Poult Sci 2007. 86:2472-2476. doi:10.3382/ps.2007-00206
© 2007 Poultry Science Association
Primary Structure and Tissue Distribution of GPR39 Messenger Ribonucleic Acid in Japanese Quail, Coturnix japonica
I. Yamamoto*,
Y. Sakaguchi
,
M. Numao,
A. Tsukada
,
N. Tsushima and
M. Tanaka*,,1
* High-Tech Research Center, Nippon Veterinary and Life Science University, Musashino, Tokyo, 180-8602, Japan; Department of Animal Science, Faculty of Applied Life Science, Nippon Veterinary and Life Science University, Musashino, Tokyo, 180-8602, Japan; and Department of Animal Physiology, Graduate School of Bioagricultural Science, Nagoya University, Nagoya, 464-8602, Japan
1 Corresponding author: mitanaka{at}nvlu.ac.jp
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ABSTRACT
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It has been found that GPR39 is an orphan receptor that belongs to the family of G protein-coupled receptors. In mammals, GPR39 has been shown to be involved in the regulation of gastrointestinal and metabolic function. In this study, we performed cDNA cloning for GPR39 in Japanese quail and characterized the tissue expression profiles of its mRNA. The cDNA encoded 462 amino acids, showing very high sequence homology to chicken GPR39 (95.5%) and moderate homology to mouse (64.7%), rat (63.7%), and human (59.9%) GPR39. Real-time PCR analysis revealed that GPR39 mRNA is expressed at high levels in the digestive tissues such as stomach, duodenum, jejunum, ileum, cecum, and colon and rectum and at moderate levels in the oviduct including infundibulum, magnum, isthmus, and uterus. These findings suggest that GPR39 may be involved in gastrointestinal and oviductal functions in Japanese quail.
Key Words: Japanese quail • GPR39 • complementary deoxyribonucleic acid • cloning • messenger ribonucleic acid expression
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INTRODUCTION
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Initially, GPR39 was identified as a G protein-coupled receptor related to the growth hormone secretagogue receptor (McKee et al., 1997; Holst et al., 2004; Kojima and Kangawa. 2005). It has been found that GPR39 is expressed in a wide range of tissues, with relatively higher levels in the gastrointestinal tract in mammals (Zhang et al., 2005; Jackson et al., 2006). Recently, obestatin, a peptide generated from proghrelin by proteolytic cleavage, has been proposed to be an endogenous ligand for GPR39 (Zhang et al., 2005). However, several other reports have denied that obestatin is a ligand for GPR39 (Chartrel et al., 2007; Holst et al., 2007; Tremblay et al., 2007). Although it has been shown that a high concentration of Zn2+ could activate GPR39 intracellular signaling in cultured cells (Holst et al., 2004, 2007; Lauwers et al., 2006), the endogenous ligand for GPR39 is still unknown (Gourcerol et al., 2007). In GPR39-deficient mice, accelerated gastric emptying of solid meal, BW gain, and high body fat composition have been observed, suggesting that GPR39 plays important roles in gastrointestinal and metabolic functions (Moechars et al., 2006).
Recently, we cloned the chicken GPR39 cDNA and characterized the primary structure of the predicted protein and tissue expression profiles of the mRNA (Yamamoto et al., 2007). Chicken GPR39 contains 7 transmembrane (TM) domains with high similarity to those of mammalian GPR39, and its mRNA is highly expressed in the oviduct as well as in the gastrointestinal tract.
In the present study, we report the cDNA sequence and mRNA expression profiles of GPR39 in another avian species, Japanese quail.
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MATERIALS AND METHODS
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Birds
Fertilized Japanese quail eggs were purchased from a commercial supplier and incubated under standard conditions. After hatching, chicks were grown in an electric brooder for up to 1 wk and thereafter in an unheated brooder. Food and water were given ad libitum. All quails were grown up to 6 wk old, which was sexually premature. All procedures were performed in accordance with the National Institutes of Health guidelines regarding the principles of animal care. The birds were killed by cervical dislocation, and tissues were immediately removed and frozen in liquid N.
RNA Extraction and cDNA Cloning of Japanese Quail GPR39
Total RNA was extracted from the various tissues of 4 individual 6-wk-old female quails by Trizol (Invitrogen, Carlsbad, CA) according to the instructions of the manufacturer. The amount of total RNA was measured by spectrophotometry. PolyA+ RNA was isolated from the duodenum of a 6-wk-old Japanese quail with Oligotex-dT 30 (Takara, Tokyo, Japan) and subjected to the preparation of a cDNA library with a Marathon cDNA amplification kit (Clontech Laboratories, Mountain View, CA). A cDNA fragment of the quail GPR39 was amplified using cGPR39-1 and cGPR39-2 designed from the chicken GPR39 cDNA sequence (Yamamoto et al., 2007), and the 5' and 3' ends of the GPR39 cDNA were amplified using jqGPR39-1 and jqGPR39-2 (Table 1
). The amplified cDNA fragments were cloned into pGEM-T EASY vector (Pro-mega Corporation, Madison, WI). Sequencing was carried out with the use of the BigDye Terminator v3.1 cycle sequencing kit and the ABI PRISM 310 Genetic Analyzer (Applied Biosystems, Foster City, CA).
Quantitative Real-Time PCR Analysis
Total RNA (500 ng) was reverse-transcribed in 10 µL of 1 x ExScript buffer containing 0.5 mM deoxynucleoside triphosphate, 50 µM random primers, and 50 units of ExScript reverse transcriptase (Takara) at 42°C for 15 min. The reaction product was subjected to quantitative PCR performed according to the user instructions of the Real-Time PCR System 7500 (Applied Biosystems). Briefly, after denaturation of the cDNA at 95°C for 2 min, PCR was carried out with a thermal protocol consisting of 95°C for 15 s and 60°C for 35 s in a 25-µL buffer containing 1 x Platinum SYBR Green qPCR SuperMix-UDG (Invitrogen) and 0.2 µM each of jqGPR39-3 and jqGPR39-4 (Table 1
). Quantitative measurement was performed by establishing a linear amplification curve from serial dilutions of Japanese quail GPR39 and S17 ribosomal protein cDNA. The jqS17-1 and jqS17-2 derived from the Japanese quail S17 cDNA sequence (Table 1) were used for S17 mRNA. The amount of GPR39 mRNA was normalized to the amount of S17 mRNA.
Statistical Analysis
Data are presented as mean ± SEM and were analyzed using Tukey’s test following ANOVA. All analyses were performed using the GraphPad Prism (GraphPad Software, San Diego, CA).
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RESULTS AND DISCUSSION
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Nucleotide and Deduced Amino Acid Sequences of Japanese Quail GPR39
The GPR39 cDNA cloned from the duodenum cDNA library consisted of 285 bp of the 5'-untranslated region (UTR), 1,389 bp of the coding region, and 1,416 bp of the 3'-UTR (Figure 1). Two potential polyadenylation signals were found at the 3'-UTR. As shown in Figure 2, the sequence of 462 amino acids encoded in the coding region showed high overall homology with those of chicken (95.5%), mouse (64.7%), rat (63.7%), and human (59.9%) GPR39 (McKee et al., 1997; Strausberg et al., 2002; Yamamoto et al., 2007). Seven TM regions were highly conserved among the compared avian and mammalian GPR39. In the cytoplasmic loop regions, highly conserved sequences were observed between TM I and II and TM III and IV, which are considered to be interaction sites with G protein. An extracellular loop region between TM VI and VII was also highly conserved, suggesting its importance for ligand binding.
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Figure 1. Complementary DNA and predicted amino acid sequence of Japanese quail GPR39. The asterisk indicates the stop codon. Two potential polyadenylation signals are shown by underline. The poly (A) tails were removed. The nucleotide sequence will appear in the GenBank database with an accession number of EF375709.
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Figure 2. Alignment of amino acid sequences of Japanese quail, chicken, human, mouse, and rat GPR39. The amino acid sequences of Japanese quail GPR39 (EF375709) are aligned with those of chicken (DQ768750), human (AF034633), mouse (BC085285), and rat (XM_222578) GPR39. The regions are boxed. Asterisks indicate amino acid residues matched to Japanese quail. Gaps are introduced to maximize the sequence identity.
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Expression Profile of GPR39 mRNA in the Tissues of Japanese Quail
Expression levels of GPR39 mRNA in the tissues of 6-wk-old female Japanese quail were examined by quantitative PCR (Figure 3). High levels of expression were observed in digestive organs such as the stomach, duodenum, jejunum, ileum, cecum, and colon and rectum, whereas levels in the gullet, crop sac, and gizzard were relatively low. Notable levels of mRNA expression were observed in the oviduct including infundibulum, magnum, isthmus, and uterus, but the level in the ovary was very low. These expression profiles in the digestive tract and oviduct are similar to those of the chicken (Yamamoto et al., 2007), suggesting that GPR39 is involved in functions of the oviduct as well as the digestive tract in these 2 Galliform species.
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Figure 3. Tissue distribution profile of GPR39 mRNA in Japanese quail. Expression levels of GPR39 mRNA in tissues of 6-wk-old female Japanese quail were determined by real-time PCR. Each value of GPR39 mRNA is normalized to that of S17 mRNA and represents means ± SEM (n = 4) of triplicate experiments. Values with different letters are significantly different (P < 0.05).
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ACKNOWLEDGMENTS
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This work was supported by the Grants-in-Aid for Scientific Research 17380176 and by the private university’s matching fund subsidy from the Ministry of Education, Culture, Sports, Science, and Technology of Japan.
Received for publication May 24, 2007.
Accepted for publication July 26, 2007.
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REFERENCES
|
|---|
Chartrel, N., R. Alvear-Perez, J. Leprince, X. Iturrioz, A. Reaux-Le Goazigo, V. Audinot, P. Chomarat, F. Coge, O. Nosjean, M. Rodriguez, J. P. Galizzi, J. A. Boutin, H. Vaudry, and C. Llorens-Cortes. 2007. Comment on "Obestatin, a peptide encoded by the ghrelin gene, opposes ghrelin’s effects on food intake". Science 315:766.Gourcerol, G., D. H. St-Pierre, and Y. Tache. 2007. Lack of obestatin effects on food intake: Should obestatin be renamed ghrelin-associated peptide (GAP)? Regul. Pept. 141:1–7.[CrossRef][Web of Science][Medline]
Holst, B., K. L. Egerod, E. Schild, S. P. Vickers, S. Cheetham, L. O. Gerlach, L. Storjohann, C. E. Stidsen, R. Jones, A. G. Beck-Sickinger, and T. W. Schwartz. 2007. GPR39 signaling is stimulated by zinc ions but not by obestatin. Endocrinology 148:13–20.[Abstract/Free Full Text]
Holst, B., N. D. Holliday, A. Bach, C. E. Elling, H. M. Cox, and T. W. Schwartz. 2004. Common structural basis for constitutive activity of the ghrelin receptor family. J. Biol. Chem. 79:53806–53817.
Jackson, V. R., H. P. Nothacker, and O. Civelli. 2006. GPR39 receptor expression in the mouse brain. Neuroreport 17:813–816.[CrossRef][Web of Science][Medline]
Kojima, M., and K. Kangawa. 2005. Ghrelin: Structure and function. Physiol. Rev. 85:495–522.[Abstract/Free Full Text]
Lauwers, E., B. Landuyt, L. Arckens, L. Schoofs, and W. Luyten. 2006. Obestatin does not activate orphan G protein-coupled receptor GPR39. Biochem. Biophys. Res. Commun. 351:21–25.[CrossRef][Web of Science][Medline]
McKee, K. K., C. P. Tan, O. C. Palyha, J. Liu, S. D. Feighner, D. L. Hreniuk, R. G. Smith, A. D. Howard, and L. H. Van der Ploeg. 1997. Cloning and characterization of two human G protein-coupled receptor genes (GPR38 and GPR39) related to the growth hormone secretagogue and neurotensin receptors. Genomics 46:426–434.[CrossRef][Web of Science][Medline]
Moechars, D., I. Depoortere, B. Moreaux, B. de Smet, I. Goris, L. Hoskens, G. Daneels, S. Kass, L. Ver Donck, T. Peeters, and B. Coulie. 2006. Altered gastrointestinal and metabolic function in the GPR39-obestatin receptor-knockout mouse. Gastroenterology 131:1131–1141.[CrossRef][Web of Science][Medline]
Strausberg, R. L., E. A. Feingold, L. H. Grouse, J. G. Derge, R. D. Klausner, F. S. Collins, L. Wagner, C. M. Shenmen, G. D. Schuler, S. F. Altschul, B. Zeeberg, K. H. Buetow, C. F. Schaefer, N. K. Bhat, R. F. Hopkins, H. Jordan, T. Moore, S. I. Max, J. Wang, F. Hsieh, L. Diatchenko, K. Marusina, A. A. Farmer, G. M. Rubin, L. Hong, M. Stapleton, M. B. Soares, M. F. Bonaldo, T. L. Casavant, T. E. Scheetz, M. J. Brownstein, T. B. Usdin, S. Toshiyuki, P. Carninci, C. Prange, S. S. Raha, N. A. Loquellano, G. J. Peters, R. D. Abramson, S. J. Mullahy, S. A. Bosak, P. J. McEwan, K. J. McKernan, J. A. Malek, P. H. Gunaratne, S. Richards, K. C. Worley, S. Hale, A. M. Garcia, L. J. Gay, S. W. Hulyk, D. K. Villalon, D. M. Muzny, E. J. Sodergren, X. Lu, R. A. Gibbs, J. Fahey, E. Helton, M. Ketteman, A. Madan, S. Rodrigues, A. Sanchez, M. Whiting, A. Madan, A. C. Young, Y. Shevchenko, G. G. Bouffard, R. W. Blakesley, J. W. Touchman, E. D. Green, M. C. Dickson, A. C. Rodriguez, J. Grimwood, J. Schmutz, R. M. Myers, Y. S. Butterfield, M. I. Krzywinski, U. Skalska, D. E. Smailus, A. Schnerch, J. E. Schein, S. J. Jones, and M. A. Marra; Mammalian Gene Collection Program Team. 2002. Generation and initial analysis of more than 15,000 full-length human and mouse cDNA sequences. Proc. Natl. Acad. Sci. USA 99:16899–16903.[Abstract/Free Full Text]
Tremblay, F., M. Perreault, L. D. Klaman, J. F. Tobin, E. Smith, and R. E. Gimeno. 2007. Normal food intake and body weight in mice lacking the G protein-coupled receptor GPR39. Endocrinology 148:501–506.[Abstract/Free Full Text]
Yamamoto, I., M. Numao, Y. Sakaguchi, N. Tsushima, and M. Tanaka. 2007. Molecular characterization of sequence and expression of chicken GPR39. Gen. Comp. Endocrinol. 151:128–134.[CrossRef][Web of Science][Medline]
Zhang, J. V., P. G. Ren, O. C. Avsian-Kretchmer, W. Luo, R. Rauch, C. Klein, and A. J. Hsueh. 2005. Obestatin, a peptide encoded by the ghrelin gene, opposes ghrelin’s effects on food intake. Science 310:996–999.[Abstract/Free Full Text]
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ABSTRACT
INTRODUCTION
