Identification of a Neuropeptide Modified with Bromine as an Endogenous Ligand for GPR7
2002; Elsevier BV; Volume: 277; Issue: 37 Linguagem: Inglês
10.1074/jbc.m205883200
ISSN1083-351X
AutoresRyo Fujii, Hiromi Yoshida, Shoji Fukusumi, Yugo Habata, Masaki Hosoya, Yuji Kawamata, Takahiko Yano, Shuji Hinuma, Chieko Kitada, Taiji Asami, M. Mori, Yukio Fujisawa, Masahiko Fujino,
Tópico(s)Regulation of Appetite and Obesity
ResumoWe 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 m NaCl in 20 mm CH3COONH4 at pH 4.7, a RESOURCE RPC column (HPLC) with 15–30% CH3CN, a TSK-gel CM-2SW column (HPLC; TOSO, Tokyo, Japan) with 0.2–0.5 m NaCl in 20 mm CH3COONH4 at pH 4.7, and a μRPC C2/C18 SC 2.1/10 column (HPLC; Amersham Biosciences) with 15–22% CH3CN (data not shown). The purified ligand for GPR7 that we thus obtained was then analyzed by N-terminal sequencing and mass spectrometry (data not shown). Our results revealed that this purified ligand was a peptide consisting of the 24 amino acid residues WYKPAAGHSSYSVGRAAGLLSGLR, indicating that this peptide was generated from the preproprotein through processing. We could not detect any modification in the peptide purified from the culture supernatant. Shimomura et al. (13Shimomura, Y., Harada, M., Goto, M., Sugo, T., Matsumoto, Y., Abe, M., Watanabe, T., Asami, T., Kitada, C., Mori, M., Onda, H., and Fujino, M. (July 18, 2002) J. Biol. Chem.10.1074/jbc.M205337200Google Scholar) have recently identified a peptide which acts as a natural ligand for both GPR7 and GPR8, and named it neuropeptide W (NPW). We found that our purified peptide and NPW shared 61% identity (Fig. 2), indicating that the two peptides are closely related, although they are derived from different genes. We analyzed the tissue distribution of NPB and GPR7 mRNAs in rats. Although the expression of NPB mRNA was detected in a variety of tissues, high levels were found in the lymphoid organs, central nervous system, mammary glands, and uterus (Fig.3, upper panel). Human NPB mRNA was highly expressed in the central nervous system (data not shown). GPR7 mRNA expression was mainly detected in the rats' central nervous system and uterus (Fig. 3, lower panel). We subsequently conducted in situhybridization to detect NPB mRNA in the rat brain. NPB mRNA was found to be widely distributed here. Dense signals from antisense NPB riboprobes were detected in the hypothalamus and hippocampus. In the hypothalamus, specific signals were detected by the antisense probe in the medial preoptic area (Fig.4A), ventromedial hypothalamic nucleus (Fig. 4B), and lateral hypothalamic area (Fig.4C). In the hippocampus, moderate expression was detected in the CA fields of the Ammon's horn but not in the dentate gyrus (Fig.4G). No hybridization signals were detected by a sense probe in the same areas (Fig. 4, D–F and H).Figure 4In situ hybridization of NPB mRNA in rat brain.A–C and G, hybridization with an antisense riboprobe. D–F andH, hybridization with a sense riboprobe. Positive signals by the antisense probe were detected in the medial preoptic area (A), ventromedial hypothalamic nucleus (B), lateral hypothalamic area (C), and CA fields of the Ammon's horn in the hippocampus (G). No hybridization signal was detected with the sense probe in the same areas (D–F andH). 3V, the third ventricle.View Large Image Figure ViewerDownload Hi-res image Download (PPT) To determine the molecular structure of endogenous NPB, we purified it from bovine hypothalamic tissue extracts by a combination of various chromatographies on the basis of cAMP production-inhibitory activity to CHO cells expressing GPR7. After boiling, bovine hypothalami (2 kg) were homogenized in 1 m acetic acid. The resultant supernatant was then fractionated by open column chromatography through a C18 column with stepwise increments of 10, 40, and 60% CH3CN in 0.05% trifluoroacetic acid. We then fractionated the 40% CH3CN fraction through a HiPrep CM-Sepharose FF column with 0–0.5 m NaCl in 20 mmCH3COONH4 at pH 4.7. The resultant fractions showing the activities were precipitated with 66% acetone and again fractionated through a RESOURCE RPC column (HPLC) with 20–30% CH3CN. After precipitation with 75% acetone to remove proteins, these were fractionated serially through a Vydac C18 218TP5415 column (HPLC) with 20–30% CH3CN, a TSK-gel CM-2SW column (HPLC) with 0.3–0.5 m NaCl in 20 mm CH3COONH4 at pH 4.7, and a μRPC C2/C18 SC 2.1/10 column (HPLC) with 16–24% CH3CN. In the final chromatography, the endogenous NPB was eluted as a single peak at 21% CH3CN (Fig. 5). As the N-terminal residue of the endogenous bovine NPB was expected to start from Trp25 in the preproprotein (Fig. 1), we analyzed its structure by nanoelectrospray ionization ion-trap mass spectrometry and MS/MS (data not shown). However, unexpectedly, the observed molecular weight (i.e. 3241.4 mass units) did not correspond to any lengths of peptides derived from the preproprotein and was 80 mass units higher than the calculated molecular weight (i.e. 3161.5 mass units) of 29-amino acid length bovine NPB. Consideration of the MS/MS spectrum and the isotope distribution of the signals suggested that the endogenous bovine NPB was modified with bromine at the first or second amino acid residue of the N-terminal. To confirm whether the endogenous NPB was indeed modified with bromine, we subjected it to electrospray ionization Fourier transform mass spectrometry. The mass values and isotope distribution of (M + 5H)5+ ions (Fig.6A, upper panel) agreed well with the theoretical profile of brominated 29-amino acid bovine NPB (Fig. 6A,lower panel). N-terminal sequencing revealedXYKPTAGQGYYSVGRAAGLLSGFHR (X not identified), which corresponded to the sequence from Tyr26 to Arg49 in the preproprotein (Fig. 1). Only the PTH-derivative at cycle 1, which was expected to be Trp from the cDNA sequence, was eluted at a different retention time from that of a standard PTH-derivative. Therefore, Trp1 was determined to be the brominated residue. Bromination was originally reported in peptides derived from marine invertebrates, where Trp residues were found to be brominated at the C-6 position of the indole moiety (14Jimenez E.C. Craig A.G. Watkins M. Hillyard D.R. Gray W.R. Gulyas J. Rivier J.E. Cruz L.J. Olivera B.M. Biochemistry. 1997; 36: 989-994Crossref PubMed Scopus (116) Google Scholar, 15Craig A.G. Jimenez E.C. Dykert J. Nielsen D.B. Gulyas J. Abogadie F.C. Porter J. Rivier J.E. Cruz L.J. Olivera B.M. McIntosh J.M. J. Biol. Chem. 1997; 272: 4689-4698Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar). After preparing PTH-5BrW and PHT-6BrW as standards, we subjected the purified NPB to N-terminal sequencing by a protocol modified to fit these standards. The PTH-derivative peak at position 1 from the endogenous NPB coincided exactly with PTH-6BrW (Fig. 6B). Our results indicate that the endogenous bovine NPB has the following structure: 6BrW-YKPTAGQGYYSVGRAAGLLSGFHRSPYA. We could not detect NPB without bromination as a major component in the fractions obtained from bovine hypothalamic tissue extracts.Figure 6Structural analyses for endogenous NPB.A, (M + 5H)5+ ions in the electrospray ionization Fourier transform mass spectrum of the purified peptide (upper panel) and the theoretical isotope distribution of (M + 5H)5+ ions for NPB-29 (lower panel). The mass values are indicated in the figure. B, elution profiles in a standard sample mixed with PTH-6BrW, PTH-5BrW, and 20-amino acid PTH standards (Applied Biosystems) (upper panel) and in cycle 1 of the purified NPB (lower panel). The purified peptide was analyzed with an ABI 491 cLC protein sequencer according to a slightly modified elution program in which elution was conducted for 6 min in isocratic mode following the usual elution for 22 min in gradient mode. PTH-derivative standards and reagent peaks are marked with asterisks. The first residue of the purified NPB was eluted at the same position as PTH-6BrW. The chemical structure of 6BrW is indicated below the elution profiles.View Large Image Figure ViewerDownload Hi-res image Download (PPT) We cloned bovine GPR7 and GPR8 cDNAs (Fig. 7) and then examined the interaction of NPB with these. Synthetic NPB specifically inhibited cAMP production in CHO cells expressing GPR7 or GPR8 in various species (Table I). NPB inhibited cAMP production in cells expressing GPR7 more efficiently than those expressing GPR8. Both bovine NPB-29 and desBr-NPB-29 efficiently suppressed the forskolin-induced production of cAMP. We also measured the effects of NPBs on forskolin-induced cAMP production in CHO cells expressing human GPR8 and found that these peptides were less effective upon GPR8. We could not detect any apparent difference in the cAMP production-inhibitory activities between authentic NPB and desBr-NPB or between human and bovine NPB.Table IInteraction of NPB with GPR7 and GPR8Peptide1-adesBr-NPB, NPB without bromination. The numbers after NPB indicate amino acid lengths.Inhibition of cAMP production1-bMedian inhibitory concentration (IC50) values were determined from dose-response curves.Binding1-cBinding potency was calculated by the competition of peptides in the binding of [125I-Tyr11]desBr-NPB-23 to human GPR7.(IC50) for human GPR7BovineHumanGPR7GPR8GPR7GPR8nmnmBovine NPB-291.94.00.589.70.34 desBr-NPB-291.25.40.438.10.31Human NPB-291.8510.44320.32 desBr-NPB-292.6520.45490.33 desBr-NPB-233.5470.58441.6 NPW-232.44.80.823.70.40Rat desBr-NPB-291.2140.868.80.341-a desBr-NPB, NPB without bromination. The numbers after NPB indicate amino acid lengths.1-b Median inhibitory concentration (IC50) values were determined from dose-response curves.1-c Binding potency was calculated by the competition of peptides in the binding of [125I-Tyr11]desBr-NPB-23 to human GPR7. Open table in a new tab To examine the binding of NPB to GPR7, we prepared human desBr-NPB-23 labeled with 125I at either of two tyrosine residues (Tyr2 and Tyr11). After iodination, we separated the two labeled peptides by HPLC using a TSK gel ODS-80TMCTR column (TOSO) with 48–58% CH3CN. The positions of the iodination were confirmed by mass spectrometry (data not shown). Since the agonistic activity of NPB was obviously reduced by iodination at Tyr2 but not at Tyr11 (data not shown), we used [125I-Tyr11]desBr-NPB-23 for binding assays. Scatchard plot analysis showed that the membrane fractions of CHO cells expressing human GPR7 had a single class of high affinity binding sites for [125I-Tyr11]desBr-NPB-23 at the dissociation constant (Kd) of 3.6 × 10−11m and maximal binding sites (Bmax) of 1.3 pmol mg−1 protein, indicating that NPB binds to GPR7 as a specific ligand with high affinity (Fig. 8). Even in the competitive binding assays, authentic NPB and desBr-NPB showed no difference in their inhibitory potency. Bovine NPB-29 and desBr-NPB-29 both efficiently inhibited the binding of [125I-Tyr11]desBr-NPB-23 (Table I), suggesting that bromination in NPB does not directly alter interaction with the receptor. In this paper, we have identified a novel brominated peptide, NPB, as an endogenous ligand for GPR7. Synthetic NPB showed very potent inhibitory activity to cAMP production in CHO cells expressing GPR7 and specifically bound to their membrane fractions with high affinity. However, we could not detect Ca2+ influx in the NPB-treated CHO cells (data not shown), suggesting that GPR7 coupled to Gi. NPB cross-reacted with GPR8, but its interaction seemed to be weaker than that with GPR7. These data indicate that NPB is a ligand principally directed to GPR7, although we do not rule out the possibility that NPB significantly acts as a ligand not only for GPR7 but also for GPR8 in some situations. In contrast, NPW shows agonistic activities to a similar extent on both GPR7 and GPR8 (13Shimomura, Y., Harada, M., Goto, M., Sugo, T., Matsumoto, Y., Abe, M., Watanabe, T., Asami, T., Kitada, C., Mori, M., Onda, H., and Fujino, M. (July 18, 2002) J. Biol. Chem.10.1074/jbc.M205337200Google Scholar), suggesting that NPB and NPW have different functions. In our structural analyses of endogenous NPB purified from bovine hypothalamus, we unexpectedly found that its N-terminal Trp was modified with bromine. Eosinophils reportedly generate a protein with brominated Tyr residues by reaction with peroxidase (16Wu W. Chen Y. d'Avignon A. Hazen S.L. Biochemistry. 1999; 38: 3538-3548Crossref PubMed Scopus (161) Google Scholar). Although the pathway to generate brominated Trp has yet to be discovered, it has been found in neurotoxic peptides (ς-conotoxin), which inactivate the excitatory serotonin-gated ion channel, derived from marine snails (14Jimenez E.C. Craig A.G. Watkins M. Hillyard D.R. Gray W.R. Gulyas J. Rivier J.E. Cruz L.J. Olivera B.M. Biochemistry. 1997; 36: 989-994Crossref PubMed Scopus (116) Google Scholar,15Craig A.G. Jimenez E.C. Dykert J. Nielsen D.B. Gulyas J. Abogadie F.C. Porter J. Rivier J.E. Cruz L.J. Olivera B.M. McIntosh J.M. J. Biol. Chem. 1997; 272: 4689-4698Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar, 17England L.J. Imperial J. Jacobsen R. Craig A.G. Gulyas J. Akhtar M. Rivier J. Julius D. Olivera B.M. Science. 1998; 281: 575-578Crossref PubMed Scopus (115) Google Scholar). This suggests that bromination in peptides occurs as a physiological phenomenon and not as an artificial event. N-terminal portions were well conserved in human, bovine, rat and mouse NPBs, even in NPWs. In addition, the agonistic activity of NPB was reduced by iodination of Tyr adjacent to N-terminal Trp. These data suggest that the N-terminal portions of these peptides are important in their interaction with receptors. However, we did not observe bromination in endogenous NPW. The reason why bromination is specifically observed in NPB is unclear at the moment. In addition, although we confirmed that all NPB samples purified from several different batches of bovine hypothalamic tissue extracts were brominated, it should be clarified in future studies whether bromination usually occurs in NPB under physiological conditions among different species. Previously there had only been two cases observed of structural additive modifications in neuropeptides. One is the sulfation of Tyr residues in cholecystokinin and gastrin, with a resulting increase in their biological activities (18Kaminski D.L. Ruwart M.J. Jellinek M. Am. J. Physiol. 1977; 233: E286-E292PubMed Google Scholar). The other is acylation in ghrelin (19Kojima M. Hosoda H. Date Y. Nakazato M. Matsuo H. Kangawa K. Nature. 1999; 403: 656-660Crossref Scopus (7101) Google Scholar). This peptide completely loses its affinity to its receptor if it is not modified with n-octanoylation at the Ser residue. Bromination in NPB is thus the third type of unique modification to be observed in neuropeptides. However, we could not detect the apparent influence of bromination on the interaction of NPB with the receptor in in vitro assays. In ς-conotoxin, the brominated Trp residues have been thought to be important to determine its pharmacological specificity (17England L.J. Imperial J. Jacobsen R. Craig A.G. Gulyas J. Akhtar M. Rivier J. Julius D. Olivera B.M. Science. 1998; 281: 575-578Crossref PubMed Scopus (115) Google Scholar). Although the physiological significance of bromination in NPB is still unclear, this modification may affect in vivo activities. Since NPW has shown in vivo activities increasing prolactin secretion and food intake (13Shimomura, Y., Harada, M., Goto, M., Sugo, T., Matsumoto, Y., Abe, M., Watanabe, T., Asami, T., Kitada, C., Mori, M., Onda, H., and Fujino, M. (July 18, 2002) J. Biol. Chem.10.1074/jbc.M205337200Google Scholar), studies to examine the in vivo effects of NPB are in progress. GPR7 has been detected even in the rat and mouse brain, but GPR8 is reportedly absent in rodents (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, 2Lee D.K. Nguyen T. Porter C.A. Cheng R. George S.R. O'Dowd B.F. Mol. Brain Res. 1999; 71: 96-103Crossref PubMed Scopus (101) Google Scholar). Our results demonstrate that NPB and GPR7 are widely distributed in various tissues in rats. Since both NPB and GPR7 mRNAs were detected in the central nervous system and uterus in rats, they might have some important functions in these tissues. In the hypothalamus, NPB mRNA was detected in the ventromedial hypothalamic nucleus and lateral hypothalamic area. GPR7 mRNA is reportedly expressed in the supraoptic nucleus and paraventricular hypothalamic nucleus (2Lee D.K. Nguyen T. Porter C.A. Cheng R. George S.R. O'Dowd B.F. Mol. Brain Res. 1999; 71: 96-103Crossref PubMed Scopus (101) Google Scholar). In addition, expressions of both NPB and GPR7 mRNAs were detected in the hippocampus. The distribution of NPB and GPR7 mRNAs in the rat brain suggests that NPB is involved in the regulation of feeding, the neuroendocrine system, memory, and learning, although these points should be examined in future studies. We believe that studies on NPB will provide new insight into not only modification in neuropeptides but also the regulatory mechanisms of brain functions. We thank Drs. Y. Sumino and H. Matsumoto for helpful discussions. We also thank Celera Genomics for the databases.
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