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Identification of a Novel Neuromedin U Receptor Subtype Expressed in the Central Nervous System

2000; Elsevier BV; Volume: 275; Issue: 50 Linguagem: Inglês

10.1074/jbc.c000522200

1083-351X

Li-Xin Shan, Xudong Qiao, James H. Crona, Jiang Behan, Suke Wang, Thomas M. Laz, Marvin Bayne, Eric L. Gustafson, Frederick J. Monsma, Joseph A. Hedrick,

Cardiovascular, Neuropeptides, and Oxidative Stress Research

Neuromedin U is a neuropeptide prominently expressed in the upper gastrointestinal tract and central nervous system. Recently, GPR66/FM-3 (NmU-R1) was identified as a specific receptor for neuromedin U. A BLAST search of the GenBankTM genomic database using the NmU-R1 cDNA sequence revealed a human genomic fragment encoding a G protein-coupled receptor that we designated NmU-R2 based on its homology to NmU-R1. The full-length NmU-R2 cDNA was subsequently cloned, stably expressed in 293 cells, and shown to mobilize intracellular calcium in response to neuromedin U. This response was dose-dependent (EC50 = 5 nm) and specific in that other neuromedins did not induce a calcium flux in receptor-transfected cells. Expression analysis of human NmU-R2 demonstrated its mRNA to be most highly expressed in central nervous system tissues. Based on these data, we conclude that NmU-R2 is a novel neuromedin U receptor subtype that is likely to mediate central nervous system-specific neuromedin U effects. Neuromedin U is a neuropeptide prominently expressed in the upper gastrointestinal tract and central nervous system. Recently, GPR66/FM-3 (NmU-R1) was identified as a specific receptor for neuromedin U. A BLAST search of the GenBankTM genomic database using the NmU-R1 cDNA sequence revealed a human genomic fragment encoding a G protein-coupled receptor that we designated NmU-R2 based on its homology to NmU-R1. The full-length NmU-R2 cDNA was subsequently cloned, stably expressed in 293 cells, and shown to mobilize intracellular calcium in response to neuromedin U. This response was dose-dependent (EC50 = 5 nm) and specific in that other neuromedins did not induce a calcium flux in receptor-transfected cells. Expression analysis of human NmU-R2 demonstrated its mRNA to be most highly expressed in central nervous system tissues. Based on these data, we conclude that NmU-R2 is a novel neuromedin U receptor subtype that is likely to mediate central nervous system-specific neuromedin U effects. neuromedin transmembrane G protein-coupled receptor adrenocorticotropic hormone polymerase chain reaction expressed sequence tag The neuromedins (Nm)1are a group of smooth muscle-stimulating peptides commonly divided into four groups: bombesin-like (NmB, NmC), kassinin-like (NmL and -K or neurokinins A and B, respectively), neurotensin-like (NmN), and neuromedin U (NmU). Among this group of peptides, neuromedin U has been the least well understood, in large part due to the lack of a known receptor. Neuromedin U was first reported in 1985 by Minamino et al. (1Minamino N. Kangawa K. Matsuo H. Biochem. Cell Biol. Commun. 1985; 130: 1078-1085Google Scholar, 2Minamino N. Sudoh T. Kangawa K. Matsuo H. Peptides. 1985; 6 (Suppl. 3): 245-248Crossref PubMed Scopus (85) Google Scholar) as a peptide isolated from porcine spinal cord. These investigators isolated two active peptides, NmU-25 and an additional cleavage product, NmU-8, and characterized them as having smooth muscle contractile activity. Neuromedin U was subsequently isolated from a variety of species including rat (3Conlon J.M. Domin J. Thim L. DiMarzo V. Morris H.R. Bloom S.R. J. Neurochem. 1988; 51: 988-991Crossref PubMed Scopus (60) Google Scholar, 4Minamino N. Kangawa K. Honzawa M. Matsuo H. Biochem. Cell Biol. Commun. 1988; 156: 355-360Google Scholar), guinea pig (5Murphy R. Turner C.A. Furness J.B. Parker L. Giraud A. Peptides. 1990; 11: 613-617Crossref PubMed Scopus (44) Google Scholar), dog (6O'Harte F. Bockman C.S. Abel P.W. Conlon J.M. Peptides. 1991; 12: 11-15Crossref PubMed Scopus (62) Google Scholar), rabbit (7Kage R. O'Harte F. Thim L. Conlon J.M. Regul. Pept. 1991; 33: 191-198Crossref PubMed Scopus (44) Google Scholar), chicken (7Kage R. O'Harte F. Thim L. Conlon J.M. Regul. Pept. 1991; 33: 191-198Crossref PubMed Scopus (44) Google Scholar, 8Domin J. Benito-Orfila M.A. Nandha K.A. Aitken A. Bloom S.R. Regul. Pept. 1992; 41: 1-8Crossref PubMed Scopus (37) Google Scholar), and frog (9Salmon A.L. Johnsen A.H. Bienert M. McMurray G. Nandha K.A. Bloom S.R. Shaw C. J. Biol. Chem. 2000; 275: 4549-4554Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar). The cDNAs for rat and human NmU have been cloned, and analysis of the nucleic acid sequence suggests that NmU is produced as a 174-amino acid precursor (10Lo G. Legon S. Austin C. Wallis S. Wang Z. Bloom S.R. Mol. Endocrinol. 1992; 6: 1538-1544PubMed Google Scholar, 11Austin C. Lo G. Nandha K.A. Meleagros L. Bloom S.R. J. Mol. Endocrinol. 1995; 14: 157-169Crossref PubMed Scopus (74) Google Scholar). The precursor contains a signal peptide and several dibasic cleavage sites that give rise to a number of possible secreted peptides, including NmU, which is present near the carboxyl terminus. Neuromedin U shows remarkable conservation throughout evolution, and a core active peptide (Phe-Leu-Phe-Arg-Pro-Arg-Asn-NH2) is absolutely conserved among mammalian species. A variety of biological activities have been reported for NmU although its role in normal physiology is unclear. The first biological activity ascribed to NmU was smooth muscle contraction (1Minamino N. Kangawa K. Matsuo H. Biochem. Cell Biol. Commun. 1985; 130: 1078-1085Google Scholar, 2Minamino N. Sudoh T. Kangawa K. Matsuo H. Peptides. 1985; 6 (Suppl. 3): 245-248Crossref PubMed Scopus (85) Google Scholar). These experiments have not been consistent among different species, however, in regard to the specific tissues that respond to NmU (1Minamino N. Kangawa K. Matsuo H. Biochem. Cell Biol. Commun. 1985; 130: 1078-1085Google Scholar, 12Bockman C.S. Abel P.W. Hicks J.W. Conlon J.M. Eur. J. Pharmacol. 1989; 171: 255-257Crossref PubMed Scopus (20) Google Scholar, 13Maggi C.A. Patacchini R. Giuliani S. Turini D. Barbanti G. Rovero P. Meli A. Br. J. Pharmacol. 1990; 99: 186-188Crossref PubMed Scopus (40) Google Scholar, 14Benito-Orfila M.A. Domin J. Nandha K.A. Bloom S.R. Eur. J. Pharmacol. 1991; 193: 329-333Crossref PubMed Scopus (28) Google Scholar, 15Brown D.R. Quito F.L. Eur. J. Pharmacol. 1988; 155: 159-162Crossref PubMed Scopus (68) Google Scholar). Neuromedin U has also been reported to increase arterial blood pressure (16Gardiner S.M. Compton A.M. Bennett T. Domin J. Bloom S.R. Am. J. Physiol. 1990; 258: R32-R38PubMed Google Scholar, 17Sumi S. Inoue K. Kogire M. Doi R. Takaori K. Suzuki T. Yajima H. Tobe T. Life Sci. 1987; 41: 1585-1590Crossref PubMed Scopus (64) Google Scholar) and modify ion transport in the intestinal tract (15Brown D.R. Quito F.L. Eur. J. Pharmacol. 1988; 155: 159-162Crossref PubMed Scopus (68) Google Scholar). Finally, NmU injected subcutaneously into rats has been reported to result in a short term increase in circulating ACTH levels and a long term increase in serum corticosterone levels (18Malendowicz L.K. Nussdorfer G.G. Nowak K.W. Mazzocchi G. In Vivo. 1993; 7: 419-422PubMed Google Scholar, 19Malendowicz L.K. Nussdorfer G.G. Markowska A. Tortorella C. Nowak M. Warchol J.B. Neuropeptides. 1994; 26: 47-53Crossref PubMed Scopus (43) Google Scholar), suggesting a role in regulation of the hypothalamo-pituitary-adrenal axis. Recently, several groups, including our own, have reported that GPR66/FM-3 is a specific receptor for NmU (20Fujii 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 (192) Google Scholar, 21Szekeres P.G. Muir A.I. Spinage L.D. Miller J.E. Butler S.I. Smith A. Rennie G.I. Murdock P.R. Fitzgerald L.R. Wu H. McMillan L.J. Guerrera S. Vawter L. Elshourbagy N.A. Mooney J.L. Bergsma D.J. Wilson S. Chambers J.K. J. Biol. Chem. 2000; 275: 20247-20250Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar, 22Hedrick J.A. Morse K. Shan L. Qiao X. Pang L. Wang S. Laz T. Gustafson E. Bayne M. Monsma F.J. J..Mol. Pharmacol. 2000; 58: 870-875Crossref PubMed Scopus (98) Google Scholar, 23Howard A.D. Wang R. Pong S.S. Mellin T.N. Strack A. Guan X.M. Zeng Z. Williams Jr., D.L. Feighner S.D. Nunes C.N. Murphy B. Stair J.N., Yu, H. Jiang Q. Clements M.K. Tan C.P. McKee K.K. Hreniuk D.L. McDonald T.P. Lynch K.R. Evans J.F. Austin C.P. Caskey C.T. Van der Ploeg L.H. Liu Q. Nature. 2000; 406: 70-74Crossref PubMed Scopus (368) Google Scholar). Originally identified as a partial mouse expressed sequence tag residing in GenBankTM, full-length mouse GPR66/FM-3 was cloned from a T cell library and subsequently used as a probe to identify a human clone (24Tan C.P. McKee K.K. Liu Q. Palyha O.C. Feighner S.D. Hreniuk D.L. Smith R.G. Howard A.D. Genomics. 1998; 52: 223-229Crossref PubMed Scopus (58) Google Scholar). Comparison of GPR66/FM-3 to other known G protein-coupled receptors (GPCRs) shows it is most similar to the human growth hormone secretagogue and neurotensin receptors (33 and 29% amino acid identity, respectively) as well as the recently described motilin receptor (25Feighner S.D. Tan C.P. McKee K.K. Palyha O.C. Hreniuk D.L. Pong S.S. Austin C.P. Figueroa D. MacNeil D. Cascieri M.A. Nargund R. Bakshi R. Abramovitz M. Stocco R. Kargman S. O'Neill G. Van Der Ploeg L.H. Evans J. Patchett A.A. Smith R.G. Howard A.D. Science. 1999; 284: 2184-2188Crossref PubMed Scopus (356) Google Scholar). This receptor (NmU-R1) is expressed in peripheral tissues, particularly in the upper gastrointestinal tract and lymphoid tissues (20Fujii 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 (192) Google Scholar, 21Szekeres P.G. Muir A.I. Spinage L.D. Miller J.E. Butler S.I. Smith A. Rennie G.I. Murdock P.R. Fitzgerald L.R. Wu H. McMillan L.J. Guerrera S. Vawter L. Elshourbagy N.A. Mooney J.L. Bergsma D.J. Wilson S. Chambers J.K. J. Biol. Chem. 2000; 275: 20247-20250Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar, 22Hedrick J.A. Morse K. Shan L. Qiao X. Pang L. Wang S. Laz T. Gustafson E. Bayne M. Monsma F.J. J..Mol. Pharmacol. 2000; 58: 870-875Crossref PubMed Scopus (98) Google Scholar, 23Howard A.D. Wang R. Pong S.S. Mellin T.N. Strack A. Guan X.M. Zeng Z. Williams Jr., D.L. Feighner S.D. Nunes C.N. Murphy B. Stair J.N., Yu, H. Jiang Q. Clements M.K. Tan C.P. McKee K.K. Hreniuk D.L. McDonald T.P. Lynch K.R. Evans J.F. Austin C.P. Caskey C.T. Van der Ploeg L.H. Liu Q. Nature. 2000; 406: 70-74Crossref PubMed Scopus (368) Google Scholar, 24Tan C.P. McKee K.K. Liu Q. Palyha O.C. Feighner S.D. Hreniuk D.L. Smith R.G. Howard A.D. Genomics. 1998; 52: 223-229Crossref PubMed Scopus (58) Google Scholar), but is essentially absent from central nervous system tissues. In the present study we report the identification and characterization of a second specific NmU receptor that is predominantly expressed in the central nervous system. A genomic fragment encoding NmU-R2 (GenBankTM accession number AC008571) was identified using the deduced amino acid sequence of NmU-R1 as "bait" for a TBLASTN search of the GenBankTM high throughput genomic database. A putative NmU-R2 open reading frame was assembled using Sequencher (Genecodes, Ann Arbor, MI). The coding region of NmU-R2 was subsequently amplified by polymerase chain reaction (PCR) employing a specific 5′ forward primer containing a consensus Kozak sequence (underlined): 5′-GCCGCCACCATGTCAGGG ATGGAA AAACTTCAGAAT-3′, a specific 3′ reverse primer: 5′-AAGAATTCAGGTTTTGTTAAAGTGGAAGCTTT-3′, and human testis Marathon-Ready cDNA as a template (CLONTECH, Palo Alto, CA). The thermal cycling profile for PCR was 95 °C, 30 s; 63 °C, 30 s; and 68 °C, 2 min (35 cycles). The resulting PCR product was cloned into pCR3.1 (Invitrogen, Carlsbad, CA), and the insert was sequenced. Expression of NmU-R2 in 293 cells (American Type Culture Collection, CRL1573) was accomplished using LipofectAMINE 2000 (Life Technologies, Inc.) according to the manufacturer's protocol. Cells stably expressing NmU-R2 were selected in Dulbecco's modified Eagle's medium, 10% fetal calf serum containing G418 at 1.0 mg/ml and subsequently maintained in Dulbecco's modified Eagle's medium, 10% fetal calf serum containing G418 at 0.5 mg/ml. Ligand screening was accomplished using the fluorometric imaging plate reader (FLIPR, Molecular Devices, Sunnyvale, CA) (26Sullivan E. Tucker E.M. Dale I.L. Methods Mol. Biol. 1999; 114: 125-133PubMed Google Scholar, 27Coward P. Chan S.D. Wada H.G. Humphries G.M. Conklin B.R. Anal. Biochem. 1999; 270: 242-248Crossref PubMed Scopus (206) Google Scholar). Briefly, 48 h before screening, 293 cells were transfected with NmU-R2 as described above. The cells were then replated 24 h before screening into clear bottom, black-walled 96-well plates precoated with poly-d-lysine (Becton-Dickinson, Franklin Lakes, NJ) at a density of 5 × 105 cells/well. On the day of screening cells were loaded for 1 h with Fluo-3AM (Sigma) according to the FLIPR manufacturer's protocol. The peptide libraries that were used in the large scale screening of NmU-R2 included more than 500 peptides that are known or suspected GPCR ligands. These were obtained from various commercial sources (RBI, Natick, MA; Bachem, King of Prussia, PA; Sigma) or were custom-synthesized (Research Genetics, Huntsville, AL). In particular, rat NmU-23, pig NmU-25, pig NmU-8, and human neuromedins B, C, L, K, and N were obtained from Bachem (King of Prussia, PA) whereas high performance liquid chromatography-purified human NmU-25 and nonamidated NmU-8 were custom-synthesized (Research Genetics). A commercial small molecule library (RBI) was also screened. This library covers various pharmacological classes including adenosines, purinergics, adrenergics, histaminergics, cholinergics, ion channel modulators, dopaminergics, glutaminergics, opioids, serotonergics, and γ-aminobutyric acid compounds (LOPAC Library, exact content available from manufacturer). 293 cells stably expressing NmU-R2 plated onto poly-d-lysine-coated coverslips that subsequently formed the base of a perfusion chamber. Cultures were loaded with fura-2 acetoxymethyl ester (5 μm, 45 min, 37 °C, Molecular Probes, Eugene, OR) in a buffered salt solution (149 mm NaCl, 3.25 mmKCl, 2.0 mm CaCl2, 2.0 mmMgCl2, 10 mm HEPES, 11 mm glucose). The perfusion chamber was continuously perfused with the buffered salt solution (3 ml/min), and drug additions were made using a pinch valve arrangement with minimal dead space. Intracellular Ca2+concentration was estimated using an Attofluor RatioVision digital fluorescence imaging system (Atto Instruments, Rockville, MD). Fura-2 was excited, alternately, at 334 and 380 nm, and the emission was collected at 510 nm and 1.25-s intervals. Calibration of the 334/380 nm fura-2 signal was performed in vitro with fura-2 pentapotassium salt in the presence of 1 mmCa2+ or 1 mm EGTA, and the 334/380 nm excitation ratio was converted to Ca2+ concentration values using the procedure of Grynkiewicz et al. (28Grynkiewicz G. Poenie M. Tsien R.Y. J. Biol. Chem. 1985; 260: 3440-3450Abstract Full Text PDF PubMed Scopus (80) Google Scholar). Expression of NmUR-2 was examined using dot blots and Northern blots obtained from a commercial source (CLONTECH). Hybridization to blots was carried out using PCR-generated DNA fragments encompassing 1200 base pairs of the coding region of NmUR-2 beginning at the 3′-end and including most of the coding region. The DNA fragments were random-prime labeled with [32P]dCTP, and the blots were hybridized for 14 h in ExpressHyb (CLONTECH) containing 2 × 106 cpm/ml of radiolabeled probe. The following day the blots were washed and exposed to Kodak Biomax MS film for 3 days at −70 °C. The dot-blot films were analyzed for NmU-R2 expression levels using the MCID M4 image analysis system (Imaging Research, Ontario, Canada), and the data were displayed as absolute optical density. A peripheral NmU receptor (NmU-R1) has been recently identified that is expressed in peripheral tissues (20Fujii 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 (192) Google Scholar, 21Szekeres P.G. Muir A.I. Spinage L.D. Miller J.E. Butler S.I. Smith A. Rennie G.I. Murdock P.R. Fitzgerald L.R. Wu H. McMillan L.J. Guerrera S. Vawter L. Elshourbagy N.A. Mooney J.L. Bergsma D.J. Wilson S. Chambers J.K. J. Biol. Chem. 2000; 275: 20247-20250Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar, 22Hedrick J.A. Morse K. Shan L. Qiao X. Pang L. Wang S. Laz T. Gustafson E. Bayne M. Monsma F.J. J..Mol. Pharmacol. 2000; 58: 870-875Crossref PubMed Scopus (98) Google Scholar). To identify potential subtypes of this receptor we performed a TBLASTN search of the high throughput genomic database subset of GenBankTM using the deduced amino acid sequence of NmU-R1 as bait. One such search identified a genomic fragment from chromosome 5 (GenBankTMAC008571) containing a region of relatively high homology (∼57%) to NmU-R1. Although the genomic sequence was unordered, a predicted open reading frame was assembled based on sequence homology to NmU-R1. PCR primers were designed based upon this predicted open reading frame, and a cDNA was subsequently obtained that was designated NmU-R2 based on its homology to NmU-R1 (Fig. 1 A). The genomic structure of NmU-R2 differs significantly from that of NmU-R1 in that the predicted open reading frame is encoded on four exons instead of the two found in the NmU-R1 gene (Fig. 1). Interestingly, the intron 2-exon 3 boundary of NmU-R2 coincides with the intron 1-exon 2 boundary of NmU-R1, and the overall homology between the open reading frames is far higher upstream of this point (TM1–TM6) than downstream. The conservation of one of the intron/exon boundaries and the relatively high homology of the two genes suggests that these two receptors arose from a duplicative event sometime in the past. The position of exon 2 of NmU-R2 is such that it encompasses only the third intracellular loop and the very beginning of transmembrane domain six. The length of this loop is also shortened in NmU-R2 as compared with NmU-R1 (Fig. 1 A). Finally, both the amino- and carboxyl-terminal sequences show considerable divergence with NmU-R2 having extended termini. In addition to the NmU-R2 genomic fragment, we also identified several expressed sequence tag (EST) sequences in the GenBankTMdatabase that proved to have identity with NmU-R2. Four of five of these ESTs originated from brain-related cDNA libraries with the fifth coming from a heart library. Three of these ESTs appear to represent an alternative NmU-R2 transcript. This transcript includes exons 2 and 3 of the NmU-R2 gene but do not splice upstream to exon 1. Instead, two novel exons are spliced in, creating a presumably sterile transcript with multiple stops in all reading frames (Fig. 1 B). Although the "aberrant" transcripts are all from fetal brain libraries, it is possible that such transcripts might exist elsewhere and suggests caution in interpreting the results of expression studies. The downstream splicing of exon 3 to exon 4 in these alternate transcripts could not be assessed from the sequence data present in GenBankTM. The expression data presented in this study were confirmed with probes that do not hybridize to the alternate NmU-R2 transcript (data not shown). To gain some insight into the possible physiological role of a second NmU receptor, the expression of this receptor was comprehensively assessed using dot blots and Northern blots of human tissues. The expression of NmU-R2 on dot blots was highest in testis and central nervous system tissues, particularly spinal cord (Fig. 2 A). This is in contrast to NmU-R1, which showed very little expression in central nervous system tissues (20Fujii 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 (192) Google Scholar, 21Szekeres P.G. Muir A.I. Spinage L.D. Miller J.E. Butler S.I. Smith A. Rennie G.I. Murdock P.R. Fitzgerald L.R. Wu H. McMillan L.J. Guerrera S. Vawter L. Elshourbagy N.A. Mooney J.L. Bergsma D.J. Wilson S. Chambers J.K. J. Biol. Chem. 2000; 275: 20247-20250Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar, 22Hedrick J.A. Morse K. Shan L. Qiao X. Pang L. Wang S. Laz T. Gustafson E. Bayne M. Monsma F.J. J..Mol. Pharmacol. 2000; 58: 870-875Crossref PubMed Scopus (98) Google Scholar). Low levels of NmU-R2 expression were detected by dot blot in stomach and duodenum (Fig. 2 A), but unlike NmU-R1, NmU-R2 expression was absent or very low in other gastrointestinal tract tissues. Similarly, expression of NmU-R2 in lymphoid tissues was either very low or undetectable (Fig. 2 A). Aside from the expression in central nervous system tissues and gastrointestinal tract, expression of NmU-R2 mRNA was also observed in kidney, lung, and thyroid (Fig. 2 A). No expression of NmU-R2 was detected in uterus, despite the fact that NmU binding has been reported in rat uterus (29Nandha K.A. Benito-Orfila M.A. Smith D.M. Bloom S.R. Endocrinology. 1993; 133: 482-486Crossref PubMed Scopus (26) Google Scholar). This may reflect a species-specific difference as discussed in the introduction. Alternatively, the expression of NmU receptor was reported to be estrogen-dependent (30Nandha K.A. Benito-Orfila M.A. Jamal H. Akinsanya K.O. Bloom S.R. Smith D.M. Peptides. 1999; 20: 1203-1209Crossref PubMed Scopus (12) Google Scholar) and may vary depending on when tissue was obtained. In any case, this is clearly an area where further investigation is warranted. The expression of NmU-R2 in the central nervous system and gastrointestinal tract was examined in more detail by Northern blot analysis. Spinal cord and corpus callosum demonstrated the highest expression with a NmU-R2 message of 2.4 kilobases (Fig. 2 B). In contrast, expression of NmU-R2 in the gastrointestinal tract as detected by Northern blot was very low and could not be detected even upon very long exposure (1 week, data not shown). Despite the inability to detect NmU-R2 in the Northern blot, the dot-blot results suggest that NmU-R2 may have some overlapping expression with NmU-R1 in the gastrointestinal tract. Thus, interpretation of the physiological effects of NmU in this tissue will need to be carefully considered in regard to which receptor is mediating a given event. The identification of NmU-R2 as a neuromedin receptor was accomplished in human embryonic kidney cells (293) that were transiently transfected with NmU-R2 cDNA. The NmU-R2 transfected cells were assessed for their ability to mobilize intracellular calcium when stimulated with each of more than 1000 known or suspected GPCR ligands including small molecules and various peptides, among them neuromedins B, C, K, L, N, and U (human NmU-25, rat NmU-23, and pig NmU-8). From among this library of potential ligands, only the NmUs generated a specific, dose-dependent calcium flux in the transfected cells (Fig. 3 A). This response was dose-dependent with an EC50 of 5 nmand a maximal response observed between 80 and 800 nm (Fig. 3 B). Maximal intracellular calcium concentration reached 600–800 nm when cells were stimulated with 100 nm NmU-25 (Fig. 3 C). We observed no significant difference in the ability of NmU-25/23 to stimulate NmU-R2 when compared with NmU-8 (data not shown). We did find amidation of NmU-8 to be necessary for activity as a nonamidated form did not activate the receptor even at concentrations in excess of 10 μm (data not shown). In addition, we found that pertussis toxin (100 ng/ml, overnight incubation) did not significantly alter NmU-R2 receptor signaling, suggesting that in 293 cells this receptor couples to calcium through the Gq subset of G proteins (Fig. 3 A, inset). Recently, a number of other investigators have also reported characterization of NmU-R2 (23Howard A.D. Wang R. Pong S.S. Mellin T.N. Strack A. Guan X.M. Zeng Z. Williams Jr., D.L. Feighner S.D. Nunes C.N. Murphy B. Stair J.N., Yu, H. Jiang Q. Clements M.K. Tan C.P. McKee K.K. Hreniuk D.L. McDonald T.P. Lynch K.R. Evans J.F. Austin C.P. Caskey C.T. Van der Ploeg L.H. Liu Q. Nature. 2000; 406: 70-74Crossref PubMed Scopus (368) Google Scholar, 31Hosoya 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 (110) Google Scholar, 32Raddatz R. Wilson A.E. Artymyshyn R. Bonini J.A. Borowsky B. Boteju L.W. Zhou S. Kouranova E.V. Nagorny R. Guevarra M.S. Dai M. Lerman G.S. Vaysse P.J. Branchek T.A. Gerald C. Forray C. Adham N. J. Biol. Chem. 2000; 275: 32452-32459Abstract Full Text Full Text PDF PubMed Scopus (177) Google Scholar). All of these manuscripts report an NmU EC50 of 1–5 nm in functional assays, which is consistent with our findings. These reports agree also that coupling appears to be primarily through Gq although Hosoya et al. (31Hosoya 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 (110) Google Scholar) reported some Gi coupling. The reports vary, however, in their findings regarding distribution of NmU-R2 mRNA. Hosoya et al. (31Hosoya 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 (110) Google Scholar) reported only rat expression and provided no data regarding the expression pattern of human NmU-R2. Furthermore, these investigators performed their analysis using only quantitative PCR and provided no other confirmation of their results. Similarly, Raddatz et al. (32Raddatz R. Wilson A.E. Artymyshyn R. Bonini J.A. Borowsky B. Boteju L.W. Zhou S. Kouranova E.V. Nagorny R. Guevarra M.S. Dai M. Lerman G.S. Vaysse P.J. Branchek T.A. Gerald C. Forray C. Adham N. J. Biol. Chem. 2000; 275: 32452-32459Abstract Full Text Full Text PDF PubMed Scopus (177) Google Scholar), while providing human expression data, relied solely on quantitative PCR for expression analysis. Howard et al. (23Howard A.D. Wang R. Pong S.S. Mellin T.N. Strack A. Guan X.M. Zeng Z. Williams Jr., D.L. Feighner S.D. Nunes C.N. Murphy B. Stair J.N., Yu, H. Jiang Q. Clements M.K. Tan C.P. McKee K.K. Hreniuk D.L. McDonald T.P. Lynch K.R. Evans J.F. Austin C.P. Caskey C.T. Van der Ploeg L.H. Liu Q. Nature. 2000; 406: 70-74Crossref PubMed Scopus (368) Google Scholar) discussed expression of human NmU-R2 but presented no data, reporting only that NmU-R2 expression was weakly observed in some tissues. Hosoya et al. (31Hosoya 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 (110) Google Scholar) found rat NmU-R2 most prominently expressed in uterus (31Hosoya 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 (110) Google Scholar); however, neither Raddatz et al. (32Raddatz R. Wilson A.E. Artymyshyn R. Bonini J.A. Borowsky B. Boteju L.W. Zhou S. Kouranova E.V. Nagorny R. Guevarra M.S. Dai M. Lerman G.S. Vaysse P.J. Branchek T.A. Gerald C. Forray C. Adham N. J. Biol. Chem. 2000; 275: 32452-32459Abstract Full Text Full Text PDF PubMed Scopus (177) Google Scholar) nor our group found any significant expression of human NmU-R2 in uterus (Fig. 2). In contrast, expression of NmU-R2 was very high in human testis (Fig. 2) (32Raddatz R. Wilson A.E. Artymyshyn R. Bonini J.A. Borowsky B. Boteju L.W. Zhou S. Kouranova E.V. Nagorny R. Guevarra M.S. Dai M. Lerman G.S. Vaysse P.J. Branchek T.A. Gerald C. Forray C. Adham N. J. Biol. Chem. 2000; 275: 32452-32459Abstract Full Text Full Text PDF PubMed Scopus (177) Google Scholar) but low in rat testis (31Hosoya 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 (110) Google Scholar). The recent reports on NmU-R2 do agree that this receptor is most prominently expressed in the brain of both human and rat; however, the exact nature of the message detected and whether it is translated remains unknown. This question becomes more important in light of our finding of alternative transcripts for NmU-R2 (Fig. 1 B). In particular, Raddatz et al. (32Raddatz R. Wilson A.E. Artymyshyn R. Bonini J.A. Borowsky B. Boteju L.W. Zhou S. Kouranova E.V. Nagorny R. Guevarra M.S. Dai M. Lerman G.S. Vaysse P.J. Branchek T.A. Gerald C. Forray C. Adham N. J. Biol. Chem. 2000; 275: 32452-32459Abstract Full Text Full Text PDF PubMed Scopus (177) Google Scholar) reported expression of neuromedin U receptor in dorsal root ganglion; however, we have been unable to demonstrate NmU responsiveness in either mouse or rat dorsal root ganglion, even at doses as high as 1 μm, although this could be species-related (data not shown). 2J. Crona, personal communication. Howard et al. (23Howard A.D. Wang R. Pong S.S. Mellin T.N. Strack A. Guan X.M. Zeng Z. Williams Jr., D.L. Feighner S.D. Nunes C.N. Murphy B. Stair J.N., Yu, H. Jiang Q. Clements M.K. Tan C.P. McKee K.K. Hreniuk D.L. McDonald T.P. Lynch K.R. Evans J.F. Austin C.P. Caskey C.T. Van der Ploeg L.H. Liu Q. Nature. 2000; 406: 70-74Crossref PubMed Scopus (368) Google Scholar) have shown that intracerebral injection of NmU altered feeding behavior in rats but did not demonstrate dose responsiveness of this effect, only that 1 μg of NmU was not sufficient to alter feeding behaviors, while 3 or 10 μg produced similar effects. These studies also failed to demonstrate that the effect observed was mediated by central nervous system receptors because no similar studies were reported using peripheral administration of NmU. Given that NmU-R1 is highly expressed in gastrointestinal tract tissue and that nothing is known about the ability of NmU to cross the blood-brain barrier, it is not unreasonable to assume that some of the observed effects upon feeding behavior might be mediated directly or indirectly via peripheral receptors. Finally, given the history of species-specific effects of NmU on muscle contraction (see introduction) and differences in the species-specific expression of the NmU receptors, it will be important to develop additional animal models for NmU function and to confirm any findings in human cells or tissues whenever possible. In summary, we have demonstrated the existence of a second specific neuromedin U receptor that we have designated NmU-R2. Cells transfected with NmU-R2 show a dose-dependent intracellular Ca2+ mobilization in response to NmU stimulation. We also show that NmU-R2 calcium signaling in 293 cells is pertussis toxin insensitive, suggesting that NmU-R2, like NmU-R1, is coupled to the Gq family of G proteins in these cells. Unlike NmU-R1, NmU-R2 mRNA is highly expressed in the central nervous system and suggests that NmU-R2 mediates the effects of NmU in these tissues. However, given that NmU-R1 and NmU-R2 expression overlap in some peripheral tissues and that NmU itself is broadly expressed it will be necessary to conduct further studies to understand the precise role each receptor plays in mediating the peripheral and central effects of neuromedin U. Targeted gene knockouts of each of the NmU receptors, as well as of NmU itself, would undoubtedly be invaluable in such studies.