Identification and Characterization of Two Neuromedin U Receptors Differentially Expressed in Peripheral Tissues and the Central Nervous System

Abstract

Two structurally related, G-protein-coupled receptors were identified as receptors for the neuropeptide, neuromedin U. This peptide is found in highest levels in the gut and genitourinary system where it potently contracts smooth muscle but is also expressed in the spinal cord and discrete regions of the brain. Binding sites for neuromedin U have been characterized in rat uterus, however, little is known about the activity of this peptide in the regions of the central nervous system where it is expressed. The receptors characterized in this report are activated by neuromedin U at nanomolar potency in heterologous expression systems and bind radiolabeled neuromedin U with high affinity. Localization of the receptor RNA by quantitative reverse transcription-polymerase chain reaction in a variety of human tissues shows distinct expression patterns for the two receptors. NMU1 is expressed predominantly in peripheral tissues, whereas NMU2 is more highly expressed in the central nervous system. Identification of neuromedin U receptor subtypes will greatly aid in the determination of the physiological roles of this peptide. Two structurally related, G-protein-coupled receptors were identified as receptors for the neuropeptide, neuromedin U. This peptide is found in highest levels in the gut and genitourinary system where it potently contracts smooth muscle but is also expressed in the spinal cord and discrete regions of the brain. Binding sites for neuromedin U have been characterized in rat uterus, however, little is known about the activity of this peptide in the regions of the central nervous system where it is expressed. The receptors characterized in this report are activated by neuromedin U at nanomolar potency in heterologous expression systems and bind radiolabeled neuromedin U with high affinity. Localization of the receptor RNA by quantitative reverse transcription-polymerase chain reaction in a variety of human tissues shows distinct expression patterns for the two receptors. NMU1 is expressed predominantly in peripheral tissues, whereas NMU2 is more highly expressed in the central nervous system. Identification of neuromedin U receptor subtypes will greatly aid in the determination of the physiological roles of this peptide. neuromedin U G-protein-coupled receptor rapid amplification of cDNA ends central nervous system inositol phosphate adrenocorticotropic hormone base pair(s) polymerase chain reaction 6-carboxy-4,7,2,7′-tetramethylrhodamine Stanford Human Genome Center guanosine 5′-3-O-(thio)triphosphate A number of neuropeptides are known to produce distinct effects within the nervous system as well as on peripheral targets. More recently, the identification and cloning of neuropeptide receptors has accelerated progress toward determining their physiological roles. Along with several other biologically active peptides, neuromedin U (NMU)1 was first isolated from porcine spinal cord (1Minamino N. Sudoh T. Kangawa K. Matsuo H. Peptides ( Elmsford ). 1985; 6: 245-248Crossref PubMed Scopus (85) Google Scholar, 2Minamino N. Kangawa K. Matsuo H. Biochem. Biophys. Res. Commun. 1985; 130: 1078-1085Crossref PubMed Scopus (263) Google Scholar) and was named for its uterine contractile activity. Indeed, receptors for this peptide and related analogues were first suggested by binding of 125I-labeled rat NMU in rat uterus membranes (3Nandha K.A. Benito-Orfila M.A. Smith D.M. Bloom S.R. Endocrinology. 1993; 133: 482-486Crossref PubMed Scopus (26) Google Scholar). Furthermore, the GTP analogue, GTPγS, inhibited binding of 125I-labeled rat NMU in this tissue suggesting that the binding site is a G-protein-coupled receptor (GPCR). Although NMU is present in various regions of the brain and spinal cord, binding sites for the peptide in the CNS have not been well characterized. Two forms of the peptide (porcine NMU and porcine NMU18–25) 2Porcine NMU18–25 has historically been referred to as NMU-8. were identified and their amino acid sequences were shown to be distinct from other known peptide families (Fig.1). NMU peptides from various species share the greatest homology in their C-terminal regions (Fig. 1). Similarly, the region of rat NMU critical for smooth muscle contractile activity was found to reside in the C terminus of the peptide (between residues 17 and 22), supporting the importance of this region for contractile activity (4Hashimoto T. Masui H. Uchida Y. Sakura N. Okimura K. Chem. Pharm. Bull. (Tokyo). 1991; 39: 2319-2322Crossref PubMed Scopus (29) Google Scholar, 5Sakura N. Ohta S. Uchida N. Kurosawa K. Okimura K. Hashimoto T. Chem. Pharm. Bull. (Tokyo). 1991; 39: 2016-2020Crossref PubMed Scopus (31) Google Scholar). Along with many other neuropeptides, NMU is present in nerves throughout the gastrointestinal tract (6Nandha K.A. Bloom S.R. Biomed. Res. 1993; 14: 71-76Google Scholar), and it stimulates contraction of isolated muscle tissue such as longitudinal muscle of human ileum (7Maggi C.A. Patacchini R. Giuliani S. Turini D. Barbanti G. Rovero P. Meli A. Br. J. Pharmacol. 1990; 99: 186-188Crossref PubMed Scopus (39) Google Scholar) and rat stomach circular muscle (8Benito-Orfila M.A. Domin J. Nandha K.A. Bloom S.R. Eur. J. Pharmacol. 1991; 193: 329-333Crossref PubMed Scopus (27) Google Scholar). Interestingly, porcine jejunum (9Brown D.R. Quito F.L. Eur. J. Pharmacol. 1988; 155: 159-162Crossref PubMed Scopus (67) Google Scholar) and guinea pig small intestine (2Minamino N. Kangawa K. Matsuo H. Biochem. Biophys. Res. Commun. 1985; 130: 1078-1085Crossref PubMed Scopus (263) Google Scholar) are not contracted by NMU, indicating species differences in gut regulation by this peptide. NMU also regulates mesenteric blood flow and ion transport in the intestine (10Sumi 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). NMU-like immunoreactivity in the intestine has been localized to both the submucosal and myenteric ganglion cells consistent with the observed effects on contractility, blood flow, and absorptive/secretory functions (11Ballesta J. Carlei F. Bishop A.E. Steel J.H. Gibson S.J. Fahey M. Hennessey R. Domin J. Bloom S.R. Polak J.M. Neuroscience. 1988; 25: 797-816Crossref PubMed Scopus (109) Google Scholar). NMU has been shown to affect hormonal systems, including a profound effect on the in vivo release of stress-related modulators from the anterior pituitary and adrenal glands (12Malendowicz L.K. Markowska A. Histol. Histopathol. 1994; 9: 591-601PubMed Google Scholar). Corticotrophs within the anterior lobe of rat and human pituitary gland contain high levels of NMU-like immunoreactivity (13Steel J.H. Van Noorden S. Ballesta J. Gibson S.J. Ghatei M.A. Burrin J. Leonhardt U. Domin J. Bloom S.R. Polak J.M. Endocrinology. 1988; 122: 270-282Crossref PubMed Scopus (108) Google Scholar), suggesting a hormonal role for NMU. NMU is present in the same secretory granules as ACTH and galanin (14Cimini V. Van Noorden S. Timson C.M. Polak J.M. Cell Tissue Res. 1993; 272: 137-146Crossref PubMed Scopus (18) Google Scholar). A small population of NMU-positive parafollicular C-cells is also found in rat thyroid gland (15Domin J. Al-Madani A.M. Desperbasques M. Bishop A.E. Polak J.M. Bloom S.R. Cell Tissue Res. 1990; 260: 131-135Crossref PubMed Scopus (19) Google Scholar, 16Lo G. Legon S. Austin C. Wallis S. Wang Z. Bloom S.R. Mol. Endocrinol. 1992; 6: 1538-1544PubMed Google Scholar). High doses of this peptide also produce a hypertensive effect when given intravenously to rats (17Gardiner S.M. Compton A.M. Bennett T. Domin J. Bloom S.R. Am. J. Physiol. 1990; 258: R32-R38PubMed Google Scholar). In addition to its roles in smooth muscle contraction and gut/genitourinary system function, NMU is also present in various brain regions, suggesting as yet unidentified central functions for this peptide (11Ballesta J. Carlei F. Bishop A.E. Steel J.H. Gibson S.J. Fahey M. Hennessey R. Domin J. Bloom S.R. Polak J.M. Neuroscience. 1988; 25: 797-816Crossref PubMed Scopus (109) Google Scholar, 18Honzawa M. Sudoh T. Minamino N. Tohyama M. Matsuo H. Neuroscience. 1987; 23: 1103-1122Crossref PubMed Scopus (50) Google Scholar, 19Domin J. Ghatei M.A. Chohan P. Bloom S.R. Peptides ( Elmsford ). 1987; 8: 779-784Crossref PubMed Scopus (108) Google Scholar). Several structures associated with sensory processing are rich in NMU-containing fibers, including spinal cord (dorsal horn > ventral horn), trigeminal sensory nuclei, vestibular nuclei, and other nuclei associated with descending spinal pathways (18Honzawa M. Sudoh T. Minamino N. Tohyama M. Matsuo H. Neuroscience. 1987; 23: 1103-1122Crossref PubMed Scopus (50) Google Scholar). Neuronal cell bodies containing NMU-like immunoreactivity have been identified in the arcuate nucleus of the hypothalamus, an area identified as important for the regulation of food intake and neuroendocrine control. Relatively high levels of NMU-like immunoreactivity were also detected in the nucleus accumbens and substantia nigra (19Domin J. Ghatei M.A. Chohan P. Bloom S.R. Peptides ( Elmsford ). 1987; 8: 779-784Crossref PubMed Scopus (108) Google Scholar), suggesting a role for NMU in the modulation of dopaminergic actions. NMU-like immunoreactivity is found in the hippocampus, amygdala, and other portions of the limbic system, suggesting a role for NMU and NMU receptors in neuropsychiatric disorders. The identification and characterization of NMU receptors expressed in these CNS regions will greatly aid in understanding the role of this peptide in brain functions. This report describes the identification of two structurally related, G-protein-coupled receptors for NMU with distinct tissue expression patterns. Identification and characterization of these receptors provide the first direct evidence for NMU receptor subtypes. Rat NMU, porcine NMU, and porcine NMU18–25 were purchased from Bachem (Torrance, CA). Human NMU was synthesized by Synaptic Pharmaceutical Corp. (Paramus, NJ) using solid phase peptide synthesis with the Fmoc (N-(9-fluorenyl)methoxycarbonyl) protection protocol, with Rink/amide methylbenzylhydryl amine resin.125I-labeled rat NMU and 125I-labeled porcine NMU18–25 were custom-labeled by NEN Life Science Products Inc. (Boston, MA). The GENEMBL data base SWPLUS was searched for G-protein-coupled receptor sequences (GPCRs) using a select set of known GPCRs and the Wisconsin Package (GCG, Genetics Computer Group, Madison, WI). One sequence, O43664, was selected for use in ligand identification screens. Before cloning the full-length receptor, the 5′- and 3′-ends of the coding sequence were verified by 5′/3′ rapid amplification of cDNA ends (RACE), using human hypothalamic Marathon-Ready cDNA (CLONTECH, Palo Alto, CA), the Marathon adaptor primers AP1 and AP2 (CLONTECH), and the following primers: for 5′ RACE: 5′-CCACGAAGATCAGCAGGTATGTGG-3′ and 5′-GGCATGAACAGCTCTGTCTGCTGG-3′; for 3′ RACE: 5′-CCAGCCGCTTCCGAGAGACCTTCC-3′ and 5′-GCCTGCTGCCATCGCCTCAGACCC-3′. A band of approximately 550 bp from the 5′ RACE reaction was isolated using the Qiaquick gel extraction kit (Qiagen, Valencia, CA) and sequenced with the primer: (5′-GCCCCAGGTACTTGAGTCTCAGTG-3′). The sequence of this band revealed an open reading frame 69 bp longer than the sequence represented in the public data base with an additional potential initiating methionine 23 amino acids upstream from the methionine indicated by O43664. In addition, this 5′ RACE sequence included some 5′-untranslated sequence and an in-frame stop codon upstream from the new methionine. Sequencing of 3′ RACE products revealed a sequence identical to the 3′-end of O43664, in addition to some 3′-untranslated sequence. The new (longer) coding sequence was named SNORF62. From the new 5′-untranslated sequence and the 3′-untranslated sequence, new primers were designed to amplify the entire SNORF62 sequence from human stomach cDNA using the Expand Long PCR system (Roche Molecular Biochemicals, Indianapolis, IN). The primers (5′-ATCTATAAGCTTCGGAGGGTGGAAGCCGGGGTCTC-3′ and 5′-ATCTATGGATCCTCAGGATGGATCGGTCTCTTGCTG-3′) were designed to incorporate restriction sites for subcloning into a mammalian expression vector modified from pcEXV (26Miller J. Germain R.N. J. Exp. Med. 1986; 164: 1478-1489Crossref PubMed Scopus (191) Google Scholar). A search of the public domain data bases revealed that entry Y52992 (Gene_P; Derwent) carried an open reading frame, which displayed 46% amino acid identity with SNORF62. A forward primer in the 5′-untranslated region with aNotI site incorporated into the 5′-end of the primer and a reverse primer in the 3′-untranslated region with an site incorporated into the 5′-end of the primer were designed and to amplify the full-length sequence from human cDNA using the Expand Long PCR system (Roche Molecular The was given the the receptor of were into at a of cells well and to for at The was and containing was to The acid and acid and the the cells were to using a The of were in and to the cell were using the and data were using the and were from and with as 1994; Scopus Google Scholar, C. K.A. J. C. J. Chem. 272: PubMed Scopus Google Scholar). NMU1 was synthesized using the polymerase from PCR products containing the coding region of the were with of receptor RNA and for at 17 were with of were at a potential of were in containing and were by from a at a of from the by from a of cells NMU1 were at a of cells to for and with at the the was with containing and the were for at were with for at The was by the addition of by at for than were isolated from the by ion the of the were to a containing of with The was with acid and was by binding were by membranes from cells the receptor in at containing Molecular and as The in the was were with 125I-labeled rat NMU activity in the of on for in a of in The ligand was from ligand by in using a addition of was using a of was to using RNA was purchased from Alto, CA) isolated from human tissues using and to the RNA was in and with to RNA was by and by using a Molecular cDNA was synthesized by reverse from RNA using and with and were carried the addition of reverse of was with and the of cDNA synthesized was of RNA and cDNA was by amplification of PCR using was to the tissue of NMU1 and NMU2 were with as the and as a The following were NMU1 primer: reverse primer: and NMU2 primer: reverse primer: PCR reaction of for forward and reverse and for the PCR were carried in using PCR were carried in a sequence system using the following for for by of for for for of human NMU1 and NMU2 were using PCR data were using sequence The from well was using an and data were to a to of NMU1 and NMU2 for human NMU1 receptor was using a of by the Stanford Human Genome Center and by Inc. The Stanford of was by PCR using the same and as for of was in PCR was to the which the NMU1 and sequences to and were for of A search of the SWPLUS data base with a search set of known GPCRs several One sequence, O43664, was found to be to the receptor as well as the identified receptor, was to be for use in ligand identification screens. In the of the 5′- and 3′-ends of the coding sequence for by an additional methionine was found upstream from the initiating methionine of O43664, which was in-frame with the of the sequence. This new receptor sequence was 69 bp coding for a 23 amino acids longer than O43664. The amino acid sequence of this is represented in are sites in the domain at amino acid and and site in the at The C-terminal two potential sites at and and potential C site at a of the amino acid sequence of SNORF62 indicated that are as shown in A receptor sequence that was 46% identical to SNORF62 was found in the public domain data The full-length receptor was from human brain cDNA and was named several PCR the sequence of was found to be from the sequence. were of which the amino acid sequence of The amino acid sequence and are shown in is a amino acid identity the two suggesting that are to be of the same receptor also amino acid identity to the receptors and identity to the hormone receptor and identity to shown in two sites in the at amino acid and and two more in the at and and are and SNORF62 also potential C sites at and as well as potential site at is and SNORF62 were to the for SNORF62 in release not were to this receptor with the structurally related of human NMU to cells SNORF62 not in in release of (Fig. A The for the of SNORF62 and by peptides are in The peptides that activated these receptors and were not and binding for peptides at NMU1 and not of NMU and related peptides for of were in and were to of the indicated peptides and release by Binding were from of 125I-labeled rat NMU binding in membranes from cells not peptide. in a new of NMU and related peptides for of were in and were to of the indicated peptides and release by Binding were from of 125I-labeled rat NMU binding in membranes from cells The high potency of of SNORF62 and for these receptors as NMU receptors. in isolated rat uterus the for contraction by rat NMU is J. A. Bloom S.R. J. Chem. PubMed Google Scholar). The potency of rat NMU observed in the and cells and be to species differences in the receptors as well as the expression system. binding was on and membranes rat NMU and 125I-labeled porcine NMU18–25 as Binding rat NMU and 125I-labeled porcine NMU18–25 to the NMU1 and NMU2 membranes was by data not and (Fig. A binding sites for were present in the cell membranes not In membranes from cells 125I-labeled rat NMU and 125I-labeled porcine NMU18–25 with high porcine NMU18–25 identified sites than rat NMU of of porcine NMU18–25 and rat NMU are both in the This be to in high of 125I-labeled porcine NMU18–25 to the binding it than 125I-labeled rat NMU. binding represented approximately and of binding for 125I-labeled rat NMU porcine In cell from binding were for 125I-labeled rat NMU porcine binding represented approximately and approximately of binding for 125I-labeled rat NMU porcine identified of sites in membranes of for 125I-labeled rat NMU of porcine Interestingly, the by human NMU was in cells than in cells (Fig. A the levels of receptor expression were on This that is more of NMU2 to in the of 125I-labeled rat NMU binding the of binding of a number of peptides (Fig. and and The binding of rat NMU in NMU1 membranes was than porcine NMU18–25 consistent with the binding for the in NMU2 membranes the binding of rat NMU and porcine NMU18–25 were and The high binding of 125I-labeled rat NMU at NMU1 and NMU2 in cell membranes is to for this in isolated rat uterus (3Nandha K.A. Benito-Orfila M.A. Smith D.M. Bloom S.R. Endocrinology. 1993; 133: 482-486Crossref PubMed Scopus (26) Google The binding site in this tissue for NMU18–25 than for rat NMU with of and (3Nandha K.A. Benito-Orfila M.A. Smith D.M. Bloom S.R. Endocrinology. 1993; 133: 482-486Crossref PubMed Scopus (26) Google Scholar). of both the and of the rat be to a the receptor subtypes and the uterine NMU NMU was to bind with high and be a for both NMU1 and these receptors to NMU in the of SNORF62 as NMU1 and as The potential of these NMU receptors were by a of to the pathways by receptor of cells not to human NMU the release of with an of (Fig. Rat NMU and porcine NMU18–25 were also in this with and The for release are than the for in This be to differences in the two of including the of In addition, the release to human NMU was present in cells following with for data not indicating that the is not predominantly by of the these that NMU1 to C a in expression of GPCRs in has been to the identity of pathways activated by R. R. Sci. PubMed Scopus Google T. Sci. U. S. A. 1987; PubMed Scopus Google Scholar). Human NMU with of in with NMU1 whereas to human NMU A was not but were observed at a human NMU of NMU also a from with NMU1 but not in not The in with NMU1 are of of C and the system. with the in the such in is by GPCRs that to the NMU quantitative reverse using for NMU1 NMU2 was to the tissue of RNA receptor Human NMU1 RNA was localized in greatest in peripheral in of the gastrointestinal and systems with highest levels in The uterus and NMU1 consistent with and of NMU in these Although NMU1 was in and the peptide has not been identified in this tissue and its role in is not the of the receptor in the be associated with effects on blood The gastrointestinal system also has of NMU1 The small and the NMU1 This is consistent with the high levels of NMU in the tract found by (19Domin J. Ghatei M.A. Chohan P. Bloom S.R. Peptides ( Elmsford ). 1987; 8: 779-784Crossref PubMed Scopus (108) Google Scholar) in both myenteric and submucosal of the gut (11Ballesta J. Carlei F. Bishop A.E. Steel J.H. Gibson S.J. Fahey M. Hennessey R. Domin J. Bloom S.R. Polak J.M. Neuroscience. 1988; 25: 797-816Crossref PubMed Scopus (109) Google Scholar) and the role of NMU as a of smooth NMU1 RNA is also present in the at levels to that in other regions of the but it is not known the NMU1 receptors are found on the are present on within the (10Sumi 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) an in blood flow in the of NMU suggesting a tissues NMU1 RNA the adrenal and with levels in muscle and This a at the of smooth muscle contraction within these nervous system structures NMU1 RNA at levels than in peripheral the NMU1 RNA has been detected in highest in the hippocampus, and spinal NMU-like immunoreactivity was identified in of these regions (19Domin J. Ghatei M.A. Chohan P. Bloom S.R. Peptides ( Elmsford ). 1987; 8: 779-784Crossref PubMed Scopus (108) Google Scholar). the it is found in levels that are to than that found in peripheral The role of NMU1 RNA in the CNS is not however, its is consistent with the of NMU found in the brain (19Domin J. Ghatei M.A. Chohan P. Bloom S.R. Peptides ( Elmsford ). 1987; 8: 779-784Crossref PubMed Scopus (108) Google Scholar). The of NMU as well as NMU1 RNA in the spinal cord, and a role in sensory NMU2 RNA was found in highest in the the spinal cord, and (Fig. This is highly of a role in sensory modulation and is in to the with which has a in peripheral The to this are the which high levels of both NMU1 and NMU2 The hippocampus, hypothalamus, and to high levels of NMU2 CNS structures NMU2 RNA the and also NMU2 RNA at levels than found in the spinal The expression of NMU2 RNA in the CNS is consistent with the that its NMU, is a sensory NMU is found in the spinal cord, and using (19Domin J. Ghatei M.A. Chohan P. Bloom S.R. Peptides ( Elmsford ). 1987; 8: 779-784Crossref PubMed Scopus (108) Google Scholar) and (11Ballesta J. Carlei F. Bishop A.E. Steel J.H. Gibson S.J. Fahey M. Hennessey R. Domin J. Bloom S.R. Polak J.M. Neuroscience. 1988; 25: 797-816Crossref PubMed Scopus (109) Google Scholar). in other regions, including hippocampus, hypothalamus, and a role in systems within the NMU2 RNA the and NMU1 RNA is found in in both the and of the whereas NMU2 RNA is found in the suggesting physiological functions for these two receptors in this is not known at this which cells in the NMU2 NMU2 is to that NMU2 RNA is expressed in levels in the gastrointestinal In both NMU1 RNA and the NMU peptide are expressed in high levels in The human NMU1 to which is localized to to which is localized to In a variety of the identification of NMU1 and NMU2 as receptors for the neuropeptide NMU. expression of predominantly in the and predominantly in the roles for these receptors in Identification of these two distinct NMU receptors also the of to CNS and peripheral effects of NMU. the of this two were which to a of this Smith A. H. S. S. J. Chem. PubMed Scopus Google R. M. S. Y. Y. S. H. M. J. Chem. PubMed Scopus Google Scholar). The and for in