| Co-Investigator(Kenkyū-buntansha) |
森下 理香 愛知県心身障害者コロニー発達障害研究所, 生化学部, 研究助手
UEDA Hiroshi Institute for Developmental Research, Aichi Human Service Center, Dept. of Biochemistry, Research Associate, 生化学部, 研究員 (50253779)
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| Research Abstract |
Heterotrimeric G proteins play a major role in signal tradsuction from cell-surfacereceptors to intracellular effectors. They are composed of α, β and γ subunits and multiple isoforms are identified for each subunits. In this study, we investigated functions of βγ subunits, particularly focused on the functional difference due to isoforms of the γ subunit. The obtained results are as follows.
1) In fibroblasts, G protein βγ subunits exert a disruptive influence on stress fibers. However, transfection of βγ induced stress fiber formation and focal adhesion assembly in epithelial-like HeLa cells. βγ-induced stress fiber and focal adhesion formation was inhibited by co-expression of a dominant negative mutant of Rho, indicating that βγ subunits regulate Rhodependent actin polymerization in HaLa cells. 2) Pertussis toxin blocks lysophosphatidic acid(LPA)-induced cell spreading of NIH 3T3 fibroblasts on fibronectin. This prevention of cell spreading was eliminated by the expression of consti
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tutively active mutants of Rho family small G proteins, Rac and Cdc42, but not by Rho. In addition, activation of the endogenous forms was suppressed by pertussis toxin, indicating that Gi-induced cell spreading is mediated through the Rac and Cdc42 pathways. Transfection of a constitutively active mutant of αi2 and βγ subunits enhanced spreading of pertussis toxin-treated cells. β1 with γ12, a major γ form in fibroblasts, was more effective for increasing cell spreading than β1γ2 or β1 plus γ12S2A, a mutant in which Ser2, a phosphorylation site for protein kinase C, is replaced with alanine. These findings indicate that both αi and βγ stimulate Rac and Cdc42 pathways with LPA-induced cell spreading. 3) Concentrations of most G protein subunits in the brain increase during development. However, expression of γ5 was high in embryonic brain and low throughout postnatal development. Immunohistochemical staining of embryonic brains showed γ5 to be specifically expressed in the prolifereative region of the ventricular zone, suggesting its specific function in undifferentiated cells. Less
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