細胞内情報伝達の生理機能解析と情報分子の可視化

• 研究課題/領域番号: 09273101
• 研究種目: 特定領域研究(A)
• 配分区分: 補助金
• 研究機関: 東京大学
• 研究代表者: 飯野 正光 東京大学, 大学院・医学系研究科, 教授 (50133939)
• 研究分担者: 萩原 正敏 東京医科歯科大, 難治研, 教授 (10208423) ; 宮崎 俊一 東京女子医大, 医学部, 教授 (80010081) ; 河西 春郎 東京大学, 大学院・医学系研究科, 助教授 (60224375)
• 研究期間 (年度): 1998
• 研究課題ステータス: 完了 (1998年度)
• 配分額: 65,400千円 (直接経費: 65,400千円); 1998年度: 29,000千円 (直接経費: 29,000千円); 1997年度: 36,400千円 (直接経費: 36,400千円)
• キーワード: カルシウム / イノシトール三燐酸 / 膵臓β細胞 / 分泌 / 受精 / 蛍光カルシウム指示薬 / cAMP / GFP / 血管平滑筋 / 転写制御

研究概要

本研究班は、設計分子の作用を解析するため、収縮、分泌、発生、免疫、転写調節と極めて重要な生体機能を制御するCa^<2+>シグナル機構研究を推進し以下の成果を得た。
ca^<2+>シグナルパターンとIP_3受容体サブタイプの発現パターンの関係を明らかにするため、遺伝子工学手法を援用して単一のIP_3受容体サブタイプのみを発現する細胞系を作成し、各サブタイプの機能を比較した。これによりサブタイプ間には大きな機能的相違が存在し、Ca^<2+>シグナルパターンの違いを形成するのに極めて重要であることを示した。
膵臓β細胞では、インスリン顆粒の開口放出にはカルシウム濃度上昇だけでなくAキナーゼによるりん酸化が必要で、それが細胞内ATP濃度に依存的であることを見出した。これがβ細胞のグルコース応答に関与する可能性を示した。
受精・初期発生の引き金になる卵細胞内Ca^<2+>濃度上昇を誘発する精子因子の解析を行った.精子抽出物がIP_3受容体を介するCa^<2+>遊離によってCa^<2+>波およびCa^<2+>オシレーションを誘発することを明らかにし、精子細胞質蛋白質の精製に着手した。
蛍光波長の異なる2つのGreen Fluorescent Protein(GFP)の蛍光共鳴エネルギー移動が蛋白質の構造変化によって変化し得ることを利用して、細胞内cAMP濃度やCa^<2+>濃度変化のモニターを可能とするような蛋白質の作製に成功した。

研究成果

• [文献書誌] Takeshima,H.: "Embryonic lethality and abnomal caridiac myocytes in mice lacking ryanodine receptor type2." EMBO J.17. 3309-3316 (1998)
• [文献書誌] Shimuta,M.: "Structure and expression of mitsugumin29 gene." FEBS Lett.431. 263-267 (1998)
• [文献書誌] Nishi,M.: "Mitsugumin23,a novel transmembrane protein on endoplasmic reticulum and nuclear membrane." FEBS Lett.432. 191-196 (1998)
• [文献書誌] Takeshima,H.: "Mitsugumin29,a novel synaptophysin family member from the triad junction in skeletal muscle." Biochem.J.331. 317-322 (1998)
• [文献書誌] Komazaki,S.: "Morphological abnomalities of adrenal grand and hypertrophy of liver in mutant mice lacking ryanodine receptors." Cell Tissue Res.294. 467-473 (1998)
• [文献書誌] Okada,H.: "Role of the inositol phosphatase SHIP in B cell receptor-induced Ca^<2+> oscillatory response." J.Immunol.161. 5129-5132 (1998)
• [文献書誌] Ikemoto,T.: "Effect of calmodulin on Ca^<2+>-induced Ca^<2+> release of skeletal muscle from mutant mice expressing either ryanodine receptor type 1 or type 3." Pflugers Arch.437. 43-48 (1998)
• [文献書誌] Hirabayashi,T.: "Critical duration of intracellularCa^<2+> response required for continuous translocation and activation of cytosolic phospholipase A_2." J.Biol.Chem.274. 5163-5169 (1999)
• [文献書誌] Hashimot,A.: "Inhibitory modulation of B cell receptor-mediated Ca^<2+> mobilization by SH2 domain containing inositol 5′-phosphatase(SHIP)." J.Biol.Chem.433(in press). (1999)
• [文献書誌] Miyakawa,T.: "Encoding of Ca^<2+> signals by differential expression of IP_3 receptor subtypes." ENBO J.18(in press). (1999)
• [文献書誌] Kasai,H.: "Ca^<2+> dependent exocytosis in endocrine,exocrine and non-secretory cells." Korean J.Physiol.Pharmacol.2. 1-7 (1998)
• [文献書誌] Kasai,H.: "Comparative biology of Ca^<2+>-dependent exocytosis:implications of kinetic diversity for secretory function." Trends Neurosci.22. 88-93 (1999)
• [文献書誌] Takahashi,N.: "Post-priming actions of ATP on Ca^<2+>-dependent exocytosis in pancreatic beta cells." Proc.Natl.Acad.Sci.USA. 96. 760-765 (1999)
• [文献書誌] Kasai,H.: "Multiple components of exocytosis and Ca^<2+> requirements." Phil.Trans.Royal Soc.B. 353. 1-5 (1999)
• [文献書誌] Eto,K.: "Role of NADH shuttle system in glucose-induced activation of mitochondrial metabolism and insulin secretion." Science. 283. 981-985 (1999)
• [文献書誌] Kyozuka,K.: "Injection of sperm extract mimics spatiotemporal dynamics of Ca^<2+> responses and progression of meiosis at fertilization of ascidian oocytes." Development. 125. 4099-4105 (1998)
• [文献書誌] Sakurai,A.: "Fertilization,embryonic development,and offspring from mouse eggs injected with round spermatids combined with Ca^<2+> oscillation-inducing sperm factor." Mol.Human Reprod.(in press). (1999)
• [文献書誌] Oda,S.: "Spatiotemporal dynamics of the 〔Ca^<2+>〕_i rise induced by microinjection of sperm extract into the mouse egg:preferential induction of a Ca^<2+> wave from the cortex." Dev.Biol.(in press). (1999)
• [文献書誌] Koizumi,J.: "The subcellular localization of SF2/ASF is regulated by the direct interaction with SR protein kinases(SRPKs)." J.Biol.Chem.(in press). (1999)
• [文献書誌] Okamoto,Y.: "cdc2 kinase-mediated phosphorylation of splicing factor SF2/ASF." Biochem.Biophys.Res.Commun.249. 872-878 (1998)
• [文献書誌] Yoshida,K.: "CREB-induced transcriptional activation depends on mGluR6 in rod bipolar cells." Mol.Brain Res.57. 241-247 (1998)
• [文献書誌] Shimomura,A.: "Dominant negative ATF1 blocks cyclic AMP-induced neurite outgrowth in PC12D cells." J.Neurochem.70. 1029-1034 (1998)
• [文献書誌] Muro,Y.: "Autoantibody to thioredoxin reductase in an ovarian cancer patient." Biochem.Biophys.Res.Commun.242. 267-271 (1998)
• [文献書誌] Kuroyanagi,N.: "Novel SR-protein-specific kinase,SRPK2,disassembles nuclear speckles." Biochem.Biophys.Res.Commun.242. 357-364 (1998)
• [文献書誌] Kamimoto,T.: "Dymple,a novel dynamin-like high molecular weigt GTPase lacking a proline-rich C-terminal domani in mammalian cells." J.Biol.Chem.273. 1044-1051 (1998)
• [文献書誌] Yamazawa,T.: "A region of the ryanodine receptor critical for excitation-contraction coupling in skeletalmuscle." J.Biol.Chem.272. 8161-8164 (1997)
• [文献書誌] Ikemoto,T.: "Functional and morphological features of skeletal muscle from mutant mice lacking both type 1 and type 3 ryanodine receptors." J.Physiol.501. 305-312 (1997)
• [文献書誌] Kasai,Y.: "Endothelium-dependent frequency modulation of Ca^<2+> signaling in individual vascular smooth muscle cells of the rat." J.Physiol.504. 349-357 (1997)
• [文献書誌] Takesako,K.: "Low-affinity fluorescent indicator for intracellular calciumions." Anal.Commun.34. 391-392 (1997)
• [文献書誌] Hirose,K.: "Allosteric regulation by cytoplasmic Ca^<2+> and IP_3 of the gating of IP_3 receptors in guineapig vascular smooth muscle cells." J.Physiol.506. 407-414 (1998)
• [文献書誌] Ito,K.: "Micromolar and submicromolar Ca^<2+> spikes regulating distinct cellular functions in pancreatic acinar cells." EMBOJ.16. 242-25110 (1997)
• [文献書誌] Ninomiya,A.: " Kinetic diversity in the fusion of exocytotic vesicles.14" EMBOJ.16. 929-934 (1997)
• [文献書誌] Takahashi,N.: "Multiple exocytotic pathways in pancreatic β-cells." J.CellBiol.138. 55-64 (1997)
• [文献書誌] Sjoedin,L.: "Cytoplasmic Ca^<2+> gradients evoked by acetylcholine and peptides in Pancreatic acinar cells of the guinea pig." Pflugers Arch.433. 397-402 (1997)
• [文献書誌] Mohri,T.: "Sperm-induced local [Ca^<2+>]_i rise separated from the Ca^<2+> wave in sea urchin eggs in the presence of a gamete fusion inhibitor,jaspisin." Development. (in press). (1998)
• [文献書誌] Nakano,Y.: "Spatiotemporal dynamics of intracellular calcium in the mouse egg injected with a spermatozoon." Mol.Human Reprod.3. 1087-1093 (1997)
• [文献書誌] Sato,Y.: "Adenophostin,a potent agonist of the inositol 1,4,5-trisphosphate receptor,is useful for fertilization of mouse eggs injected with round spermatids leading to normal offspring." Biol.Reprod.(in press). (1998)
• [文献書誌] Kyozuka,K.: "Intracellular calcium oscillations and meiosis resumption from metaphase 1 induced by injection of the sperm extract in the acidian Ciona savignyi." Zool.Sci.(in press). (1998)
• [文献書誌] Kamimoto,T.: "Dymple a novel dynamin-like high molecular weight GTPase lacking a proline-rich carboxyl-terminal domain in mammallian cells." J.Biol.Chem.273. 1044-1051 (1998)
• [文献書誌] Muro,Y.: "Low frequency of autoantibies against Ki-67 antigen in Japanese patients with systemic autoimmune" J.Autoimm.10. 499-503 (1997)
• [文献書誌] Nomoto,S.: "Functional analyses of mammalian protein kinase C isozymes in budding yeast and mammalian fibroblasts." Genes to Cells. 2. 601-614 (1997)
• [文献書誌] Nagai,Y.: "Identification of a novel nuclear speckle-type protein,SPOP." FEBS lett.418. 23-26 (1997)
• [文献書誌] Muro,Y.: "Autoantibody to thioredoxin reductase in an ovarian cancer patient." Biochem.Biophys.Res.Commun.(in press). (1997)
• [文献書誌] Shimomura,A.: "Dominant negative ATF1 blocks cyclic AMP-induced neurite outgrowth in PC12D cells." J.Neurochem.(in press). (1997)
• [文献書誌] Kuroyanagi,N,: "Novel SR-protein-specific kinase,SRPK2,disassembles nuclear speckles." Biochem,Biophys.Res.Commun.(in press). (1997)
• [文献書誌] Masunaga,R.: "Alteration of platelet aggregation in patients with thyroid disorders." Metabolism. 46. 1128-1131 (1997)
• [文献書誌] Watsuji,T.: "Controlled gene expression with a reverse tetracycline regulated retroviral vector(RTRV)system" Biochem.Biophys.Res.Commun.234. 769-773 (1997)
• [文献書誌] Watanabe,Y.: "Characterization of a serum response factor-like protein in saccharomyces cerevisiae,Rlmi,which has transcriptional activity regulated by the Mpk1(Slt2)mitogen-activated protein kinase pathway." Mol.Cell.Biol.17. 2615-2623 (1997)
• [文献書誌] Sugiura,K.: "Expression cloning and intracellular localization of a human ZF5 homologue." Biochem.Biophys.Acta.1352. 23-26 (1997)