-coupled receptorQRFPpyroglutamylated arginine-phenylalanine-amide peptideRF-amidearginine-phenylalanine-amideRFRPRFamide-related peptideRTreverse transcriptionCHOChinese hamster ovaryFLIPRfluorometric imaging plate readerHEKhuman embryonic kidneyMALDI-TOFmatrix-assisted-laser desorption/ionization time-of-flightACTHadrenocorticotropic hormoneTAMRA5-carboxytetramethylrhodamineFAM5-carboxyfluorescein. 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 (1Hinuma S. Onda H. Fujino M. J. Mol. Med. 1999; 77: 495-504Crossref PubMed Scopus (54) Google Scholar). 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 (2Hinuma S. Habata Y. Fujii R. Kawamata Y. Hosoya M. Fukusumi S. Kitada C. Masuo Y. Asano T. Matsumoto H. Sekiguchi M. Kurokawa T. Nishimura O. Onda H. Fujino M. Nature. 1998; 393: 272-276Crossref PubMed Scopus (541) Google Scholar, 3Tatemoto K. Hosoya M. Habata Y. Fujii R. Kakegawa T. Zou M.X. Kawamata Y. Fukusumi S. Hinuma S. Kitada C. Kurokawa T. Onda H. Fujino M. Biochem. Biophys. Res. Commun. 1998; 251: 471-476Crossref PubMed Scopus (1363) Google Scholar). Through this method we succeeded in identifying various orphan GPCR ligands including peptides with arginine-phenylalanine-amide (RFamide) structure at their C termini (2Hinuma S. Habata Y. Fujii R. Kawamata Y. Hosoya M. Fukusumi S. Kitada C. Masuo Y. Asano T. Matsumoto H. Sekiguchi M. Kurokawa T. Nishimura O. Onda H. Fujino M. Nature. 1998; 393: 272-276Crossref PubMed Scopus (541) Google Scholar, 4Hinuma S. Shintani Y. Fukusumi S. Iijima N. Matsumoto Y. Hosoya M. Fujii R. Watanabe T. Kikuchi K. Terao Y. Yano T. Yamamoto T. Kawamata Y. Habata Y. Asada M. Kitada C. Kurokawa T. Onda H. Nishimura O. Tanaka M. Ibata Y. Fujino M. Nat. Cell Biol. 2000; 10: 703-708Crossref Scopus (508) Google Scholar). G protein-coupled receptor pyroglutamylated arginine-phenylalanine-amide peptide arginine-phenylalanine-amide RFamide-related peptide reverse transcription Chinese hamster ovary fluorometric imaging plate reader human embryonic kidney matrix-assisted-laser desorption/ionization time-of-flight adrenocorticotropic hormone 5-carboxytetramethylrhodamine 5-carboxyfluorescein. 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 (5Price D.A. Greenberg M.J. Science. 1977; 197: 670-671Crossref PubMed Scopus (920) Google Scholar). It has been found that more than 20 RFamide peptide genes encode over 50 distinctive peptides in the nematode Caenorhabditis elegans (6Li C. Kim K. Nelson L.S. Brain Res. 1999; 848: 26-34Crossref PubMed Scopus (181) Google Scholar). In mammals, four RFamide peptide genes have so far been identified, that is neuropeptide FF (7Perry S.J. Yi-Kung H.E. Cronk D. Bagust J. Sharma R. Walker R.J. Wilson S. Burke J.F. FEBS Lett. 1997; 409: 420-430Crossref Scopus (197) Google Scholar, 8Elshourbagy N.A. Ames R.S. Fitzgerald L.R. Foley J.J. Chambers J.K. Szekeres P.G. Evans N.A. Schmidt D.B. Buckley P.T. Dytko G.M. Murdock P.R. Milligan G. Groarke D.A. Tan K.B. Shabon U. Nuthulaganti P. Wang D.Y. Wilson S. Bergsma D.J. Sarau H.M. J. Biol. Chem. 2000; 275: 25965-25971Abstract Full Text Full Text PDF PubMed Scopus (237) Google Scholar), prolactin-releasing peptide (2Hinuma S. Habata Y. Fujii R. Kawamata Y. Hosoya M. Fukusumi S. Kitada C. Masuo Y. Asano T. Matsumoto H. Sekiguchi M. Kurokawa T. Nishimura O. Onda H. Fujino M. Nature. 1998; 393: 272-276Crossref PubMed Scopus (541) Google Scholar), RFamide-related peptide (RFRP) (4Hinuma S. Shintani Y. Fukusumi S. Iijima N. Matsumoto Y. Hosoya M. Fujii R. Watanabe T. Kikuchi K. Terao Y. Yano T. Yamamoto T. Kawamata Y. Habata Y. Asada M. Kitada C. Kurokawa T. Onda H. Nishimura O. Tanaka M. Ibata Y. Fujino M. Nat. Cell Biol. 2000; 10: 703-708Crossref Scopus (508) Google Scholar), and metastin (9Ohtaki T. Shintani Y. Honda S. Matsumoto H. Hori A. Kanehashi K. Terao Y. Kumano S. Takatsu Y. Masuda Y. Ishibashi Y. Watanabe T. Asada M. Yamada T. Suenaga M. Kitada C. Usuki S. Kurokawa T. Onda H. Nishimura O. Fujino M. Nature. 2001; 411: 613-617Crossref PubMed Scopus (1195) Google Scholar). 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 (8Elshourbagy N.A. Ames R.S. Fitzgerald L.R. Foley J.J. Chambers J.K. Szekeres P.G. Evans N.A. Schmidt D.B. Buckley P.T. Dytko G.M. Murdock P.R. Milligan G. Groarke D.A. Tan K.B. Shabon U. Nuthulaganti P. Wang D.Y. Wilson S. Bergsma D.J. Sarau H.M. J. Biol. Chem. 2000; 275: 25965-25971Abstract Full Text Full Text PDF PubMed Scopus (237) Google Scholar). Another group has recently reported the identification of a new RFamide peptide and its receptor (10Jiang Y. Luo L. Gustafson E.L. Yadav D. Laverty M. Murgolo N. Vassileva G. Zeng M. Laz T.M. Behan J. Qiu P. Wang L. Wang S. Bayne M. Greene J. Monsma Jr., F. Zhang F.L. J. Biol. Chem. 2003; 278: 27652-27657Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar). 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. Cloning of QRFP cDNA—To isolate a human QRFP cDNA by reverse transcription (RT)-PCR, we designed primers (5′-ATGGTAAGGCCTTACCCCCTGATCTAC-3′ and 5′-CAAATCCTTCCAAGGCGTCCTGGCCCT-3′) based on the Celera Discovery Systems and Celera Genomics-associated data base. PCR was performed in a reaction mixture (25 μl in total) containing a 0.2 μm concentration of the primers, a template cDNA synthesized from human brain poly(A)+ RNA (Clontech) using a Marathon cDNA amplification kit (Clontech), 0.1 mm dNTPs, 1.25 units of KlenTaq DNA polymerase (Clontech), and 2.5 μl 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 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)+ RNA. We subsequently synthesized primers based on this cDNA sequence, and then isolated rat QRFP cDNA with a full coding region by 5′- and 3′-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)+ RNA fractions prepared from mouse brain and bovine hypothalamus, respectively. Cloning of AQ27 cDNAs—We designed primers (5′-TGTCAGCATGCAGGCGCTTAACATTACCCCGGAGCAG-3′ and 5′-GACTAGTTTAATGCCCACTGTCTAAAGGAGAATTCTC-3′) to isolate a human AQ27 cDNA by RT-PCR. The PCR was performed in a reaction mixture (25 μl in total) containing a 0.2 μm 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 μl 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 α promoter in the expression vector pAKKO-111H (11Hinuma S. Hosoya M. Ogi K. Tanaka H. Nagai Y. Onda H. Biochim. Biophys. Acta. 1994; 1219: 251-259Crossref PubMed Scopus (36) Google Scholar). The resultant expression vector plasmid was transfected into dhfr– CHO cells, and then dhfr+ CHO cells were selected as described previously (11Hinuma S. Hosoya M. Ogi K. Tanaka H. Nagai Y. Onda H. Biochim. Biophys. Acta. 1994; 1219: 251-259Crossref PubMed Scopus (36) Google Scholar). Reporter Gene Assays—An expression vector with the human AQ27 cDNA (pAKKO-hAQ27) was constructed by inserting the AQ27 coding region into pAKKO-111H (11Hinuma S. Hosoya M. Ogi K. Tanaka H. Nagai Y. Onda H. Biochim. Biophys. Acta. 1994; 1219: 251-259Crossref PubMed Scopus (36) Google Scholar). 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 μm). 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 α-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– CHO cells and established CHO cells expressing each GPCR. Synthetic QRFPs were prepared as described previously (2Hinuma S. Habata Y. Fujii R. Kawamata Y. Hosoya M. Fukusumi S. Kitada C. Masuo Y. Asano T. Matsumoto H. Sekiguchi M. Kurokawa T. Nishimura O. Onda H. Fujino M. Nature. 1998; 393: 272-276Crossref PubMed Scopus (541) Google Scholar). The suppression of forskolin-induced cAMP production in CHO cells was determined as described previously (12Fukusumi S. Kitada C. Takekawa S. Kizawa H. Sakamoto J. Miyamoto M. Hinuma S. Kitano K. Fujino M. Biochem. Biophys. Res. Commun. 1997; 232: 157-163Crossref PubMed Scopus (145) Google Scholar). Ca2+Mobilization Assays—Ca2+ mobilization assays using a stable CHO cell line expressing AQ27 (CHO-hAQ27) cells and mock CHO cells were conducted as described elsewhere (13Hosoya M. Moriya T. Kawamata Y. Ohkubo S. Fujii R. Matsui H. Shintani Y. Fukusumi S. Habata Y. Hinuma S. Onda H. Nishimura O. Fujino M. J. Biol. Chem. 2000; 275: 29528-29532Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar). Changes in intracellular Ca2+ 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 (8Elshourbagy N.A. Ames R.S. Fitzgerald L.R. Foley J.J. Chambers J.K. Szekeres P.G. Evans N.A. Schmidt D.B. Buckley P.T. Dytko G.M. Murdock P.R. Milligan G. Groarke D.A. Tan K.B. Shabon U. Nuthulaganti P. Wang D.Y. Wilson S. Bergsma D.J. Sarau H.M. J. Biol. Chem. 2000; 275: 25965-25971Abstract Full Text Full Text PDF PubMed Scopus (237) Google Scholar). In brief, a tyrosine residue of synthetic human QRFP (i.e. <EDEGSEATGFLPAAGEKTSGPLGNLAEELNGYSRKKGGFSFRFamide, where <E indicates pyroglutamic acid) was radioiodinated with Na125I (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 [125I-Tyr32]QRFP and incubated at room temperature for 90 min. To determine the amount of nonspecific binding, 1 μm unlabeled QRFP was added to the mixture. After filtration, the radioactivity of [125I-Tyr32]QRFP bound to the membrane fractions was determined. Quantification of Rat QRFP and AQ27 mRNAs by RT-PCR— Poly(A)+ RNA fractions were prepared from rat tissues, and their expression levels of QRFP and AQ27 mRNAs were quantitatively analyzed by means of RT-PCR using an ABI Prism 7700 sequence detector as described elsewhere (14Fujii R. Hosoya M. Fukusumi S. Kawamata Y. Habata Y. Hinuma S. Onda H. Nishimura O. Fujino M. J. Biol. Chem. 2000; 275: 21068-21074Abstract Full Text Full Text PDF PubMed Scopus (190) Google Scholar). The following primer sets and probes were used: 5′-AGCACACTGGCTTCCGTCTAG-3′, 5′-CGCTGGCCTTCTCTGAGTCA-3′, and 5′(FAM)-AGGCAGGACAGTGGCAGTGAAGCC-(TAMRA)3′ for rat QRFP mRNA and 5′-CGGAAGCCTGGGAATTCTG-3′, 5′-ATGTGTCTCCTTTGGTTTCTTCCA-3′, and 5′(FAM)-AGCAAAGTTATCTCGACCACAGCGTCCA-(TAMRA)3′ 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 (15Fujiwara K. Maruyama M. Usui K. Sakai T. Matsumoto H. Hinuma S. Kitada C. Inoue K. Neurosci. Lett. 2003; 338: 127-130Crossref PubMed Scopus (13) Google Scholar). 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 μl 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 μl 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 (8Elshourbagy N.A. Ames R.S. Fitzgerald L.R. Foley J.J. Chambers J.K. Szekeres P.G. Evans N.A. Schmidt D.B. Buckley P.T. Dytko G.M. Murdock P.R. Milligan G. Groarke D.A. Tan K.B. Shabon U. Nuthulaganti P. Wang D.Y. Wilson S. Bergsma D.J. Sarau H.M. J. Biol. Chem. 2000; 275: 25965-25971Abstract Full Text Full Text PDF PubMed Scopus (237) Google Scholar). 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 (8Elshourbagy N.A. Ames R.S. Fitzgerald L.R. Foley J.J. Chambers J.K. Szekeres P.G. Evans N.A. Schmidt D.B. Buckley P.T. Dytko G.M. Murdock P.R. Milligan G. Groarke D.A. Tan K.B. Shabon U. Nuthulaganti P. Wang D.Y. Wilson S. Bergsma D.J. Sarau H.M. J. Biol. Chem. 2000; 275: 25965-25971Abstract Full Text Full Text PDF PubMed Scopus (237) Google Scholar). Signal sequences were predicted with the SignalP Version 2.0 software program (16Nielsen H. Brunak S. Heijne V.G. Protein Eng. 1999; 12: 3-9Crossref PubMed Scopus (536) Google Scholar). 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. GGFSFRF-amide) 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.Fig. 2Comparison of amino acid sequences of known RFamide peptides and QRFP. Residues with C-terminal RFamide structure are boxed. PrRP, prolactin-releasing peptide; NPFF, neuropeptide FF; NPAF, neuropeptide AF; MetEnk-amide, Met-enkephalinamide.View Large Image Figure ViewerDownload Hi-res image Download (PPT) 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 (2Hinuma S. Habata Y. Fujii R. Kawamata Y. Hosoya M. Fukusumi S. Kitada C. Masuo Y. Asano T. Matsumoto H. Sekiguchi M. Kurokawa T. Nishimura O. Onda H. Fujino M. Nature. 1998; 393: 272-276Crossref PubMed Scopus (541) Google Scholar, 3Tatemoto K. Hosoya M. Habata Y. Fujii R. Kakegawa T. Zou M.X. Kawamata Y. Fukusumi S. Hinuma S. Kitada C. Kurokawa T. Onda H. Fujino M. Biochem. Biophys. Res. Commun. 1998; 251: 471-476Crossref PubMed Scopus (1363) Google Scholar). 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)+ RNA based on public genome information (GenBank™ accession number AQ270411), and has been found to be identical to GPR103 as recently reported (17Lee D.K. Nguyen T. Lynch K.R. Cheng R. Vanti W.B. Arkhitko O. Lewis T. Evans J.F. George S.R. O'Dowd B.F. Gene (Amst.). 2001; 275: 83-91Crossref PubMed Scopus (166) Google Scholar). We isolated its rat and mouse counterparts from their respective brain poly(A)+ 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 (18Joost, P., and Methner, A. (2002) Genome Biol., http://genomebiology.com/2002/3/11/RESEARCH/0063,Google Scholar). 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 (19Yoshida H. Habata Y. Hosoya M. Kawamata Y. Kitada C. Hinuma S. Biochim. Biophys. Acta. 2003; 1593: 151-157Crossref PubMed Scopus (147) Google Scholar). 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. (20Durocher Y. Perret S. Thibaudeau E. Gaumond M.H. Kamen A. Stocco R. Abramovitz M. Anal. Biochem. 2000; 284: 316-326Crossref PubMed Scopus (96) Google Scholar, 21Chen W. Shields T.S. Stork P.J. Cone R.D. Anal. Biochem. 1995; 226: 349-354Crossref PubMed Scopus (181) Google Scholar). They demonstrated that the activation of GPCRs coupled with Gs and/or Gq causes an increase in the transcription of the cAMP-responsive element promoter. We modified their method for detecting Gq signals so that, by our technique, Gq signals were detected as the increase of luciferase activity in the presence of forskolin. As AQ27 coupled to Gq, 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 10–5 to 10–6m (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 (22Fukusumi S. Habata Y. Yoshida H. Iijima N. Kawamata Y. Hosoya M. Fujii R. Hinuma S. Kitada C. Shintani Y. Suenaga M. Onda H. Nishimura O. Tanaka M. Ibata Y. Fujino M. Biochim. Biophys. Acta. 2001; 1540: 221-232Crossref PubMed Scopus (155) Google Scholar) 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 CH3CN, 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 μRPC 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 <EDEGSEATGFLPAAGEKTSGPLGNLAEELNGYSRKKGGFSFRFamide (<E indicates pyroglutamic acid). Although we could not determine from the mass spectrometric data whether or not the C-terminal 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 (23Mekler D.J. Enzyme Microb. Technol. 1994; 16: 450-456Crossref PubMed Scopus (195) Google Scholar). (iii) We confirmed with synthetic peptides that the nonamidated form of this peptide showed drastically reduced agonistic activity (Table I). (iv) The M + H+ calculated average masses of the amidated and nonamidated form were 4504.9 and 4505.9, respectively. Although the M + H+ observed average mass (m/z 4505.7) seemed to be closer to the M + H+ calculated average mass of the nonamidated peptide than that of the amidated form, both M + H+ calculated average masses were within the allowable error limit (0.05%) of delayed extraction MALDITOF mass spectrometry in linear mode using external calibration. Therefore, the mass spectrometric data did not contradict the predicted RFamide structure. We determined the N-terminal 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 (24Busby Jr., W.H. Quackenbus
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