| Project/Area Number |
05454322
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| Research Category |
Grant-in-Aid for General Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Research Field |
内分泌・代謝学
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| Research Institution | KOBE UNIVERSITY |
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Principal Investigator |
CHIHARA Kazuo KOBE UNIV MED,PROFESSOR, 医学部, 教授 (00107955)
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| Co-Investigator(Kenkyū-buntansha) |
KAJI Hidesuke KOBE UNIV MED,ASSIST.PROF., 医学部, 助手 (90224401)
MATSUI Toshimitsu KOBE UNIV MED,LECTURER, 医学部・付属病院, 講師 (10219371)
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| Project Period (FY) |
1993 – 1994
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| Project Status |
Completed (Fiscal Year 1994)
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| Budget Amount *help |
¥6,900,000 (Direct Cost: ¥6,900,000)
Fiscal Year 1994: ¥1,200,000 (Direct Cost: ¥1,200,000)
Fiscal Year 1993: ¥5,700,000 (Direct Cost: ¥5,700,000)
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| Keywords | CHOLECYSTOKININ / GASTRIN / G PROTEIN-COUPLED RECEPTOR / TYROSINE KINASE / KNOCKOUT MOUSE / RAS / CYTOSKELTON / RECEPTOR ANTAGONIST / Ras / 遺伝子 / FAK / 中枢神経系 |
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| Research Abstract |
We have cloned a human brain cholecystokinin (CCK) -B receptor cDNA and characterized its function by introducing it into Chinese hamster ovary (CHO) cells. The deduced amino acid sequence was highly conserved as compared with those of the gastrin receptors in Mastomys enterochromaffin-like (ECL) cells (90%) and canine parietal cells (89%). Human brain CCK-B receptors possessed slightly but significantly higher affinities for CCK-8 than for gastrin I,while both ligands equally bound to Mastomys ECL cell-derived gastrin receptors. Both CCK-8 and gastrin I markedly augmented phosphoinositide hydrolysis and cytosolic free calcium in the CHO transfectants, indicating that the cloned CCK-B receptor could functionally couple with intracellular signalling molecules. Moreover, CCK-8 and gastrin I dose-dependently increased the [^3H] thymidine incorporation of the CHO transfectants in serum-free medium, and promoted the cell growth. The CCK-B receptor mRNA was abundantly expressed in particular
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areas of human brain and stomach, such as cerebral cortex and mucosa of the gastric fundus. This is the first demonstration of trophic effects of CCK and gastrin through the normal human brain CCK-B receptor.
The human CCK-B/gastrin receptor was expressed in mouse NIH3T3 fibroblasts to investigate the molecular basis of signal transduction pathway of the guanine nucleotide regulatory protein (G protein) -coupled receptor. CCK-8 induced tyrosine phosphorylation of several protein species. Among them, mitogen-activated protein (MAP) kinase was tyrosine phosphorylated and activated in response to CCK-8, as was induced by platelet derived growth factor (PDGF). In contrast, tyrosine phosphorylation of p125^<FAK> (focal adhesion kinase) was induced by CCK-8 but not by PDGF.CCK-8 as well as gastrin I induced the expression of early responsive genes such as c-fos and c-myc. CCK-8 as well PDGF activated the c-src proto-oncogene product (p60^<c-src>) as well as a small GTP-binding protein, ras p21. Moreover, CCK-8 rapidly reorganized actin stress fibers, while PDGF initially induced membrane ruffling. The micro-injection of either rho GDP dissociation inhibitor or Clostridium botulinum ADP-ribosyltransferase C3 which impairs the function of another small GTP-binding prorein, rho p21 inhibited thr stress hiber formations by CCK-8. These results suggest that the CCK-B/gastrin receptors expressed might regulate not only cell growth but also cell motility or adhesion by reorganizing actin cytoskeletons by cross-talking with the tyrosine kinase cascades. Less
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