Molecular identification of GHS-R and GPR38 in Suncus murinus

Peptides 36 (2012) 29–38

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Molecular identification of GHS-R and GPR38 in Suncus murinus Airi Suzuki a , Yuko Ishida a , Sayaka Aizawa a , Ichiro Sakata a , Chihiro Tsutsui b , Anupom Mondal a , Koike Kanako a , Takafumi Sakai a,c,∗ a b c

Area of Regulatory Biology, Division of Life Science, Graduate School of Science and Engineering, Saitama University, 255 Shimo-ohkubo, Sakura-ku, Saitama 338-8570, Japan Nano Carbon Bio Device Research Center, Tokyo City University, 1-28-1 Tamadsudsumi, Setagaya-ku, Tokyo 158-8557, Japan Saitama University Brain Science Institute, Saitama University, 255 Shimo-ohkubo, Sakura-ku, Saitama 338-8570, Japan

a r t i c l e

i n f o

Article history: Received 1 March 2012 Received in revised form 23 April 2012 Accepted 23 April 2012 Available online 30 April 2012 Keywords: Ghrelin Motilin GHS-R GPR38 Suncus murinus

a b s t r a c t We previously identified ghrelin and motilin genes in Suncus murinus (suncus), and also revealed that motilin induces phase III-like strong contractions in the suncus stomach in vivo, as observed in humans and dogs. Moreover, repeated migrating motor complexes were found in the gastrointestinal tract of suncus at regular 120-min intervals. We therefore proposed suncus as a small laboratory animal model for the study of gastrointestinal motility. In the present study, we identified growth hormone secretagogue receptor (GHS-R) and motilin receptor (GPR38) genes in the suncus. We also examined their tissue distribution throughout the body. The amino acids of suncus GHS-R and GPR38 showed high homology with those of other mammals and shared 42% amino acid identity. RT-PCR showed that both the receptors were expressed in the hypothalamus, medulla oblongata, pituitary gland and the nodose ganglion in the central nervous system. In addition, GHS-R mRNA expressions were detected throughout the stomach and intestine, whereas GPR38 was expressed in the gastric muscle layer, lower intestine, lungs, heart, and pituitary gland. These results suggest that ghrelin and motilin affect gut motility and energy metabolism via specific receptors expressed in the gastrointestinal tract and/or in the central nervous system of suncus. © 2012 Elsevier Inc. All rights reserved.

1. Introduction Ghrelin is a peptide hormone that was first identified in rat and human stomachs in 1999 by Kojima et al. [23]. The ghrelin gene of various species, including not only mammals but also the chicken, turtle, bullfrog, and rainbow trout, has been cloned since then [18–21]. It is well established that ghrelin is involved in many physiological actions, including regulation of growth hormone secretion [3,23,54], food intake [32,51], energy metabolism [39,48], gastrointestinal motility [31,38,55], gastric acid secretion [11,25,52], cardiovascular function [35], and cell proliferation [8,24]. Motilin was originally purified and sequenced from porcine intestinal mucosa as a 22-amino acid polypeptide in the 1970s [1,2,43], and it is known to induce interdigestive migrating motor complexes (MMCs) with typical phase III contractions. Since the discovery of porcine motilin, mRNA and amino acid sequences of motilin from several other species have been identified, including humans, cows, dogs, cats, and chickens [16]. Motilin-producing

∗ Corresponding author at: Area of Regulatory Biology, Division of Life Science, Graduate School of Science and Engineering, Saitama University, 255 Shimo-ohkubo, Sakura-ku, Saitama 338-8570, Japan. Tel.: +81 48 858 3869; fax: +81 48 858 3422. E-mail address: [email protected] (T. Sakai). 0196-9781/$ – see front matter © 2012 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.peptides.2012.04.019

cells are mainly present in the upper small intestine, especially in the duodenal mucosa [22,42]. Specific receptors for ghrelin and motilin have been identified and were named growth hormone secretagogue receptor (GHS-R) [15] and motilin receptor (GPR38) [10], respectively. Both belong to the class A rhodopsin-like G protein-coupled seventransmembrane receptor family, and they have a similar structure that is not observed in other GPCR subfamilies [28]. In humans, the ghrelin and motilin ligands share about 21% amino acid identity, and about 50% of their precursor mRNAs are identical [37]. In addition, their receptors show marked sequence homology with an overall identity of 52% with 86% in the transmembrane region. The binding sites of their ligands have been revealed by binding assays and by studies of intracellular calcium response using cultured cells expressing mutated receptors. It has been suggested that five amino acid residues of GHS-R [9,36,44,50] and nine amino acid residues of GPR38 [26,27] are important for ligand binding or receptor activity. GHS-R has been identified in many animals, and it is reportedly expressed in various tissues, including the gastrointestinal tract, hypothalamus, and pituitary gland [12]. However, the detailed distribution of GPR38 in the body remains unclear despite motilin having been discovered decades ago. In addition, considering the sequence similarity of their ligands and receptors, it would be reasonable to assume that interactions between ghrelin and motilin

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A. Suzuki et al. / Peptides 36 (2012) 29–38

Table 1 Primers used in cloning for GHS-R and GPR38. Primer name

Direction

Sequence

sGHSR FWD3 sGHSR BWD5 sGHSR FWD6 sGHSR BWD3 sGHSR FWD2 sGHSR FWD4 Anchor in Anchor out sGHSR 5 -UTR FWD1 sGHSR 5 -UTR BWD2 StraightWalk WP-1 StraightWalk WP-2 StraightWalk GHSR SP-1 StraightWalk GHSR SP-2 sGPR38 FWD8 sGPR38 BWD7 sGPR38 FWD18 sGPR38 BWD14 sGPR38 FWD19 sGPR38 FWD20 sGPR38 5 -UTR FWD37 sGPR38 5 -UTR BWD26 StraightWalk GPR38 SP-1 StraightWalk GPR38 SP-2

Sense Anti-sense Sense Anti-sense Sense Sense Sense Sense Sense Anti-sense Sense Sense Anti-sense Anti-sense Sense Anti-sense Sense Anti-sense Sense Sense Sense Anti-sense Anti-sense Anti-sense

CACCACCACCAACCTCTACC CTGGCAGGAAGAAGAAGACG CTACTTCGCCATCTGCTTCC AGCCCGAGAACTTTCATCCT TCTTCCTGCCAGTCTTCTGC CCCTTCCACGTAGGACGATA AGGACTCGAGCTCAAGC CCAGTGAGCAGAGTGACG TCTCCGCTCCAAGCTTCA TGGAAGAGTTTGCAGAGCAG CGCAGGCTGGCAGTCTCTTTAG ATGCGGCCGCTCTCTTTAGGGTTACACGATTGCTT GTGACCACCACTTTGGCCCGCA CTCGCTCACGAACTGGAAGAGTTTGC TGTACTTCTCYCAGTACTTTAACATYGT CTTCTTKGARATGAGGTTGTAGAGGA CCATGTGGGCAGAATCATCT AGAGGATGGGGTTGATGGAG AAGGACACCCGGACAATGTA CATTGTTGCTTTGCAACTTTTC GAGCAGACCCTGGATTGAAC AGGAAGAAGTAGGCGGTGGT CAGACGGCCAGGTAGCGCTCGAC ATGCTGCACAGGTACGAGTTGGTGGC

are coordinated. However, ghrelin–motilin interactions could not be studied well as motilin-producing small laboratory animal models have not been available. Rodents such as rats and mice are the classical and most widely used laboratory animals, but they are not suitable for motilin experiments because in these species, motilin and GPR38 became inactivated through evolution and are now pseudogenes [14]. We previously reported on the suitability of house musk shrews (Suncus murinus; suncus), which belongs to the order Insectivora, as a new model animal for studying motilin- and ghrelin-family peptides. We previously cloned the complete cDNA sequences and determined the tissue distributions of motilin and ghrelin in suncus [17,49]. We also reported that a reproducible contractile response of the suncus stomach can be induced by the in vitro and in vivo administration of suncus motilin or human motilin [29,40,49]. In the current study, in order to further elucidate the mechanisms of motilin and ghrelin physiology, we determined the nucleotide and amino acid sequences of suncus GHS-R and GPR38 and examined their tissue distribution. 2. Materials and methods 2.1. Animals The experiments were performed using male and female adult suncus (age: 12–22 weeks) of an outbred KAT strain established from a wild population in Kathmandu, Nepal [33]. The animals were housed individually in plastic cages equipped with an empty can for a nest box and under controlled conditions (23 ± 2 ◦ C, lights on from 08:00 to 20:00 h), with a free access to water and commercial trout feed pellets (Nippon Formula Feed Manufacturing Co. Ltd., Yokohama, Japan). The metabolizable energy content of the pellets was 344.3 kcal/100 g, and they consisted of 54.1% protein, 30.1% carbohydrates, and 15.8% fat. All procedures were approved and performed in accordance with the guidelines of the Saitama University Committee on Animal Research. 2.2. Bioinformatics and primer design The Ensembl Genome Browser from the Sanger Institute (http://uswest.ensembl.org/index.html) and the Nucleotide

Database from the National Center for Biotechnology Information (NCBI; http://www.ncbi.nlm.nih.gov/nuccore) were used for searches related to suncus GPR38 and GHS-R. Primers used in the cloning for GHS-R and GPR38 were designed using Primer3 software (http://frodo.wi.mit.edu/primer3/), and are shown in Table 1. 2.3. Extraction of genomic DNA and RNA Suncus genomic DNA from the liver and total RNA from the stomach and hypothalamus were extracted using ISOGEN (Nippon gene, Tokyo, Japan). Poly A+ RNA was then purified from the total RNA from the hypothalamus with the Oligotex dT30 Super mRNA Purification Kit (Takara Bio, Shiga, Japan) according to the manufacturer’s instructions. Total RNA was used for the synthesis of stomach cDNA and poly A+ RNA was used for the synthesis of hypothalamus cDNA. Using an oligo(dT) anchor primer, 5 CCAGTGAGCAGAGTGACGAGGACTCGAGCTCAAGCTTTTTTTTTTTTT TTTTTTTVN-3 was synthesized from 180 ng of hypothalamic poly A+ RNA and 1 ␮g of DNase I (Promega, Madison, WI)-treated stomach total RNA using a M-MLV reverse transcriptase (Invitrogen Co., CA, USA) and Prime Script (Takara Bio), respectively. The suncus genomic DNA and the hypothalamic cDNA were used to clone suncus GHS-R, and the suncus genomic DNA and stomach cDNA were used to clone suncus GPR38. 2.4. Cloning of partial suncus GHS-R To obtain the sequence of suncus GHS-R exon 1, we designed the appropriate primers (sense: sGHS-R FWD3, FWD6; anti-sense: sGHS-R BWD5, BWD3) by referring to the GHS-R sequence of the shrew (accession no. ENSSARG00000002887) in Ensembl genome database. PCR was performed using the suncus genomic DNA and primers FWD3 and BWD5 with Ex Taq polymerase (Takara Bio). The reaction cycle was as follows: 94 ◦ C for 5 min; and then 35 cycles of 94 ◦ C for 1 min, 68 ◦ C for 1 min, and 74 ◦ C for 1 min. The amplified fragments were run on a 2% agarose gel (Bio-Rad Laboratories, CA, USA) and visualized on UV illuminator (Atto, Tokyo, Japan) after ethidium bromide staining. The amplified fragments were then cloned into pGEM T-easy vectors (Promega) and sequenced with a CEQ 8000 Genetic Analysis system (Beckman Coulter Inc., CA, USA). The sequences obtained were then submitted to a BLAST search

A. Suzuki et al. / Peptides 36 (2012) 29–38

(NCBI). After confirming sequence homology, PCR was performed using suncus hypothalamic cDNA under the same PCR conditions. The amplified fragments were then cloned and their sequences analyzed as detailed above. We confirmed that the partial GHS-R mRNA sequence obtained matched the genomic sequence. 2.5. 3 -RACE (Rapid Amplification of cDNA Ends) for GHS-R Hypothalamic cDNA was amplified with sGHS-R FWD2 (sense) and an adapter primer for 3 -RACE (anchor out, used as the backward primer) using AmpliTaq Gold (Roche Molecular Systems, NJ, USA). The amplification reaction was performed under the following conditions: 95 ◦ C for 5 min, followed by 40 cycles at 94 ◦ C for 1 min, 64 ◦ C for 1 min, and then 60 ◦ C for 10 min. The second PCR was performed using the products from the primary PCR as a template and sGHS-R FWD4 and 3 -RACE anchor (anchor in) primers along with Ex Taq polymerase. The amplification reaction was performed under the following conditions: 94 ◦ C for 5 min, followed by 40 cycles at 94 ◦ C for 1 min, 56 ◦ C for 1 min, and 74 ◦ C for 1 min. The third PCR was performed using diluted products from the second PCR as templates and sGHS-R FWD5 and 3 -RACE anchor (anchor in) primers with Ex Taq polymerase. The amplification reaction was performed under the following conditions: 94 ◦ C for 5 min, followed by 40 cycles at 94 ◦ C for 1 min, 65 ◦ C for 1 min, and 74 ◦ C for 1 min. The amplified fragments from the third PCR were then cloned into pGEM T-easy vectors and sequenced. 2.6. 5 -RACE for GHS-R For determination of the 5 region, we used the Straight Walk Kit (Bex Co. Ltd., Tokyo, Japan) and suncus genomic DNA according to manufacturer’s instructions. BamHI, Bg/II, Bc/I, MboI, Sau3AI, SpeI, NheI, XbaI, and AvrII were used to cut the genomic DNA and bind the adaptor sequence. The one nucleotide extension and the addition of the adaptor sequence are shown below (RWA-1 was used for BamHI, Bg/II, Bc/I, MboI, and Sau3AI; RWA-2 was used for SpeI, NheI, XbaI, and AvrII). RWA-1: 5 CGCAGGCTGGCAGTCTCTTTAGGGTTACACGATTGCTT-3 , 5 -(PO4 ) ATCAAGCAATCGTGT (NH2 )-3 ; RWA-2: 5 -CGCAGGCTGGCAGTCT CTTTAGGGTTACACGATTGCTT-3 , 5 -(PO4 ) TAGAATCAATCGTGT (NH2 )-3 . After the addition of the adaptor sequence, we amplified and sequenced the fragments. The adaptor (WP-1 and WP-2, sense) and specific primers (SP-1 and SP-2, anti-sense) were designed using Primer Express v1.0 (ABI). Primary PCR using primers WP-1 and SP-1 along with KOD-Plus-Taq polymerase was performed under the following conditions: 94 ◦ C for 2 min, followed by 35 cycles at 94 ◦ C for 30 s, 65 ◦ C for 30 s, and 68 ◦ C for 5 min. The second PCR was performed using the SpeI treatment products and primers WP2 and SP2. The PCR conditions were the same as those for the primary PCR. The products from the second PCR were then sequenced. We obtained the sequences of the 5 region of suncus GHS-R using the Straight Walk technique, therefore we performed PCR to confirm that the sequences exist in suncus hypothalamic cDNA. We designed the primers (sGHS-R 5 -UTR FWD1 and sGHS-R 5 -UTR BWD2) and amplified suncus hypothalamic cDNA using AmpliTaq Gold. The amplification reaction was performed under the following conditions: 95 ◦ C for 5 min, followed by 40 cycles at 94 ◦ C for 1 min, 56 ◦ C for 1 min, and 60 ◦ C for 10 min. The amplified fragments were then sequenced as described previously. 2.7. Cloning of suncus GPR38 To obtain the partial suncus GPR38 sequence, we designed degenerate primers (sGPR38 FWD8, sGPR38 FWD18, sGPR38 BWD7, sGPR38 BWD14) using Primer3 software and referring to the GPR38 sequence in humans (accession no. ENSG00000102539),

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rabbits (accession no. ENSOCUG00000014737), cats (accession no. ENSFCAG00000014268), and monkeys (accession no. ENSMMUG00000007691) in Ensembl database. PCR was performed using suncus genomic DNA with sGPR38 FWD8 and sGPR38 BWD7 primers and Ex Taq polymerase. The amplification reaction was performed under the following conditions: 94 ◦ C for 5 min, followed by 35 cycles at 94 ◦ C for 1 min, 68 ◦ C for 1 min, and 74 ◦ C for 1 min. The amplified fragments were run on 2% agarose gels and visualized on a UV illuminator after ethidium bromide staining. The amplified fragments were cloned and sequenced as for GHS-R. The obtained sequences were then submitted to a BLAST search (NCBI). After confirming the sequence homology, PCR was performed using suncus stomach cDNA under the same PCR conditions. The amplification reaction was performed under the following conditions: 94 ◦ C for 5 min, followed by 40 cycles at 94 ◦ C for 1 min, 63 ◦ C for 1 min, and 74 ◦ C for 1 min. The amplified fragments were then cloned into pGEM T-easy vectors and sequenced. 2.8. 3 -RACE for GPR38 In the primary PCR, cDNA was amplified with sGPR38 FWD19 (sense) and the adapter primer for 3 -RACE (anchor out) using Ex Taq polymerase. The amplification reaction was performed under the following conditions: 94 ◦ C for 5 min, followed by 40 cycles at 94 ◦ C for 1 min, 62 ◦ C for 1 min, and 74 ◦ C for 1 min. The second PCR was performed using diluted products from the primary PCR as templates with sGPR38 FWD20 and 3 RACE anchor (anchor in) primers along with Ex Taq polymerase. The amplification reaction was performed under the following conditions: 94 ◦ C for 5 min, followed by 40 cycles at 94 ◦ C for 1 min, 65 ◦ C for 1 min, and 74 ◦ C for 1 min. The amplified fragments from the third PCR were then cloned into pGEM T-easy vectors and sequenced. 2.9. 5 -RACE for GPR38 The Straight Walk Kit (Bex Co. Ltd.) was also used to determine the 5 region of GPR38. BamHI, Bg/II, Bc/I, MboI, Sau3AI, SpeI, NheI, XbaI, and AvrII restriction enzymes were used. After the addition of the adaptor sequence, the amplified fragments were sequenced. The adaptor primers (WP-1 and WP-2, sense) and specific primers (SP-1 and SP-2, anti-sense) were designed using Primer Express v1.0 (ABI). Primary PCR using primers WP-1 and SP-1 and KOD-Plus-Taq polymerase was performed under the following conditions: 94 ◦ C for 2 min, followed by 35 cycles at 94 ◦ C for 30 s, 65 ◦ C for 30 s, and 68 ◦ C for 5 min. The second PCR was performed using products amplified after Bc/I treatment and primers WP2 and SP2. The PCR conditions were the same as those for the primary PCR. The products from the second PCR were then sequenced. We obtained the sequences of the 5 region of suncus GPR38 using the Straight Walk technique and therefore performed PCR to confirm that the sequences exist in suncus cDNA. We designed the primers (sGPR38 5 -UTR FWD37 and sGPR38 5 -UTR BWD26) and amplified suncus hypothalamic cDNA using PrimeSTAR GXL DNA polymerase (Takara Bio). The amplification reaction was performed under the following conditions: 94 ◦ C for 1 min, followed by 30 cycles at 98 ◦ C for 10 s, 65 ◦ C for 50 s, and 72 ◦ C for 10 min. The amplified fragments were then reamplified using LA Taq polymerase (Takara Bio) to add an “A-tail” under the following PCR conditions: 94 ◦ C for 1 min, followed by 30 cycles at 98 ◦ C for 10 s, 67 ◦ C for 45 s, and 72 ◦ C for 10 min. The final amplified fragments were cloned and sequenced as described previously. 2.10. RT-PCR for GHS-R, GPR38, and their ligands in tissues Total RNA was extracted from the tissues of nine regions of the gastrointestinal tract (gastric fundus, gastric corpus, gastric

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TCCCTGCGCCCCGAGCTCTAGCCTCCCTCAAACTCTAAAGGGACCCCCTCGGGAGCCTCG CCGGAGCCCCTCGGCAGCCCAAGAGCCCCGCTCCCCAATTCTAGCAGCATGTGGAACGCG M W N A ACCCCGAGCGCCAATGAATCGGTCCCCAACTTCACACTGCCCGAGCCCGACTGGGACGCG T P S A N E S V P N F T L P E P D W D A GCCGACAACAACGCGTCGCTGTCTGACGAGCTGTTGCATCTCTTCCCCGCGCCGCTCCTG A D N N A S L S D E L L H L F P A P L L GCCGGGGTGACGGCCACCTGCGTGGCGCTCTTCGTGGTGGGCATCGCGGGCAACCTGCTC A G V T A T C V A L F V V G I A G N L L ACCATGCTGGTGGTGTCGCGTTTCCGCGAGCTGCGGACCACCACCAACCTCTACCTGTCC T M L V V S R F R E L R T T T N L Y L S AGCATGGCCTTCTCCGACCTGCTCATCTTCCTCTGCATGCCGCTCGACCTGGTGCGCCTC S M A F S D L L I F L C M P L D L V R L TGGCAGTACCGACCCTGGAACTTGGGCGACCTGCTCTGCAAACTCTTCCAGTTCGTGAGC W Q Y R P W N L G D L L C K L F Q F V S GAGAGCTGCACCTACGCCACGGTGCTCACCATCACCGCGCTCAGCGTCGAGCGCTACTTC E S C T Y A T V L T I T A L S V E R Y F GCCATCTGCTTCCCGCTGCGGGCCAAAGTGGTGGTCACCAAGGGCCGGGTGAAGCTGGTC A I C F P L R A K V V V T K G R V K L V ATCCTGGTCATCTGGGCCGTGGCCTTCTGCAGCGCCGGGCCCATCTTCATGCTGGTCGGG I L V I W A V A F C S A G P I F M L V G GTGGAACACGAGAACGGCACCGACCCCCGGGACACCAACGAGTGCCGCGCCACCGAGTTT V E H E N G T D P R D T N E C R A T E F GCGGTGCGCTCGGGGCTCCTCACGGTCATGGTGTGGGTGTCCAGCGTGTTCTTTTTCCTG A V R S G L L T V M V W V S S V F F F L CCCGTCTTCTGCCTCACCGTCCTTTACAGCCTCATCAGCAGGAAGCTGTGGCGCCGAGGG P V F C L T V L Y S L I S R K L W R R G CGCGGAGAAGCGGCGGTGGGCACCTCTGTCCGAGATCAGAACCACAAGCAAACTGTGAAG R G E A A V G T S V R D Q N H K Q T V K ATGCTGGCTGTCGTGGTATTTGCTTTCATCCTATGCTGGTTGCCCTTCCATGTAGGACGA M L A V V V F A F I L C W L P F H V G R TATTTATTTTCCAAATCCTTTGAACCTGGTTCCGTTGAGATTGCTCAGATCAGCCAATAC Y L F S K S F E P G S V E I A Q I S Q Y TGCAACCTGGTATCTTTTGTCCTCTTCTACCTCAGTGCAGCCATCAACCCCATTCTGTAC C N L V S F V L F Y L S A A I N P I L Y AACATCATGTCAAAGAAGTACCGGGTGGCAGTGTTCAAACTTCTGGGACTTGAACCTTTC N I M S K K Y R V A V F K L L G L E P F TCCCAGAGGAAGCTTTCCACGCTGAAGGATGAAAGTTCTCGTGCTTGGATAGAATCTAGT S Q R K L S T L K D E S S R A W I E S S ATTAATACGTGACCAATATAGCTGAGCAAAGTCATTACTTATTATTCTAAACCAGAAGCC I N T * ATAGCCCAGCAGGACTTGGGAGGAAGTTGAAGGTTAATTTTGGAATTAGTAATACATAGA AAACAATTGTAAACAATTGGAAGAAGTGAGAAGATAGAATTTACAGTGTGTGAGCAGTTG GGTTAAATTGCACACTCATCCGTGTTCTAATACACTCTGCATATTCTGAATTTTGCATTC TATGATTTTGCATTCTGCTTTTGGTGCTAGTCAGGGCTTTGTAAGGTTAAGAATGATGGA AGTGCAGAGAGGGCAATTATAGCTTTTATTACCAAGTAATTATTAAATCCAATTTCCTTT TTCATAATAGTAAAACAATTTTGATTAAAAAAAAAAAAAAAAA 1603

60 120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 1560

Fig. 1. cDNA and the predicted amino acid sequences of suncus GHS-R. The arrowhead indicates the position of the intron. A potential polyadenylation signal has been underlined.

antrum, and intestine 1–6), the hypothalamus, medulla oblongata, lungs, heart, liver, pancreas, kidneys, adrenal glands, spleen, pituitary gland, nodose ganglion and testes from male adult suncus using ISOGEN and in accordance with the manufacturer’s instructions. In the gastric fundus, gastric corpus, and gastric antrum, the mucosal layers were separated from the muscle layers with a glass slide; only the muscle layers were frozen with liquid nitrogen and broken using CRYO PLUS (Microtech Co. Ltd., Chiba, Japan) before being dipped in ISOGEN. First-strand cDNA was synthesized from 1 ␮g (nodose ganglion, 150 ng) of total RNA with random primers using ReverTra Ace reverse transcriptase (Toyobo, Osaka, Japan). The tissue distribution of mRNA expression of GHS-R, GPR38, ghrelin, and motilin was analyzed using the primers shown in Table 2. The amplification reactions of GPR38, ghrelin, motilin and ␤-actin were performed under the following conditions using AmpliTaq Gold: 95 ◦ C for 5 min; 40 cycles of 94 ◦ C for 1 min, and then 55 ◦ C (GPR38, motilin), 60 ◦ C (␤-actin) or 63 ◦ C (ghrelin) for 1 min; followed by 60 ◦ C for 10 min. The mRNA expression of

GHS-R was amplified using One-step RT-PCR kit (QIAGEN) using 250 ng (hypothalamus, medulla oblongata, lungs, heart, liver, pancreas, kidneys, adrenal glands, spleen, pituitary gland, nodose ganglion and testes) or 500 ng (gastrointestinal tract) total RNA

Table 2 Primers used in RT-PCR for tissue distribution. Primer name

Direction

Sequence

sGhrelinF sGhrelinR sMotilinF sMotilinR sGHS-RF sGHS-RR sGPR38F sGPR38R sActinF sActinR

Sense Anti-sense Sense Anti-sense Sense Anti-sense Sense Anti-sense Sense Anti-sense

GAAAGGAGCCCAAGAAGC ACTGGGGTCACGGTGAATAG GGACCCCACAGATGGAGAAGAA CAGCTTAGTGCACCTTCTCCCT CAGTCAGGAGGAGCTTGGAG CTGGCCTTGCTGATGGTACT CGAGATCAGAACCACAAGCA CTACATGGAAGGGCAACCAG TGCGTGACATCAAGGAGAAG GACAGCACTGTGTTGGCATA

A. Suzuki et al. / Peptides 36 (2012) 29–38

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TM 1 suncus human rat

1 MWNATPSANESVPNFTLPEPDWDAADNNASLSDELLHLFPAPLLAGVTATCVALFVVGIA 1 MWNATPSE-EPGFNLTLADLDWDASPGNDSLGDELLQLFPAPLLAGVTATCVALFVVGIA 1 MWNATPSE-EPEPNVTL-DLDWDASPGNDSLPDELLPLFPAPLLAGVTATCVALFVVGIS ******* *. *.** : ****: .* ** **** **********************:

60 59 58

TM 2

suncus human rat

61 GNLLTMLVVSRFRELRTTTNLYLSSMAFSDLLIFLCMPLDLVRLWQYRPWNLGDLLCKLF 120 60 GNLLTMLVVSRFRELRTTTNLYLSSMAFSDLLIFLCMPLDLVRLWQYRPWNFGDLLCKLF 119 59 GNLLTMLVVSRFRELRTTTNLYLSSMAFSDLLIFLCMPLDLVRLWQYRPWNFGDLLCKLF 118 ***************************************************:********

suncus human rat

121 QFVSESCTYATVLTITALSVERYFAICFPLRAKVVVTKGRVKLVILVIWAVAFCSAGPIF 180 120 QFVSESCTYATVLTITALSVERYFAICFPLRAKVVVTKGRVKLVIFVIWAVAFCSAGPIF 179 119 QFVSESCTYATVLTITALSVERYFAICFPLRAKVVVTKGRVKLVILVIWAVAFCSAGPIF 178 *********************************************:**************

suncus human rat

181 MLVGVEHENGTDPRDTNECRATEFAVRSGLLTVMVWVSSVFFFLPVFCLTVLYSLISRKL 240 180 VLVGVEHENGTDPWDTNECRPTEFAVRSGLLTVMVWVSSIFFFLPVFCLTVLYSLIGRKL 239 178 VLVGVEHENGTDPRDTNECRATEFAVRSGLLTVMVWVSSVFFFLPVFCLTVLYSLIGRKL 238 :************ ******.******************:****************.***

suncus human rat

241 WRRGRGEAAVGTSVRDQNHKQTVKMLAVVVFAFILCWLPFHVGRYLFSKSFEPGSVEIAQ 300 240 WRRRRGDAVVGASLRDQNHKQTVKMLAVVVFAFILCWLPFHVGRYLFSKSFEPGSLEIAQ 299 239 WRRR-GDAAVGASLRDQNHKQTVKMLAVVVFAFILCWLPFHVGRYLFSKSFEPGSLEIAQ 298 *** *:*.**:*:*****************************************:****

suncus human rat

301 ISQYCNLVSFVLFYLSAAINPILYNIMSKKYRVAVFKLLGLEPFSQRKLSTLKDESSRAW 360 300 ISQYCNLVSFVLFYLSAAINPILYNIMSKKYRVAVFRLLGFEPFSQRKLSTLKDESSRAW 359 299 ISQYCNLVSFVLFYLSAAINPILYNIMSKKYRVAVFKLLGFESFSQRKLSTLKDESSRAW 358 ************************************:***:*.*****************

suncus human rat

361 IESSINT 367 360 TESSINT 366 359 TKSSINT 364 :*****

TM 4

TM 3

TM 5

TM 6

TM 7

Fig. 2. Multiple amino acid sequence comparisons of GHS-R in suncus, humans, and rats. Asterisks indicate amino acids identical across all species. Dots indicate that more than half of the amino acids are identical across all species. The amino acid sequences have been sourced from the DDBJ/EMBL/GenBank databases: humans (NM 198407) and rats (NM 032075). The deduced protein shared high identity with those of rats (91%) and humans (93%).

as template. The amplification reactions of GHS-R were performed under the following conditions: 50 ◦ C for 30 min and then 95 ◦ C for 15 min; 40 cycles of 94 ◦ C for 1 min, 60 ◦ C for 1 min and then 72 ◦ C for 1 min; followed by 72 ◦ C for 10 min. The PCR products were run on 2% agarose gels and visualized by ethidium bromide staining. 3. Results 3.1. Identification of suncus GHS-R mRNA Suncus GHS-R mRNA was cloned from hypothalamic cDNA. The sequence of suncus GHS-R (1603 bp) was found to consist of a 108-bp 5 -untranslated region (UTR), a 1104-bp coding region, and a 391-bp 3 -UTR. The results of sequence analysis of suncus genomic DNA indicated that there is an approximately 2000-bp intron between the positions 907 and 908 in suncus GHS-R, which consists of 2 exons (Fig. 1). The open reading frame (ORF) of the cDNA encoded a 367-amino acid polypeptide. TMHMM analysis showed that suncus GHS-R has seven transmembrane structures: TM1, 45–67 (23 AA); TM2, 80–102 (23 AA); TM3, 28–150 (23 AA); TM4, 162–185 (24 AA); TM5, 215–236 (22 AA); TM6, 265–288 (24 AA); and TM7, 307–326 (20 AA). These transmembrane regions were consistent with those of other species (Fig. 2). At the amino acid level, the deduced protein shared high identity with that of the rats (91%) and humans (93%) (Fig. 2), and the alignment score of the transmembrane regions showed extremely high identity with that of humans (98%), rats (99%), rabbits (98%), cows (99%), pigs (99%), and dogs (93%) (data not shown).

3.2. Identification of suncus GPR38 mRNA Suncus GPR38 mRNA was cloned from cDNA derived from the hypothalamus and stomach. The sequence of suncus GPR38 (1437 bp) was found to consist of a 138-bp 5 -UTR, a 1143-bp coding region, and a 156-bp 3 -UTR. Sequence analysis of suncus genomic DNA showed that there was an approximately 1000-bp intron between the positions 946 and 947 of suncus GPR38, which consisted of 2 exons (Fig. 3). The ORF of the GPR38 cDNA encoded a 380-amino acid polypeptide. TMHMM analysis indicated that suncus GPR38 has seven transmembrane structures: TM1, 32–54 (23 AA); TM2, 61–83 (23 AA); TM3, 103–125 (23 AA); TM4, 146–168 (23 AA); TM5, 216–238 (23 AA), TM6, 268–290 (23 AA); and TM7, 305–327 (23 AA). These transmembrane regions were almost consistent with those of other species (Fig. 4). At the amino acid level, the deduced protein shared high identity with that of humans (76%), dogs (70%) (Fig. 4), rabbits (74%), and cows (77%) (data not shown). The alignment score of the transmembrane region shared extremely high identity with that of humans (87%), dogs (91%) (Fig. 4), rabbits (90%), and cows (91%) (data not shown). The alignment score between GHS-R and GPR38 was 42% for the total sequence region and 62% for the transmembrane region (Fig. 5).

3.3. Distribution of GHS-R and GPR38 mRNA expression in male suncus tissues RT-PCR was performed to determine the distribution of ghrelin and GPR38 in various tissues. In the gastrointestinal tract, ghrelin

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AGCAGACCCTGGATTGAACTGAAGAAGAGCTCGGGTCCCTCCGCACAACGCGCGGGGCCT 60 GGGTTTGGAAAGCTTCCCCAGTGCGTGCGGGGCTTCTCGGGGTGCCGCGGGCTAAGGGAA 120 GCCCCCTCCCGAGGGCTCATGGACAGTCCCTGGAACCGGAGCGCCCCGGCCCCGCTGCCT 180 M D S P W N R S A P A P L P TGCGACGAGCGCCGCTGCTCGGCCTTCCCGCTGGGCGCGCTGGTGCCGGTGACCGCCGTG 240 C D E R R C S A F P L G A L V P V T A V TGCCTCGGGCTGTTCGCCGTCGGGGTGAGCGGCAACGCGGTGACGGTGCTGCTGGTGTGG 300 C L G L F A V G V S G N A V T V L L V W CGCGCCCGGGCGCTGCGGAGCGCCACCAACTCGTACCTGTGCAGCATGGCCGCGTCGGAC 360 R A R A L R S A T N S Y L C S M A A S D CTGCTCATCCTGCTGGGGCTGCCCTGGGACCTGTACCGCCTGTGGCGCTCGCGGCCCTGG 420 L L I L L G L P W D L Y R L W R S R P W GTCTTCGGCTCGCTGCTCTGCCGCCTGTCGCTCTACCTGGGCGAGGGCGGCACCTACGCC 480 V F G S L L C R L S L Y L G E G G T Y A ACGCTGCTGCACATGACGGCGCTCAGCGTCGAGCGCTACCTGGCCGTCTGCCGCCCGCTC 540 T L L H M T A L S V E R Y L A V C R P L CGGGCCCGCCGCGTCCTGGGCAGCCGGCGGCGCGTCCGCGCGCTCATCGCGGCGCTCTGG 600 R A R R V L G S R R R V R A L I A A L W GCCGTGGCGCTGCTCTCCGCCGCCCCCTTCTTCTTCCTCGTGGGCGTCGAGCAGGACCCC 660 A V A L L S A A P F F F L V G V E Q D P GCGTCCTCCGACGGGGCCCCCCGGCTCAACGCCACCGCCCCGGGCTCCGACGCCGCCCGG 720 A S S D G A P R L N A T A P G S D A A R GCTGCGGCCGCGCTCTTCAGCCGCGAGTGCCGGCCCAGCCCCGAGCAGGAGGTCGCCCTG 780 A A A A L F S R E C R P S P E Q E V A L CGCGTCATGCTGTGGGTCACCACCGCCTACTTCTTCCTCCCTTTTCTGTGCCTCAGCGTC 840 R V M L W V T T A Y F F L P F L C L S V CTCTACGGGCTCATCGGGCGCGAGCTCTGGAGGACCCGGGGCCCCCTGGAAGGCATCGCT 900 L Y G L I G R E L W R T R G P L E G I A GCCCCCCGGCGGGAGAAGGACCACAGGCAGACCATCCGCGTCCTGCTGGTGGTGGTTATA 960 A P R R E K D H R Q T I R V L L V V V I GCGTTTGTAGTTTGCTGGTTGCCTTTCCATGTGGGAAGAATCATCTACATAAACACCAAG 1020 A F V V C W L P F H V G R I I Y I N T K GACACCCGGACAATGTACTTCTCTCAGTACTTCAACATTGTTGCTTTGCAACTTTTCTAC 1080 D T R T M Y F S Q Y F N I V A L Q L F Y TTGAGCGCCTCCATTAATCCCATCCTCTACAACCTCATCTCAAAAAAGTATAGAGCTGCT 1140 L S A S I N P I L Y N L I S K K Y R A A GCCTCCAAACTACTATGGGCCCGAAAGTCCCCACACAGAGTTTTCTGCAGTGGTGGACAC 1200 A S K L L W A R K S P H R V F C S G G H AAGGAGGACAGAAGAAGAGACACTGCAAGCCATACAGAGACCAGCTCTAACATAAAGACA 1260 K E D R R R D T A S H T E T S S N I K T TTGGCTACCAGCACAATCTAGCTTGTCACCTCCTTGCATGGTCCTGGAACTTCAGAAAAA 1320 L A T S T I * AACAGATCTGTAGGGAAATAAACATGGAAAGGGAGGAGGTGACACTGTGAAGAATAGAGA 1380 GTGCGATATTTTGCAATTTTAGCAGAAACTGTGCATAATTATCAGGGTACTGAAAGG 1437 Fig. 3. cDNA and the predicted amino acid sequences of suncus GPR38. The arrowhead indicates the position of the intron. A potential polyadenylation signal has been underlined.

mRNA was expressed in the mucosal layer of the stomach fundus, corpus, and antrum, and was also expressed in intestine (1–2 and 5–6) (Fig. 6A). GHS-R mRNA expression was detected throughout the stomach and intestine (Fig. 6A). In terms of the extra-GI tract tissues, GHS-R mRNA was found in the hypothalamus, medulla oblongata, adrenal gland, nodose ganglion and pituitary gland (Fig. 6B). Motilin mRNA was expressed throughout all intestinal tissues (intestine 1–6), but was restricted to the intestine (Fig. 6A). GPR38 mRNA was expressed in the muscle layer of the stomach fundus, corpus, and antrum, as well as in the mucosal layer of the fundus and in all intestinal tissues (intestine 1–6) (Fig. 6A). GPR38 mRNA was also found in the hypothalamus, medulla oblongata, lungs, heart, pituitary gland, and nodose ganglion (Fig. 6B). 4. Discussion Common laboratory rodents such as rats and mice lack genes for motilin and its receptor (GPR38) [14], and thus are not considered suitable animals for the study of motilin. We recently focused on the S. murinus, which belongs to the order Insectivora, and reported that fasted suncus showed clear repeated MMCs like those found in

humans and dogs. We also identified the complete cDNA sequences and tissue distribution of motilin and ghrelin [17,49]. Moreover, we demonstrated that suncus motilin stimulates gastric contractions in a dose-dependent manner [40,49]. However, the tissue distribution of GHS-R and GPR38 in suncus is not known. To study the mechanisms of motilin and ghrelin in the suncus, we determined the genes for suncus GHS-R and GPR38 and examined their distribution in tissues. The putative suncus GHS-R consists of 367 amino acids and seven transmembrane structures that are well known in other species. The deduced amino acid sequence showed high homology with the GHS-R identified in humans, rats, rabbits, and cows (91%, 93%, 91%, and 93%, respectively), and the transmembrane region showed extremely high homology (>98%). The relationship between protein sequence and function in GHS-R has been studied previously, and it has been reported that the Cys Cys disulfide bonds at Cys116 in extracellular loop 1, Cys198 in extracellular loop 2 [9,36], and Met213 , Gln124 , and Glu184 are important for ligand binding [50]. These amino acids are all conserved in suncus GHS-R. On the other hand, two types of GHS-R, an active form (GHS-R1a) and an inactive form (GHS-R1b), have been identified in mammals

A. Suzuki et al. / Peptides 36 (2012) 29–38

35

TM 1 suncus human dog

1 MDSPWNRSAPAP----------LPCDERRCSAFPLGALVPVTAVCLGLFAVGVSGNAVTV 1 MGSPWNGSDGPEGAREPPWPALPPCDERRCSPFPLGALVPVTAVCLCLFVVGVSGNVVTV 1 MGGPGNSSDGAEGAQLP-------CDERLCSPFPLGALVPVTAVCLGLFAVGVSGNLVTV **** **.************** **.****** *** * .* * * .

50 60 53

TM 3

TM 2 suncus human dog

51 LLVWRARALRSATNSYLCSMAASDLLILLGLPWDLYRLWRSRPWVFGSLLCRLSLYLGEG 110 61 MLIGRYRDMRTTTNLYLGSMAVSDLLILLGLPFDLYRLWRSRPWVFGPLLCRLSLYVGEG 120 54 LLIGRYRDMRTTTNLYLGSMAVSDLLILLGLPLDLYRLWRSRPWVFGQLLCRLSLYLGEG 112 :*: * * :*::** ** ***.********** ************** ********:***

suncus human dog

111 GTYATLLHMTALSVERYLAVCRPLRARRVLGSRRRVRALIAALWAVALLSAAPFFFLVGV 170 121 CTYATLLHMTALSVERYLAICRPLRAR-VLVTRRRVRALIAVLWAVALLSAGPFLFLVGV 179 114 CTYATLLHVTALSVERYLAVCRPLRAR-ALLSRRRARALIAALWAVALLSAAPFFFLVGV 172 *******:******************* ** ***.*****.*********.**:*****

suncus human dog

171 EQDPASSDGAPRLN----------------------ATAPGSDAARAAAALFSRECRPSP 208 180 EQDPGISVVPGLNGTARIASSPLASSPPLWLSRAPPPSPPSGPETAEAAALFSRECRPSP 239 173 EQDAGG---PGLNGSARLAR---------------APSPPPGPE----AALFSRECRPSP 210 . . .:.* . *********** ****..

suncus human dog

209 EQEVALRVMLWVTTAYFFLPFLCLSVLYGLIGRELWRTRGPLEGIAAPRREKDHRQTIRV 268 240 AQLGALRVMLWVTTAYFFLPFLCLSILYGLIGRELWSSRRPLRGPAASGRERGHRQTVRV 299 211 SQLGALRVMLWVTTAYFFLPFLCLCVLYGRIGRELRRRRGPLRGRAASGRERGHRQAVRV 270 * **.* **. **:.***::** * ********************.:*** *****

suncus human dog

269 LLVVVIAFVVCWLPFHVGRIIYINTKDTRTMYFSQYFNIVALQLFYLSASINPILYNLIS 328 300 LLVVVLAFIICWLPFHVGRIIYINTEDSRMMYFSQYFNIVALQLFYLSASINPILYNLIS 359 271 LLAVVLAFLVCWLPFHVGRIIYINTEDSRMMHFSQYFNIVALQLFYLSASINPILYNLIS 330 **.**:**::***************:*:* *:****************************

suncus human dog

329 KKYRAAASKLLWARKSPHRVFCSGG-------------HKEDRRRDTASHTETSSNIKTL 375 360 KKYRAAAFKLLLARKSRPRGFHRS--------RDTAGEVAGDTGGDTVGYTETSANVKTM 411 331 KKYRAAARKLLLPRRPTRRRVCRSGAVEGGPGRAAAGLAETGPHGHTAASPSAARQVASR 390 . .*.. ..:: :: : ******* *** .*:. * . .

suncus human dog

376 ATSTI------ 380 412 G---------- 412 391 HDPGTSGKTGV 401

TM 4

TM 5

TM 6

TM 7

Fig. 4. Multiple amino acid sequence comparisons of GPR38 in suncus, humans, and dogs. Asterisks indicate amino acids identical across all species. Dots indicate that more than half of the amino acids are identical across all species. The amino acid sequences have been sourced from the DDBJ/EMBL/GenBank databases: humans (NM 001507) and dogs (AB436535). The deduced protein shared high identity with those of humans (76%) and dogs (70%).

[5]. GHS-R1a shows strong biological activity against synthesized GHS and a natural ligand, acylated ghrelin [5]. However, a splicing variant of GHS-R, GHS-R1b, consists of five transmembrane structures caused by a stop codon at the end of the third intracellular loop and is believed to be a non-functional receptor [50]. In this study, the GHS-R 1a type receptor, but not GHS-R1b, was cloned. However, because GHS-R1b has been found to be expressed in certain regions only, further identification and examination of GHS-R 1b in suncus may be required. The deduced suncus GPR38 consists of 380 amino acids and seven transmembrane structures, and the GPR38 protein sequence was homologous with that of other animals, including humans, dogs, rabbits, and cows (76%, 70%, 74%, and 77%, respectively). The transmembrane region showed especially high homology (>87%). The suncus GPR38 has a long second extracellular loop that is identical to that of other species and is a characteristic of GPR38 [26]. Matsuura et al. reported that both ends of this loop (Val170 , Leu214 , Arg215 ) represent conserved domains and are functionally important for the binding and activity of motilin, whereas the non-conserved residues in the middle region of the loop are not necessary for binding [26]. Gly26 , Pro93 , Leu99 , and Phe301 are also important for motilin binding [27]. These seven amino acids are not important for erythromycin binding, whereas it has been reported that glutamine (Glu109 ) in the third transmembrane region is important for the binding of both motilin and erythromycin [27].

GPR38 also forms a Cys Cys disulfide bond at cysteine residues in extracellular loops 1 and 2 (Cys101 and Cys204 ), which are important for the physiological activity of motilin [27,47]. We revealed that these important amino acid residues for ligand binding are all conserved in suncus GPR38. In the gastrointestinal tract, suncus GHS-R was expressed in the stomach and intestine, and this distribution of GHS-R was almost consistent with that of other species, suggesting that GHS-R in the gastrointestinal tract is involved in the regulation of gastric motility [46] or cell proliferation, as has been reported in other animals [7]. GHS-R was also detected in the hypothalamus, medulla oblongata, pituitary gland and nodose ganglion in suncus. It has also shown that GHS-R is involved in feeding behavior and growth hormone secretion in rats. The effect of increased food intake and growth hormone secretion by ghrelin was blocked in vagotomized rats [4,41]. Thus, it would be possible that GHS-R in the nodose ganglion of Suncus plays a role in growth hormone secretion and feeding behavior as in rodents. It is well known that GHS-R is expressed in the arcuate nucleus of the hypothalamus and is important for feeding behavior in rodents [56,57]. Although further studies are needed to elucidate the effect of ghrelin on food intake in suncus, it is possible that ghrelin stimulates food intake by activating GHS-R in the arcuate nucleus in the suncus. It has also been reported that GHS-R in the nodose ganglion is involved in the regulation of growth hormone release and food intake [4,41]; thus, GHS-R expression in

36

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GPR38 GHS-R

GPR38 GHS-R

GPR38 GHS-R

GPR38 GHS-R

GPR38 GHS-R

GPR38 GHS-R

GPR38 GHS-R

TM 1 ----------------MDSPWNRSAPAPLPCDERRCSAFPLGALVPVTAVCLGLFAVGVS MWNATPSANESVPNFTLPEPDWDAADNNASLSDELLHLFPAPLLAGVTATCVALFVVGIA : .* :* . .:. ** *. ***.*:.**.**:: TM 2 TM 3 GNAVTVLLVWRARALRSATNSYLCSMAASDLLILLGLPWDLYRLWRSRPWVFGSLLCRLS GNLLTMLVVSRFRELRTTTNLYLSSMAFSDLLIFLCMPLDLVRLWQYRPWNLGDLLCKLF ** :*:*:* * * **::** **.*** *****:* :* ** ***: *** :*.***:* TM 4 LYLGEGGTYATLLHMTALSVERYLAVCRPLRARRVLGSRRRVRALIAALWAVALLSAAPF QFVSESCTYATVLTITALSVERYFAICFPLRAKVVVTKG-RVKLVILVIWAVAFCSAGPI ::.*. ****:* :********:*:* ****: *: . **: :* .:****: **.*: TM 5 FFLVGVEQDPASSDGAPRLNATAPGSDAARAAAALFSRECRPSPEQEVALRVMLWVTTAY FMLVGVEHENG----------TDPRDTNECRATEFAVRSG--------LLTVMVWVSSVF *: : *. * **:**::.: *:*****:: . * * . TM 6 FFLPFLCLSVLYGLIGRELWRTRGPLEGIAAPRREKDHRQTIRVLLVVVIAFVVCWLPFH FFLPVFCLTVLYSLISRKLWRRGRGEAAVGTSVRDQNHKQTVKMLAVVVFAFILCWLPFH ****.:**:***.**.*:*** .:.:. *:::*:**:::* ***:**::****** TM 7 VGRIIYINTKDTR---TMYFSQYFNIVALQLFYLSASINPILYNLISKKYRAAASKLLWA VGRYLFSKSFEPGSVEIAQISQYCNLVSFVLFYLSAAINPILYNIMSKKYRVAVFKLLGL *** :: :: :. :*** *:*:: ******:*******::*****.*. *** RKSPHRVFCSGGHKEDRRRDTASHTETSSNIKTLATSTI EPFSQRKLSTLKDESSRAWIESSINT------------:* . . .:* :.: .:..*

Fig. 5. Comparison of GHS-R and GPR38. Asterisks show identical amino acids. The alignment score was 42% for the total sequence region and 62% for the transmembrane region.

the nodose ganglion of the suncus may act in the same manner. On the other hand, suncus GPR38 was expressed in the gastric muscle layer, gastric fundus mucosa, and lower intestine, which is also consistent with the observation in other species [9,34,53]. In addition, it has been reported that motilin stimulates intestinal contraction in some species, suggesting that motlin-GPR38 system is involved in the contraction not only in the stomach but also in

suncus intestines [6,13,45]. We previously studied the effect of motilin on gastric contraction using an organ bath system, and found that motilin-induced contractions were completely abolished by tetrodoxin treatment. It appears that motilin directly stimulates GPR38 expressed in the gastric myenteric plexus [29]. As small laboratory animals have not been previously available for motilin studies, there is currently little information on the effect of

Fig. 6. Distributions of GHS-R, ghrelin, GPR38, and motilin in the gastrointestinal tract, as detected by RT-PCR. (A) In the gastrointestinal tract, GHS-R mRNA was detected in all regions of stomach and intestine. GPR38 mRNA was found in all regions of the muscle layer of the stomach and throughout the intestine. Expression of ghrelin and motilin mRNA was restricted to the gastric mucosa and intestine, respectively. (B) In terms of other central and peripheral tissues, GHS-R and GPR38 mRNA expression were detected in the hypothalamus and medulla oblongata. In addition, GPR38 mRNA was expressed in the lungs, heart, pituitary gland, and nodose ganglion. GHS-R mRNA expression was detected in the adrenal gland, pituitary gland and nodose ganglion. NC; negative control.

A. Suzuki et al. / Peptides 36 (2012) 29–38

motilin on the central nervous system. However, it would be very interesting to examine the effect of motilin on the brain in terms of the regulation of food intake or energy homeostasis because of the observed high expression of GPR38 in the hypothalamus and nodose ganglion. As many researchers have believed for several decades that motilin from the duodenum binds to GPR38 expressed in the axonal terminals of afferent nerves and then its signals are transmitted to the brainstem for the regulation of MMC, the nodose ganglion may play a role in mediating the effect of motilin in some physiological functions. On the other hand, GPR38 expression was also found in the lungs and heart, suggesting that motilin has an unknown physiological function in the respiratory and cardiovascular systems, and thus further studies are required. These would be relatively easy to perform using a suncus model. In summary, we cloned the GHS-R and GPR38 genes in suncus and revealed their tissue distribution. This is the first report of a small laboratory animal model that expresses GHS-R and GPR38. As we have already identified the amino acid sequences of motilin and ghrelin and shown their physiological effect on gastric contraction in suncus [30], identification of their specific receptors should enable us to undertake a more detailed analysis of their physiological mechanisms.

[17]

[18]

[19]

[20]

[21]

[22]

[23]

[24]

[25]

Acknowledgments

[26]

We thank Prof. Minoru Tanaka and Mr. Satoya Hoshino for their technical assistance.

[27]

[28]

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