Identification and Functional Characterization of a ...

THE JOURNAL OF BIOLOGICAL CHEMISTRY © 2000 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 275, No. 38, Issue of September 22, pp. 29528 –29532, 2000 Printed in U.S.A.

Identification and Functional Characterization of a Novel Subtype of Neuromedin U Receptor* Received for publication, May 18, 2000, and in revised form, July 6, 2000 Published, JBC Papers in Press, July 7, 2000, DOI 10.1074/jbc.M004261200

Masaki Hosoya, Takeo Moriya, Yuji Kawamata, Shoichi Ohkubo, Ryo Fujii, Hideki Matsui, Yasushi Shintani, Shoji Fukusumi, Yugo Habata, Shuji Hinuma‡, Haruo Onda, Osamu Nishimura, and Masahiko Fujino From the Pharmaceutical Discovery Research Division, Takeda Chemical Industries, Ltd., Wadai 10, Tsukuba, Ibaraki 300-4293, Japan

The bioactive peptides neuromedin U-8 and U-25 were originally isolated from the porcine spinal cord based on their powerful contractile activity in the uterine smooth muscle of rats (1, 2). Since then, neuromedin U has been found to show various biological activities including the elevation of blood pressure and the induction of adrenocorticotropin and corticosterone release in vivo in rats (1, 3– 6). Applying our previously described strategy to identify endogenous ligands for orphan G protein-coupled receptors (GPCRs)1 (7–9), we recently succeeded in demonstrating that the orphan GPCR, FM-3, a homologue of the neurotensin (NT) and growth hormone secreta* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. The nucleotide sequence reported in this paper has been submitted to the DDBJ/GenBankTM/EBI Data Bank with accession numbers AB041228 and AB041229. ‡ To whom correspondence should be addressed. Tel.: 81-298-645035; Fax: 81-298-64-5000; E-mail: [email protected]. 1 The abbreviations used are: GPCR, G protein-coupled receptor; NT, neurotensin; CHO, Chinese hamster ovary; AA, arachidonic acid metabolite; RT-PCR, reverse transcription-polymerase chain reaction; PCR, polymerase chain reaction; CHO-hTGR-1, CHO cells expressing human TGR-1 cDNA; FLIPR, fluorometric imaging plate reader system; PP, pancreatic polypeptide.

gogue receptors, is a specific and functional receptor for neuromedin U (10). When FM-3 was expressed in Chinese hamster ovary (CHO) cells, neuromedin U induced specific and clear elevation of extracellular acidification rates, arachidonic acid metabolite (AA) release, and intracellular Ca2⫹ mobilization. Radiolabeled neuromedin U specifically bound with high affinity to membrane fractions prepared from these cells. Our analyses for FM-3 mRNA expression levels using quantitative reverse transcription-polymerase chain reaction (RT-PCR) indicated that FM-3 mRNA was expressed highly in the small intestine and lung of rats, but only very low levels were detected in the uterus. Nonetheless neuromedin U has reportedly shown very strong contractile activity (1, 2), and numerous binding sites have been detected in rat uterine tissue (11, 12). We considered that this discrepancy might be due to the existence of an unknown receptor subtype for neuromedin U in the rat uterus. We therefore searched for another neuromedin U receptor subtype in addition to FM-3. In this paper, we report on the isolation of human and rat cDNA encoding a novel GPCR, TGR-1, which has significant homology with FM-3. We found that, like FM-3, TGR-1 functioned as a specific receptor for neuromedin U. Our analyses for TGR-1 mRNA by quantitative RT-PCR in rat tissues demonstrated that it was extensively expressed in the uterus. EXPERIMENTAL PROCEDURES

Cloning of Human and Rat TGR-1 cDNAs—Two regions with high sequence similarity to FM-3 were identified in the GenBankTM genomic DNA data base between base pairs 204,900 and 205,550 and between base pairs 138,706 and 138,828 in a single human genomic DNA sequence (accession number AC008571). The deduced amino acid sequences of both regions were found to have 56% identity with corresponding FM-3 amino acid sequences. A cDNA fragment encompassing these sequences was isolated from a human testis cDNA library (Life Technologies, Inc.) by PCR using the specific primers 5⬘-CGCCCACCAACTACTACCTCT-3⬘ and 5⬘-ACAAAGCTGAAGAAGAGTCGG-3⬘. The resultant PCR product of about 700 base pairs contained the two expected regions plus an unknown 126-base pair sequence between them, corresponding to the coding region spanning transmembrane domains 2– 6. Both ends of this DNA fragment were further extended using human brain and testis Marathon ready cDNA libraries (CLONTECH) by 5⬘- and 3⬘-rapid amplification of cDNA ends procedures with adapter primers provided with the cDNA libraries in addition to the following specific primers: 5⬘-ACAAAGCTGAAGAAGAGTCGG-3⬘ and 5⬘-CGCCCACCAACTACTACCTCT-3⬘ for the first PCR and 5⬘-ATGGGCTTGATGACCGTACAG-3⬘ and 5⬘-TGTGGCGCAACTACCCTTTCT-3⬘ for the nested PCR. The GPCR encoded by the human cDNA thus obtained was designated as TGR-1. Rat TGR-1 cDNA was isolated from rat uterine poly(A)⫹ RNA. For PCR, we designed primers (i.e. 5⬘-GCTATGAAGACGCCCACCAACTACTA-3⬘ and 5⬘-CACCACATGGACGAGGTTGAACACAGC-3⬘) and prepared a 25-␮l reaction mixture containing 0.2 ␮M of each primer, a template cDNA synthesized from rat uterine poly(A)⫹ RNA, 0.4 mM dNTPs, 1.25 unit of KlenTaq DNA polymerase (CLONTECH), and 2.5

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Neuromedin U is a bioactive peptide isolated originally from the porcine spinal cord. We recently identified neuromedin U as the cognate ligand for the orphan G protein-coupled receptor FM-3. In this study, we isolated cDNA coding for a novel G protein-coupled receptor, TGR-1, which was highly homologous with FM-3. We found that neuromedin U specifically and clearly elevated the extracellular acidification rates, arachidonic acid metabolite release, and intracellular Ca2ⴙ mobilization in Chinese hamster ovary cells expressing TGR-1. Radiolabeled neuromedin U specifically bound with high affinity to membrane fractions prepared from these cells. These results show that TGR-1, like FM-3, is a specific and functional receptor for neuromedin U. We analyzed TGR-1 mRNA tissue distribution in rats using quantitative reverse transcription-polymerase chain reaction and found it to considerably differ from that of FM-3 mRNA. TGR-1 mRNA was primarily expressed in the uterus, suggesting that TGR-1 mediates the contractile activity of neuromedin U in this tissue. The identification of specific and functional receptor subtypes for neuromedin U will facilitate the study of their physiological roles and the search for their specific agonists and antagonists.

Neuromedin U Receptor Subtype

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FIG. 1. Amino acid sequences of human and rat TGR-1 aligned with human FM-3. Residues identical in at least two sequences are boxed. The predicted seven transmembrane domains (TM 1–7) are indicated in bars above the sequences. Nucleotide and amino acid sequence data for human and rat TGR-1 cDNAs appear in the DDBJ/EMBL/GenBankTM data bases with accession numbers AB041228 and AB041229, respectively.

Quantitative Analyses for Rat TGR-1 mRNA—Poly(A)⫹ RNAs were prepared from different tissues of 8 –12-week-old Wistar rats, and cDNAs were synthesized as described elsewhere (14). Poly(A)⫹ RNAs of the placenta and mammary gland were prepared from female rats 17 days pregnant. We quantified rat TGR-1 mRNAs using a Prism 7700 Sequence Detector (PE Biosystems) with a primer set (i.e. 5⬘-ATCCATGTGGTATCAGGTGTCTTCT-3⬘ and 5⬘-CGCCGAGACAGGAGGTTATAGA-3⬘) and a fluorescence-labeled probe, 5⬘-FAM-TATCTGAGCTCCGCGGTCAACCCC-TAMRA-3⬘. For PCR, a 25-␮l reaction mixture containing a cDNA solution synthesized from 4 ng of poly(A)⫹ RNA, a 0.2 ␮M concentration of each primer, and 0.1 ␮M probe was prepared with a TaqMan Universal PCR Master Mix (PE Biosystems). PCR was conducted at 50 °C for 10 min in order for the reaction of uracil-Nglycosylase to prevent amplification of carried over PCR products and then at 95 °C for 15 min to activate AmpliTaq Gold DNA polymerase and finally for 40 cycles at 95 °C for 15 s and at 60 °C for 60 s for amplification. To obtain calibration curves, we amplified the known amounts of rat TGR-1 cDNA fragments in the same manner as the samples. Good linear relationships were obtained between the amount of rat TGR-1 cDNA introduced and the release of reporter dye within 1–106 copies. As an internal control, rat glyceraldehyde-3-phosphate dehydrogenase mRNA expression was also measured using Rodent GAPDH Control Reagents (PE Biosystems) according to the manufacturer’s instructions. In most tissues, the expression of glyceraldehyde3-phosphate dehydrogenase mRNA was 0.7 ⫻ 105 to 1.3 ⫻ 106 copies/ng of poly(A)⫹ RNA, except in the skeletal muscle (4.6 ⫻ 106 copies/ng poly(A)⫹ RNA)) and costal cartilage (2.2 ⫻ 106 copies/ng of poly(A)⫹ RNA). RESULTS

Cloning of Human and Rat cDNAs Encoding TGR-1—In our data base search, we found human DNA sequences that coded for a novel GPCR. Based on sequence information, we designed primers for PCR, and then from human brain and testis we isolated cDNA encoding a GPCR, which we designated as TGR-1. The predicted amino acid sequence encoded by TGR-1 had an amino acid length of 413 (Fig. 1). In comparing amino acid sequence, human TGR-1 shared 52% identity overall with


␮l of the buffer provided by the manufacturer. PCR was conducted at 94 °C for 2 min, followed by 38 cycles at 98 °C for 10 s, at 64 °C for 20 s, and at 72 °C for 20 s. Based on the partial rat cDNA sequence obtained, we synthesized different primers and then isolated cDNA fragments covering a fully coded region by 5⬘- and 3⬘-rapid amplification of cDNA ends using these primers and a Marathon cDNA Amplification Kit. We isolated rat TGR-1 cDNA with a fully coded region from rat uterine cDNA synthesized with a Marathon cDNA Amplification Kit (CLONTECH) by PCR using the specific primers 5⬘-CTGATGCTATCCTTTCACTCTCTCAGACC-3⬘ and 5⬘-TCCTTGCAGTTTTGGCACATAGATGGA-3⬘. Preparation of CHO Cells Expressing Human TGR-1—The entire region encoding human TGR-1 (corresponding to positions 1–1245 in the nucleotide sequence deposited in the DDBJ/EMBL/GenBankTM data base under accession number AB041228) was inserted downstream from an SR␣ promoter in the expression vector, pAKKO-111H (13). The resultant expression vector plasmid was transfected into dhfr⫺ CHO cells, and transformed dhfr⫹ CHO cells (CHO-hTGR-1) were selected as described elsewhere (13). Peptides—Porcine neuromedin U-8 and U-25 and rat U-23 were purchased from Bachem AG (Bubendorf, Switzerland). Human U-25 was synthesized by Peptide Institute (Osaka, Japan). Other peptides were purchased commercially. AA Release Assays—AA release assays were conducted as described elsewhere (10). Ca2⫹ Mobilization Assays—Ca2⫹ mobilization assays using CHOhTGR-1 cells and mock-transfected CHO cells were conducted as described elsewhere (10). Changes in intracellular Ca2⫹ concentrations induced by neuromedin U were measured with a fluorometric imaging plate reader (FLIPR; Molecular Devices Corp.). Receptor Binding Assays—Preparation of 125I-labeled porcine U-8 and membrane fractions, and receptor-binding assays were conducted as described elsewhere (10). The labeled ligand (concentration 100 pM) and membrane fractions prepared from CHO-hTGR-1 cells were mixed and then incubated in 100 ␮l of the binding buffer at room temperature for 1.5 h. To determine the amount of nonspecific binding, a separate mixture was also prepared with the addition of unlabeled porcine U-8 (concentration 1 ␮M) and incubated under the same conditions. After incubation, bound and free radioactivities were separated, and the amount of bound ligand was counted with a liquid scintillation counter.

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FIG. 2. AA release assay on CHO-hTGR-1 cells with neuromedin U and related peptides. CHO-hTGR-1 cells were incubated overnight with 3H-labeled arachidonic acid, washed, and then incubated for 30 min with human U-25 (Œ), rat U-23 (⽧), porcine U-25 (䡺), and porcine U-8 (E) at the indicated concentrations. Related peptides, including human ghrelin (●), motilin (‚), NT (f), and PP (〫), were also assayed at 10⫺7 M. After the cells were washed, radioactivity released in each supernatant was measured. Symbols represent the mean with S.E. in triplicate determinations.

FIG. 4. Scatchard analysis of radiolabeled porcine U-8 binding to TGR-1. Membrane fractions prepared from CHO-hTGR-1 cells were incubated with increasing concentrations of 125I-labeled porcine U-8. Bound and free radiolabeled U-8 were separated when binding reached equilibrium. Data are plotted as bound (B, pmol mg⫺1) versus bound/ free (B/F, pmol mg⫺1 nM⫺1) radiolabeled U-8. Each symbol represents the mean with S.E. in triplicate determinations.

FIG. 5. Competitive inhibition assay for 125I-labeled porcine U-8 binding to TGR-1. Membrane preparations from CHO-hTGR-1 cells and 125I-labeled porcine U-8 were incubated for 1.5 h in the presence of human U-25 (Œ), rat U-23 (⽧), porcine U-25 (䡺), and porcine U-8 (E) at the indicated concentrations. Binding assays were also conducted in the presence of human ghrelin (●), motilin (‚), NT (f), and PP (〫) at 10⫺7 M. Bound and free ligands were separated by rapid filtration, and radioactivity remaining on the filters was measured. Each symbol represents the mean with S.E. in triplicate determinations.

concentration (IC50) of 2.2 ⫻ 10⫺10 M. IC50 values of rat U-23 and porcine U-8 were both 3.5 ⫻ 10⫺10 M. Human U-25 was the least potent, with an IC50 of 6.2 ⫻ 10⫺10 M. Human ghrelin showed slight inhibition at 10⫺7 M, but motilin, NT, and PP displayed no obvious inhibitory activity. Tissue Distribution of TGR-1 mRNA in Rats—We analyzed


human FM-3. We subsequently isolated rat TGR-1 cDNA from rat uterus, which encoded an open reading frame with a 395amino acid length and showed 78% amino acid sequence identity with human TGR-1 (Fig. 1). The positions of supposed initiator methionine in human and rat TGR-1 cDNAs were determined based on analyses for optimal nucleic acid sequences for translation (15) and comparison of conserved amino acid sequences between humans and rats. Specific Signal Transduction by Neuromedin U in CHO Cells Expressing TGR-1—Since TGR-1 has considerable homology with FM-3, we studied the effect of porcine neuromedin U-8 on CHO-hTGR-1 cells by microphysiometric assay and found that it induced a specific, dose-dependent promotion of the extracellular acidification rates in these cells (data not shown). We investigated what cellular changes were induced in CHOhTGR-1 cells by neuromedin U. As with CHO cells expressing FM-3, CHO-hTGR-1 cells showed specific, dose-dependent AA release when neuromedin U was added (Fig. 2). In the AA release assays, rat U-23 and porcine U-25 and U-8 were almost equally potent. Their median effective concentration (EC50) was 1.4 –2.0 ⫻ 10⫺9 M. However, that of human U-25 was seen to be slightly lower (4.0 ⫻ 10⫺9 M). To confirm the specific interaction of TGR-1 and neuromedin U, we applied human ghrelin, motilin, NT, and pancreatic polypeptide (PP) at 10⫺7 M, but these did not cause obvious AA release in CHO-hTGR-1 cells. Since extracellular AA release is caused by the activation of phospholipase A2, which is closely linked to Ca2⫹ influx, we studied changes in the intracellular Ca2⫹ concentration of CHO-hTGR-1 cells by FLIPR assay. We found that porcine U-8 induced rapid, evident mobilization of intracellular Ca2⫹ at doses of 10⫺7 and 10⫺8 M. Porcine U-8 did not induce Ca2⫹ mobilization even at 10⫺7 M in mock-transfected CHO cells (Fig. 3). We examined the effect of neuromedin U on intracellular cAMP levels of CHO-hTGR-1 cells and found that neuromedin U partially inhibited forskolin-induced cAMP production dose-dependently (data not shown). Specific Binding of Neuromedin U to TGR-1—125I-Labeled porcine U-8 bound efficiently to membrane fractions prepared from CHO-hTGR-1 cells. Scatchard analysis showed that CHOhTGR-1 cells expressed a single class of high affinity binding sites for 125I-labeled porcine U-8 (Fig. 4). The dissociation constant (Kd) was 2.2 ⫻ 10⫺11 M, and the number of maximal binding sites (Bmax) was 6.8 pmol mg⫺1 protein. In the competitive binding assay, human, porcine, and rat neuromedin U were found to inhibit this binding dose-dependently (Fig. 5). Porcine U-25 was the most potent, with a median inhibition

FIG. 3. Neuromedin U-induced changes in intracellular Ca2ⴙ concentration in CHO-hTGR-1 cells. CHO-hTGR-1 cells and mocktransfected CHO cells were cultured in black-walled 96-well plates overnight. The cells were loaded with Fluo 3-AM, following which fluorescent change was measured by FLIPR. Porcine U-8 was added to the cells after 10 s. Concentrations of porcine U-8 used were 10⫺7 M (E) and 10⫺8 M (Œ) for CHO-hTGR-1 cells and 10⫺7 M (䡺) for the mocktransfected CHO cells. Data are shown as representative traces.


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FIG. 6. Tissue distribution of TGR-1 mRNA in rats analyzed by RT-PCR. Poly(A)⫹ RNA preparations obtained from the indicated rat tissues were subjected to quantitative RT-PCR using an ABI Prism 7700 Sequence Detector. Each column represents the mean in duplicate determinations.

DISCUSSION

We isolated TGR-1 as a novel GPCR showing considerable homology with FM-3, which has been recently shown to be the cognate receptor for neuromedin U (10). Neuromedin U showed a potent agonistic activity to CHO cells expressing human TGR-1 and specifically bound with high affinity to their membrane fractions at a Kd of 22 pM. Our data clearly show that TGR-1 is a subtype of neuromedin U receptors. Since neuromedin U caused Ca2⫹ influx and AA release in CHO-hTGR-1 cells, TGR-1 is considered to couple to Gq in the signal transduction pathway similar to FM-3. Since slight inhibition of forskolin-stimulated cAMP production was also detected in CHO-hTGR-1 cells treated with neuromedin U, TGR-1 may couple not only to Gq but also to Gi in CHO cells. Since it was first purified from the porcine spinal cord (1, 2), neuromedin U has been isolated from a number of species (11). In comparing neuromedin U peptides derived from different species, the C-terminal five amino acid residues were found to be totally conserved, suggesting that this region is of major importance. It has also been reported that amidation of the C-terminal Asn is necessary for neuromedin U to exhibit biological activities (1). These facts suggest that the C-terminal region is vital for neuromedin U to interact with its receptor. TGR-1 shows significant homology with FM-3, which is reported to resemble NT and growth hormone secretagogue receptors (16). NT and ghrelin show slight sequence homology with neuromedin U overall but do not show significant homology at their C termini (10). In our competitive binding experiment, ghrelin showed slight but significant inhibition on 125Ilabeled neuromedin U-8 binding to TGR-1 at 10⫺7 M, although it did not show agonistic activity on TGR-1. Our results suggest that these ligands and receptors are closely related both structurally and functionally. We previously analyzed FM-3 mRNA distribution and found it to be highly expressed in the small intestine of rats (10). It has been reported that neuromedin U-like immunoreactivity is widely detected in the gastrointestinal tract (17–23) and that neuromedin U promotes motor response (3, 4), blood flow (5), and ion transport in the gut (6). It has been proposed that neuromedin U exerts influence as a neuropeptide or neuro-

modulator rather than as a circulating hormone, because its levels in plasma are fairly low (17). Taken together, this suggests that the reported action of neuromedin U on the gastrointestinal tract is primarily mediated by FM-3. In contrast, the expression of FM-3 mRNA was found to be low in the rat uterus (10). In this paper, we found an extremely high level of TGR-1 mRNA expressed in the rat uterus. This is consistent with reports that, in uterine tissue, neuromedin U shows strong contractile activity (1, 2) and numerous binding sites for neuromedin U are detected (11, 12). However, we found TGR-1 mRNA expression levels to be lower in the gastrointestinal tract. Considering these results together, the reported action of neuromedin U on uterine tissue would appear to be primarily mediated by TGR-1. It has been reported that both neuromedin U mRNA and neuromedin U-like immunoreactivity are abundantly detected in the human and rat pituitary gland (17, 24, 25). We detected the highest level of neuromedin U mRNA expression in the rat pituitary gland (10). It may be that neuromedin U is originally secreted from the pituitary and thereby put into circulation. However, further investigation is necessary to clarify the pathway of neuromedin U’s action on the uterus. Significant expression of TGR-1 mRNA was detected in the central nervous tissues (i.e. hypothalamus, medulla oblongata, and spinal cord), the lung, and the ovary, suggesting that TGR-1 plays important roles here. In the rat brain, neuromedin U-positive nerve fibers have been observed in the hypothalamic paraventicular and supraoptic nuclei (19, 24). The subcutaneous administration of neuromedin U into rats has been shown to increase plasma adrenocorticotropin and corticosterone concentrations (26). These facts suggest the involvement of neuromedin U in the control of the hypothalamo-pituitary-adrenocortical axis. We have detected only low levels of FM-3 mRNA expression in the hypothalamus, pituitary, and adrenal gland (10). However, in the present investigation, a relatively high level of TGR-1 mRNA expression was found in the rat hypothalamus, suggesting that neuromedin U functions in the hypothalamus via TGR-1. Our results suggest that TGR-1 and FM-3 possess different distributions and functions among tissues in vivo. The discovery of neuromedin U receptor subtypes provides valuable insight into the physiological roles of neuromedin U and the identification of neuromedin U receptor agonists and antagonists. Acknowledgment—We thank K. Kanehashi for technical assistance in FLIPR assays. REFERENCES 1. Minamino, N., Kangawa, K., and Matsuo, H. (1985) Biochem. Biophys. Res. Commun. 130, 1078 –1085 2. Minamino, N., Sudoh, T., Kangawa, K., and Matsuo, H. (1985) Peptides 6,


TGR-1 mRNA distribution in various rat tissues. The highest level of TGR-1 mRNA was detected in the uterus (25 ⫻ 103 copies/ng of poly(A)⫹ RNA) (Fig. 6). In the central nervous system, high expression levels were found in the hypothalamus and moderate levels in both the medulla oblongata and spinal cord (2.6, 0.7, and 1.5 ⫻ 103 copies/ng of poly(A)⫹ RNA, respectively). Expression was low in the gastrointestinal tract. In other peripheral tissues, moderate expression was observed in the lung and ovary (0.6 and 1.1 ⫻ 103 copies/ng of poly(A)⫹ RNA, respectively).

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Identification and Functional Characterization of a Novel Subtype of Neuromedin U Receptor Masaki Hosoya, Takeo Moriya, Yuji Kawamata, Shoichi Ohkubo, Ryo Fujii, Hideki Matsui, Yasushi Shintani, Shoji Fukusumi, Yugo Habata, Shuji Hinuma, Haruo Onda, Osamu Nishimura and Masahiko Fujino J. Biol. Chem. 2000, 275:29528-29532. doi: 10.1074/jbc.M004261200 originally published online July 7, 2000

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