A novel functional genomics approach and ligand hunting for orphan seven transmembrane receptors.

• Project/Area Number: 15590095
• Research Category: Grant-in-Aid for Scientific Research (C)
• Allocation Type: Single-year Grants
• Section: 一般
• Research Field: Biological pharmacy
• Research Institution: Kyoto University (2004); National Research Institute for Child Health and Development (2003)
• Principal Investigator: HIRASAWA Akira Kyoto University, Graduate School of Pharmaceutical Sciences, Department of Genomic Drug Discovery Sciences, Associate Professor, 薬学研究科, 助教授 (70242633)
• Project Period (FY): 2003 – 2004
• Project Status: Completed (Fiscal Year 2004)
• Budget Amount: ¥3,700,000 (Direct Cost: ¥3,700,000); Fiscal Year 2004: ¥1,100,000 (Direct Cost: ¥1,100,000); Fiscal Year 2003: ¥2,600,000 (Direct Cost: ¥2,600,000)
• Keywords: orphan receptor / ligand hunting / GPR120 / GLP-1 / enteroendocrine cells / obesity / diabetes / GFP / データベース / 細胞内局在

Research Abstract

This research aims to establish a novel functional genomics approach and ligand hunting method for orphan receptor by combining technology of subcellular localization analysis for receptor, signal transduction analysis, and expression profile analysis.
Following three experiments were done.
1.Ligand hunting for orphan receptor GPR120
To identify endogenous ligands for GPR120, we performed receptor internalization assay established in this research project and we found that long-chain free fatty acid evoke specific internalization of GPR120. In agreement with internalization assay, long-chain fatty acids were found to evoke specific rise in [Ca^<2+>]_i and ERK activation.
2.Expression profile analysis for GPR120
To clarify the function of the orphan receptor GPR120 I tried the gene expression profile analysis. Using microarray analysis, I found that GPR120 express abundantly in intestines and enteroendocrine cell line STC-1.
3.Functional analysis for GPR120 in GPR120 natively expressing cell line STC-1
In STC-1 cells, free fatty acids stimulation evokes GLP-1 secretion, rise in [Ca^<2+>]_i and ERK activation. Transfection of RNA_i specific for GPR120, inhibited GLP-1 secretion and [Ca^<2+>]_i rise. These results indicate that fatty ac_id induced Ca^<2+> increase and GLP-1 secretion in STC-1 cell is mediated through GPR120.
The discovery of ligand and physiological role for GPR120 prove the effectiveness of the technique established in this research. The purpose of this research was achieved.

Research Products

• [Journal Article] Free fatty acids regulate gut incretin glucagon-like peptide-1 secretion through GPR120. (2005)
  • Author(s): Hirasawa A et al.
  • Journal Title: Nature Medicine 11(1) Pages: 90-94
  • Description: 「研究成果報告書概要(和文)」より
• [Journal Article] Bile acids promote glucagon-like peptide-1 secretion through TGR5 in a murine enteroendocrine cell line STC-1. (2005)
  • Author(s): Katsuma S, Hirasawa A, Tsujimoto G.
  • Journal Title: Biochem Biophys Res Commun. 329(1) Pages: 386-390
  • Description: 「研究成果報告書概要(和文)」より
• [Journal Article] Free fatty acids inhibit serum deprivation-induced apoptosis through GPR120 in a murine enteroendocrine cell line STC-1. (2005)
  • Author(s): Katsuma S, Hatae N, Yano T, Ruike Y, Kimura M, Hirasawa A et al.
  • Journal Title: J Biol Chem 280(20) Pages: 19507-19515
  • Description: 「研究成果報告書概要(和文)」より
• [Journal Article] Free fatty acids regulate gut incretin glucagon-like peptide-1 secretion through GPR120. (2005)
  • Author(s): Hirasawa A, Tsumaya K, Awaji T, Katsuma S, Adachi T, Yamada M, Sugimoto Y, Miyazaki S, Tsujimoto G.
  • Journal Title: Nature Medicine. 11 Pages: 90-94
  • Description: 「研究成果報告書概要(欧文)」より
• [Journal Article] Bile acids promote glucagon-like peptide-1 secretion through TGR5 in a murine enteroendocrine cell line STC-1. (2005)
  • Author(s): Katsuma S, Hirasawa A, Tsujimoto G.
  • Journal Title: Biochem Biophys Res Commun. 329 Pages: 386-390
  • Description: 「研究成果報告書概要(欧文)」より
• [Journal Article] Related Articles, Links Free fatty acids inhibit serum deprivation-induced apoptosis through GPR120 in a murine enteroendocrine cell line STC-1. (2005)
  • Author(s): Katsuma S, Hatae N, Yano T, Ruike Y, Kimura M, Hirasawa A, Tsujimoto G.
  • Journal Title: J Biol Chem. 280(20) Pages: 19507-19515
  • Description: 「研究成果報告書概要(欧文)」より
• [Journal Article] Free fatty acids inhibit serum deprivation-induced apoptosis through GPR120 in a murine enteroendocrine cell line STC-1. (2005)
  • Author(s): Katsuma S, Hatae N, Yano T, Ruike Y, Kimura M, Hirasawa A et al.
  • Journal Title: J Biol Chem in press
• [Journal Article] cDNA microarray analysis of bovine embryo gene expression profiles during the pre-implantation period. (2004)
  • Author(s): Ushizawa K, Herath CB, Kaneyama K, Shiojima S, Hirasawa A, et al.
  • Journal Title: Reprod Biol Endocrinol 2 Pages: 77-92
  • Description: 「研究成果報告書概要(和文)」より
• [Journal Article] Pregnancy-associated changes in genome-wide gene expression profiles in the liver of cow throughout pregnancy. (2004)
  • Author(s): Herath CB, Shiojima S, Ishiwata H, Katsuma S, Kadowaki T, Ushizawa K, Imai K, Takahashi T, Hirasawa A, Tsujimoto G, Hashizume K.
  • Journal Title: Biochem Biophys Res Commun 313 Pages: 666-680
  • Description: 「研究成果報告書概要(和文)」より
• [Journal Article] cDNA microarray analysis of bovine embryo gene expression profiles during the pre-implantation period. (2004)
  • Author(s): Ushizawa K, Herath CB, Kaneyama K, Shiojima S, Hirasawa A, Takahashi T, Imai K, Ochiai K, Tokunaga T, Tsunoda Y, Tsujimoto G, Hashizume K.
  • Journal Title: Reprod Biol Endocrinol. 2 Pages: 77-92
  • Description: 「研究成果報告書概要(欧文)」より
• [Journal Article] Pregnancy-associated changes in genome-wide gene expression profiles in the liver of cow throughout pregnancy. (2004)
  • Author(s): Herath CB, Shiojima S, Ishiwata H, Katsuma S, Kadowaki T, Ushizawa K, Imai K, Takahashi T, Hirasawa A, Tsujimoto G, Hashizume K.
  • Journal Title: Biochem Biophys Res Commun. 313 Pages: 666-680
  • Description: 「研究成果報告書概要(欧文)」より
• [Journal Article] cDNA microarray analysis of bovine embryo gene expression profiles during the pre-implantation period. (2004)
  • Author(s): Ushizawa K, Herath CB, Kaneyama K, Shiojima S, Hirasawa A et al.
  • Journal Title: Reprod Biol Endocrinol. 2(1) Pages: 77-77
• [Journal Article] Gene expression profile in experimental mesangial proliferative glomerulonephritis. (2004)
  • Author(s): Izumi Y, Izumiya Y, Shiota M, Yukimura T, Shiojima S, Yamada M, Kadowaki T, Katsuma S, Hirasawa A et al.
  • Journal Title: J Pharmacol Sci. 96(1) Pages: 91-94
  • NAID: 10014166433
• [Journal Article] Carvedilol selectively inhibits oscillatory intracellular calcium changes evoked by human alpha1D- and alpha1B-adrenergic receptors. (2004)
  • Author(s): Koshimizu TA, Tsujimoto G, Hirasawa A, Kitagawa Y, Tanoue A.
  • Journal Title: Cardiovasc Res. 63(4) Pages: 662-672
• [Journal Article] Granzyme A mediates glucocorticoid-induced apoptosis in leukemia cells. (2003)
  • Author(s): Yamada M, Hirasawa A, Shiojima S, Tsujimoto G.
  • Journal Title: FASEB J 17 Pages: 1712-1714
  • Description: 「研究成果報告書概要(和文)」より
• [Journal Article] Granzyme A mediates glucocorticoid-induced apoptosis in leukemia cells. (2003)
  • Author(s): Yamada M, Hirasawa A, Shiojima S, Tsujimoto G.
  • Journal Title: FASEB J. 17 Pages: 1712-1714
  • Description: 「研究成果報告書概要(欧文)」より
• [Publications] Katsuma S, Shiojima S, Hirasawa A, Tanoue A, Yano J, Tsujimoto G, et al.: "Transcriptional profiling of gene expression patterns during sphingosine 1-phosphate-induced mesangial cell proliferation."Biochem Biophys Res Commun.. 300. 577-584 (2003)
• [Publications] Shibata K, Katsuma S, Koshimizu T, Shinoura H, Hirasawa A, Tanoue A, Tsujimoto G: "Alpha 1-adrenergic receptor subtypes differentially control the cell cycle of transfected CHO cells through a cAMP-dependent mechanism involving p27Kip1."J Biol Chem.. 278. 672-678 (2003)
• [Publications] Yoshida N, Sugiyama M, Tanoue A, Hirasawa A, Tsujjimoto G: "Determination of a novel haplotype of beta 2 adrenergic receptor in the Japanese population by the combination of the electronic microchip assay using the nanoChip system with allelespecific PCR."Drug Metabolism and Pharmacokinetics.. 17. 532-539 (2003)
• [Publications] Ishiwata H, Katsuma S, Hirasawa A, Shiojima S, Tsujimoto G., et al.: "Characterization of Gene Expression Profiles in Early Bovine Pregnancy Using a Custom cDNA Microarray."Mol Reprod Dev.. 65. 9-18 (2003)
• [Publications] Koshimizu TA, Tanoue A, Hirasawa A, Yamauchi J, Tsujimoto G: "Recent advances in alpha1-adrenoceptor pharmacology."Pharmacology & Therapeutics.. 98. 235-244 (2003)
• [Publications] Nakamura T, Shiojima S, Hirasawa A, Katsuma S, Tsujimoto G, et al.: "Temporal gene expression changes of adipogenesis in human mesenchymal stem cells."Biochem.Biophys.Res.Commun.. 303. 306-312 (2003)
• [Publications] Fukuhara Y, Hirasawa A, Tsujimoto G, et al.: "Gene expression profiles in the regenerating rata liver after partial hepatectomy."J Hepatology. 38. 784-792 (2003)
• [Publications] Yamada M, Hirasawa A, Shiojima S, Tsujimoto G: "Granzyme A mediates glucocorticoid-induced apoptosis in leukemia cells."FASEB Journal.. 17(12). 1712-1714 (2003)
• [Publications] Richardson J, Chatwin H, Hirasawa A, Tsujimoto G, Evans PD: "Agonist-specific coupling of a cloned human alpha(1A)-adrenoceptor to different second messenger pathways."Naunyn Schmiedebergs Arch Pharmacol.. 367. 333-341 (2003)