Identification of a Neuropeptide Modified with Bromine as an Endogenous Ligand for GPR7

We isolated a novel gene in a search of the Celera data base and found that it encoded a peptidic ligand for a G protein-coupled receptor, GPR7 (O’Dowd, B. F., Scheideler, M. A., Nguyen, T., Cheng, R., Rasmussen, J. S., Marchese, A., Zastawny, R., Heng, H. H., Tsui, L. C., Shi, X., Asa, S., Puy, L., and George, S. R. (1995)Genomics 28, 84–91; Lee, D. K., Nguyen, T., Porter, C. A., Cheng, R., George, S. R., and O’Dowd, B. F. (1999) Mol. Brain Res. 71, 96–103). The expression of this gene was detected in various tissues in rats, including the lymphoid organs, central nervous system, mammary glands, and uterus. GPR7 mRNA was mainly detected in the central nervous system and uterus. In situ hybridization showed that the gene encoding the GPR7 ligand was expressed in the hypothalamus and hippocampus of rats. To determine the molecular structure of the endogenous GPR7 ligand, we purified it from bovine hypothalamic tissue extracts on the basis of cAMP production-inhibitory activity to cells expressing GPR7. Through structural analyses, we found that the purified endogenous ligand was a peptide with 29 amino acid residues and that it was uniquely modified with bromine. We subsequently determined that the C-6 position of the indole moiety in the N-terminal Trp was brominated. We believe this is the first report on a neuropeptide modified with bromine and have hence named it neuropeptide B. In in vitro assays, bromination did not influence the binding of neuropeptide B to the receptor. We isolated a novel gene in a search of the Celera data base and found that it encoded a peptidic ligand for a G protein-coupled receptor, GPR7 (O’Dowd, B. F., Scheideler, M. A., Nguyen, T., Cheng, R., Rasmussen, J. S., Marchese, A., Zastawny, R., Heng, H. H., Tsui, L. C., Shi, X., Asa, S., Puy, L., and George, S. R. (1995)Genomics 28, 84–91; Lee, D. K., Nguyen, T., Porter, C. A., Cheng, R., George, S. R., and O’Dowd, B. F. (1999) Mol. Brain Res. 71, 96–103). The expression of this gene was detected in various tissues in rats, including the lymphoid organs, central nervous system, mammary glands, and uterus. GPR7 mRNA was mainly detected in the central nervous system and uterus. In situ hybridization showed that the gene encoding the GPR7 ligand was expressed in the hypothalamus and hippocampus of rats. To determine the molecular structure of the endogenous GPR7 ligand, we purified it from bovine hypothalamic tissue extracts on the basis of cAMP production-inhibitory activity to cells expressing GPR7. Through structural analyses, we found that the purified endogenous ligand was a peptide with 29 amino acid residues and that it was uniquely modified with bromine. We subsequently determined that the C-6 position of the indole moiety in the N-terminal Trp was brominated. We believe this is the first report on a neuropeptide modified with bromine and have hence named it neuropeptide B. In in vitro assays, bromination did not influence the binding of neuropeptide B to the receptor. G protein-coupled receptor neuropeptide B Chinese hamster ovary phenylthiohydantoin 5-bromotryptophan 6-bromotryptophan high performance liquid chromatography nonbrominated neuropeptide W A large number of new genes have been discovered in the progress of analyses for the human genome. How to determine the functions of these genes is an important issue. G protein-coupled receptors (GPCRs)1 play important roles in the regulation of physiological phenomena including sense, growth, reproduction, metabolism, and homeostasis. In the human genome, numerous genes have been found encoding GPCRs with as yet unknown ligands. The identification of ligands for these “orphan” GPCRs is a key to revealing their functions. In addition, since GPCRs have been historically important as drug targets, it is hoped that the identification of ligands for orphan GPCRs will bring new drug targets. We have developed our own unique methods to determine ligands for such orphan GPCRs on the basis of detecting specific signal transduction in cells expressing targeted receptors (3Hinuma S. Onda H. Fujino M. J. Mol. Med. 1999; 77: 495-504Crossref PubMed Scopus (54) Google Scholar). First, we sought for ligands in tissue extracts and, subsequently, in a library with synthetic compounds. Recently, we have developed a method to search for genes encoding ligands in databases providing genomic and cDNA sequences. By utilizing these methods, we have already succeeded in the identification of several ligands for orphan GPCRs (4Hinuma 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 (526) Google Scholar, 5Tatemoto 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 (1287) Google Scholar, 6Fujii 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 (186) Google Scholar, 7Hosoya 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 (107) Google Scholar, 8Hinuma 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; 2: 703-708Crossref PubMed Scopus (498) Google Scholar). GPR7 has been cloned as an orphan GPCR resembling opioid or somatostatin receptors (1O'Dowd B.F. Scheideler M.A. Nguyen T. Cheng R. Rasmussen J.S. Marchese A. Zastawny R. Heng H.H. Tsui L.C. Shi X. Asa S. Puy L. George S.R. Genomics. 1995; 28: 84-91Crossref PubMed Scopus (114) Google Scholar). Another GPCR, GPR8, sharing 59% amino acid identity with GPR7, has been also reported, but ligands for GPR7 and GPR8 have not been identified (1O'Dowd B.F. Scheideler M.A. Nguyen T. Cheng R. Rasmussen J.S. Marchese A. Zastawny R. Heng H.H. Tsui L.C. Shi X. Asa S. Puy L. George S.R. Genomics. 1995; 28: 84-91Crossref PubMed Scopus (114) Google Scholar). By utilizing the Celera data base, we searched for candidate genes encoding ligands for orphan GPCRs. In this paper, we report on the identification of a novel gene encoding a ligand for GPR7. In addition, we show here that this endogenous GPR7 ligand purified from tissue extracts is a peptide modified with bromine. Celera Discovery Systems- and Celera Genomics-associated databases were used to search for genes encoding proteins with the motif of the secretory signal sequence. In our search, we found a gene, NPB, encoding a novel secretory protein. Based on the sequence information provided by the data base, we isolated a cDNA from human brain cDNAs by PCR using a primer set (5′-GTCGACATGGCCCGGTCCGCGACACTGGCGGCC-3′ and 5′-GCTAGCAGCGGTGCCAGGAGAGGTCCGGGCTCA-3′). We subsequently designed several primers on the basis of this human NPB cDNA and isolated rat, mouse, and bovine NPB cDNAs from brain cDNAs by the rapid amplification of cDNA ends method using a Marathon cDNA amplification kit (CLONTECH, Palo Alto, CA). Utilizing several primers designed from the published sequences of human GPR7 and GPR8 cDNAs (1O'Dowd B.F. Scheideler M.A. Nguyen T. Cheng R. Rasmussen J.S. Marchese A. Zastawny R. Heng H.H. Tsui L.C. Shi X. Asa S. Puy L. George S.R. Genomics. 1995; 28: 84-91Crossref PubMed Scopus (114) Google Scholar), 2The nucleotide sequences of the human GPR7, GPR8, and prepro-NPW cDNAs can be accessed through the GenBankTM/EBI Data Bank with accession numbers U22491,U22492, and AB084276, respectively. we isolated bovine GPR7 and GPR8 cDNAs by rapid amplification of cDNA ends. The complete coding regions of bovine GPR7 and GPR8 were amplified from bovine hypothalamus cDNAs by PCR with primer sets (5′-GTCGACCGAGTGTCTGTCCTCGCCAGGATG-3′ and 5′-GCTAGCTCCTTGTTATCGGGCTCAGGAGGTGGT-3′ for GPR7 and 5′-GTCGACCATGATGGAGGCCACTGGGCTGGAAGG-3′ and 5′- GCTAGCTTATGCCCCCTGGCACCGACATGCGGT-3′ for GPR8). The entire coding regions of NPB, GPR7, and GPR8 cDNAs were cloned, respectively, into the downstream region of an SR α promoter in the expression vector pAKKO-111H (9Hinuma 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 plasmids were transfected intodhfr− CHO cells, following whichdhfr+ CHO cells were selected, respectively, as previously described (9Hinuma S. Hosoya M. Ogi K. Tanaka H. Nagai Y. Onda H. Biochim. Biophys. Acta. 1994; 1219: 251-259Crossref PubMed Scopus (36) Google Scholar). The inhibition of forskolin-induced cAMP production in CHO cells was determined as previously described (10Fukusumi 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 (144) Google Scholar). Poly(A)+ RNA fractions were prepared from tissues of 8–12-week-old Wistar rats, and cDNAs were synthesized from these (11Fujii R. Fukusumi S. Hosoya M. Kawamata Y. Habata Y. Hinuma S. Sekiguchi M. Kitada C. Kurokawa T. Nishimura O. Onda H. Sumino Y. Fujino M. Regul. Pept. 1999; 83: 1-10Crossref PubMed Scopus (99) Google Scholar). Poly(A)+ RNAs were prepared from placenta, mammary gland, and whole fetus tissue of female rats 17 days pregnant. Rat NPB and GPR7 mRNA expressions were determined with a Prism 7700 sequence detector (Applied Biosystems) (6Fujii 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 (186) Google Scholar) with primers and fluorescence-labeled probes (5′-CTGTCGAGTTTCCACAGGTTCC-3′, 5′-TTGCGCAGAGGTACGGTTCC-3′, and 5′-6-carboxyfluorescein-ATCCACGCGACGTTCCGAGTCTCCA-6- carboxytetramethylrhodamine-3′ for NPB and 5′-TGCGTGCTATCCAGCTAGACAG-3′, 5′-AGAGGAGGCACACAGCCAGAAT-3′, and 5′-6-carboxyfluorescein-CGTGCCAAGAAACGCGTGACCTTGTT-6-carboxytetramethylrhodamine-3′ for GPR7). A fragment of rat NPB cDNA (corresponding to nucleotides 224–393) was cloned into pBluescriptII KS+ (Stratagene, La Jolla, CA). Digoxigenin-labeled antisense and sense riboprobes were constructed with T7 RNA polymerase or T3 RNA polymerase, respectively (12Iijima N. Kataoka Y. Kakihara K. Bamba H. Tamada Y. Hayashi S. Matsuda T. Tanaka M. Honjyo H. Hosoya M. Hinuma S. Ibata Y. Neuroreport. 1999; 10: 1713-1726Crossref PubMed Scopus (74) Google Scholar). NPB mRNA was visualized with alkaline phosphatase (8Hinuma 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; 2: 703-708Crossref PubMed Scopus (498) Google Scholar). To determine the molecular structure of endogenous bovine NPB, mass spectrometry was performed with a Fourier transform mass spectrometer (Apex II, Bruker Daltonics, Bremen, Germany) equipped with an electrospray ion source. To discriminate 6-bromotryptophan (6BrW) from 5-bromotryptophan (5BrW), peptide sequencing was performed on a protein sequencer (491 cLC; Applied Biosystems) with a modified gradient program. Phenylthiohydantoin (PTH)-6BrW and PTH-5BrW were prepared, respectively, by coupling phenylisothiocyanate (Sigma) withdl-6BrW (Biosynth AG, Staad, Switzerland) ordl-5BrW (Aldrich, Steinheim, Germany) and converting to PTH-derivatives. To summarize briefly, the amino acids were reacted in 7:1:1:1 (v/v/v/v) ethanol/triethylamine/water/phenylisothiocyanate at room temperature for 20 min, dried, and then treated with trifluoroacetic acid at 50 °C for 10 min. After again drying, the reaction mixtures were treated with 1:1 (v/v) methanol, 2 nHCl at 50 °C for 10 min. PTH-derivatives were purified by high performance liquid chromatography (HPLC) with a C18 column (218TP5415; Vydac, Hesperia, CA). Peptides were chemically synthesized with an automatic peptide synthesizer (model 433; PerkinElmer Biosystems) according to an Fmoc (N-(9-fluorenyl)methoxycarbonyl)/N,N′-dicyclohexylcarbodiimide/1-hydroxybenzotriazole protocol. l-6BrW was introduced into the peptides through enantiopure Boc-l-6BrW prepared by chiral separation. Receptor-binding assays were conducted principally according to our method previously described (4Hinuma 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 (526) Google Scholar). Briefly, the synthetic human nonbrominated form of NPB-23 (desBr-NPB-23) was labeled with Na125I using lactoperoxidase. Membrane fractions (1 μg) from CHO cells expressing human GPR7 were mixed with 125I-labeled human desBr-NPB-23 (100 pm) and incubated at room temperature for 90 min. To determine the amount of nonspecific binding, 1 μm unlabeled human desBr-NPB-23 was added to the mixture. The amounts of 125I-labeled human desBr-NPB-23 bound to the membrane fractions were measured after rapid filtration. In order to find novel secretory protein genes, we searched for candidate proteins with possible secretory signal peptides in hypothetical proteins deduced from human genomic sequences in the Celera data base. Since one of these candidates appeared to be derived from a novel secretory protein gene, NPB, we cloned a cDNA on the basis of the sequence information. We subsequently cloned NPB cDNAs in other species also. As shown in Fig. 1, these cDNAs encoded proteins with secretory signals. Homology among the proteins ranged from 53 to 92%, and they seemed to possess preproprotein structures. For example, human NPB contained an N-terminal secretory signal peptide with 24-amino acid length and potential sites (i.e.Arg48-Arg49 and Arg54-Arg55) for cleaving with proteases. We anticipated that the preproprotein we found might produce a ligand for orphan GPCRs. We therefore expressed the human NPB cDNA in CHO cells and examined whether ligands were secreted in the culture supernatants. The culture supernatants were screened by adding them to CHO cells expressing orphan GPCRs. The existence of ligands in the culture supernatants was determined by detecting specific signal transduction in the CHO cells. We detected specific cAMP production-inhibitory activities to CHO cells expressing GPR7 in the culture supernatant. Based on this, we purified a ligand for GPR7 from the culture supernatant. Open column chromatography was applied wherein the culture supernatant (2 liters) was boiled and then eluted through a C18 column (Prep C18125A; Waters) with stepwise increments of 10, 40, and 60% CH3CN in 0.05% trifluoroacetic acid. Activity was detected in the fraction eluted with 40% CH3CN. Therefore, this fraction was purified serially through a HiPrep CM-Sepharose FF column (Amersham Biosciences) with 0–0.5 in 20 at a column (HPLC) with a column with in 20 at and a column Biosciences) with CH3CN not The purified ligand for GPR7 that we was then by N-terminal sequencing and mass spectrometry not that this purified ligand was a peptide of the amino acid residues that this peptide was from the preproprotein through We not in the peptide purified from the culture supernatant. T., T., T., C., H., and M. J. Biol. Scholar) have identified a peptide as a ligand for GPR7 and GPR8, and named it neuropeptide W We found that our purified peptide and identity that the peptides they derived from We the tissue of NPB and GPR7 in rats. the expression of NPB mRNA was detected in a of high were found in the lymphoid organs, central nervous system, mammary glands, and NPB mRNA was expressed in the central nervous system not GPR7 mRNA expression was mainly detected in the central nervous system and We subsequently conducted in to NPB mRNA in the rat NPB mRNA was found to be from antisense NPB riboprobes were detected in the hypothalamus and In the specific were detected by the antisense in the hypothalamic and hypothalamic In the expression was detected in the of the but not in the hybridization were detected by a sense in the and situ hybridization of NPB mRNA in rat and hybridization with an antisense hybridization with a sense by the antisense were detected in the hypothalamic hypothalamic and of the in the hippocampus hybridization signal was detected with the sense in the the To determine the molecular structure of endogenous NPB, we purified it from bovine hypothalamic tissue extracts by a of various on the basis of cAMP production-inhibitory activity to CHO cells expressing GPR7. After bovine (2 were in 1 acid. The resultant supernatant was then by column chromatography through a C18 column with stepwise increments of 10, 40, and 60% CH3CN in 0.05% trifluoroacetic acid. We then the 40% CH3CN fraction through a HiPrep CM-Sepharose FF column with 0–0.5 in 20 at The resultant fractions the activities were with and again through a column (HPLC) with CH3CN. After with to these were serially through a C18 column (HPLC) with a column (HPLC) with in 20 at and a column (HPLC) with CH3CN. In the the endogenous NPB was eluted as a at CH3CN As the N-terminal of the endogenous bovine NPB was to from in the preproprotein we structure by mass spectrometry and not the molecular mass did not to of peptides derived from the preproprotein and was mass the molecular mass of acid length bovine of the and the of the that the endogenous bovine NPB was modified with bromine at the first or amino acid of the To whether the endogenous NPB was modified with we it to electrospray Fourier transform mass The mass and of with the of acid bovine NPB N-terminal sequencing not to the sequence from to in the preproprotein the at 1, was to be Trp from the cDNA was eluted at a from that of a Therefore, was determined to be the was in peptides derived from Trp residues were found to be at the C-6 position of the indole moiety M. J. 1997; PubMed Scopus Google Scholar, J. J. J. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar). After PTH-5BrW and as we the purified NPB to N-terminal sequencing by a modified to these The at position 1 from the endogenous NPB with that the endogenous bovine NPB has the following We not NPB bromination as a in the fractions from bovine hypothalamic tissue analyses for endogenous in the electrospray Fourier transform mass of the purified peptide and the of for The mass in the in a mixed with and acid (Applied Biosystems) and in 1 of the purified NPB The purified peptide was with an protein sequencer according to a modified in was conducted for in following the for in gradient and with The first of the purified NPB was eluted at the position as The structure of is the We cloned bovine GPR7 and GPR8 cDNAs and then examined the of NPB with NPB cAMP production in CHO cells expressing GPR7 or GPR8 in various species NPB cAMP production in cells expressing GPR7 expressing bovine and the forskolin-induced production of We also measured the of on forskolin-induced cAMP production in CHO cells expressing human GPR8 and found that these peptides were We not in the cAMP production-inhibitory activities NPB and or human and bovine of NPB with GPR7 and NPB The numbers after NPB amino acid of cAMP were determined from was by the of peptides in the binding of to human for human NPB The numbers after NPB amino acid were determined from was by the of peptides in the binding of to human GPR7. Open in a new To the binding of NPB to GPR7, we prepared human desBr-NPB-23 labeled with at of residues and After we the labeled peptides by using a column with CH3CN. The of the were by mass spectrometry not Since the activity of NPB was by at but not at not we used for binding showed that the membrane fractions of CHO cells expressing human GPR7 a of high binding sites for at the of and binding sites of that NPB to GPR7 as a specific ligand with high in the binding assays, NPB and showed in their and the binding of that bromination in NPB not with the receptor. In this paper, we have identified a novel NPB, as an endogenous ligand for GPR7. NPB showed activity to cAMP production in CHO cells expressing GPR7 and bound to their membrane fractions with high we not in the CHO cells not that GPR7 to NPB with GPR8, but seemed to be that with GPR7. data that NPB is a ligand principally to GPR7, we not the that NPB as a ligand not for GPR7 but also for GPR8 in In activities to a on GPR7 and GPR8 T., T., T., C., H., and M. J. Biol. Scholar), that NPB and have functions. In our structural analyses of endogenous NPB purified from bovine we found that N-terminal Trp was modified with bromine. a protein with residues by reaction with Y. A. 1999; PubMed Scopus Google Scholar). the to Trp has yet to be it has been found in peptides the ion derived from M. J. 1997; PubMed Scopus Google J. J. J. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar, J. R. J. M. J. D. 1998; PubMed Scopus Google Scholar). that bromination in peptides as a physiological and not as an N-terminal were in rat and in In addition, the activity of NPB was by of to N-terminal data that the N-terminal of these peptides important in their with we did not bromination in endogenous The bromination is in NPB is at the In addition, we that NPB purified from several of bovine hypothalamic tissue extracts were it be in whether bromination in NPB physiological among been of structural in is the of residues in and with a in their activities M. J. Google Scholar). The other is in M. H. Y. M. H. K. Nature. 1999; Scopus Google Scholar). peptide to receptor it is not modified with at the in NPB is the of unique to be in we not the influence of bromination on the of NPB with the receptor in in vitro In the Trp residues have been to be important to determine J. R. J. M. J. D. 1998; PubMed Scopus Google Scholar). the physiological of bromination in NPB is this in Since has shown in activities and T., T., T., C., H., and M. J. Biol. Scholar), to the in of NPB in GPR7 has been detected in the rat and but GPR8 is in (1O'Dowd B.F. Scheideler M.A. Nguyen T. Cheng R. Rasmussen J.S. Marchese A. Zastawny R. Heng H.H. Tsui L.C. Shi X. Asa S. Puy L. George S.R. Genomics. 1995; 28: 84-91Crossref PubMed Scopus (114) Google Scholar, Nguyen T. Cheng R. George S.R. B.F. Mol. Brain Res. 1999; PubMed Scopus Google Scholar). that NPB and GPR7 in various tissues in rats. Since NPB and GPR7 were detected in the central nervous system and in rats, they might have important functions in these In the NPB mRNA was detected in the hypothalamic and hypothalamic GPR7 mRNA is expressed in the and hypothalamic Nguyen T. Cheng R. George S.R. B.F. Mol. Brain Res. 1999; PubMed Scopus Google Scholar). In addition, expressions of NPB and GPR7 were detected in the The of NPB and GPR7 in the rat brain that NPB is in the regulation of the system, and these be examined in We believe that on NPB will new into not in but also the of brain functions. We Y. Sumino and H. Matsumoto for We also Celera for the