| Co-Investigator(Kenkyū-buntansha) |
KONTANI Kenji The University of Tokyo, Graduate School of Pharmaceutical Sciences, Dept. of Physiol. Chem., Research fellow, 大学院・薬学系研究科, 助手 (30302615)
HOSHINO Shin-ichi The University of Tokyo, Graduate School of Pharmaceutical Sciences, Dept. of Physiol. Chem., Research fellow, 大学院・薬学系研究科, 助手 (40219168)
NISHINA Hiroshi The University of Tokyo, Graduate School of Pharmaceutical Sciences, Dept. of Physiol. Chem., Associate Professor, 大学院・薬学系研究科, 助教授 (60212122)
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| Research Abstract |
Stimulation of G protein-coupled receptors (GPCRs) by agonists induces the dissociation of the signal coupling proteins into GTP-bound α and βγ subunits (Gβγ). In addition to the α subunit, Gβγ has recently been shown to regulate activities of effector enzymes or ion channels. In the present study, we investigated the molecular mechanisms by which βγ subunits regulate the effector activities. Moreover, we studied phosphatidylinositol 3-OH kinase (PI 3-kinase) consisting of p110β catalytic and p85 regulatory subunits, which is synergistically activated by the stimulation of GPCRs and tyrosine-kinase receptors. 1. The first WD motif of Gβγ appeared to be involved in the stimulation of GィイD2sィエD2-dependent activation of type-2 adenylyl cyclase. In contrast, the third and sixth motifs of Gβγ interacted with Raf-1 kinase in yeast two-hybrid system. 2. Lipid kinase activity of p110β/p85 PI 3-kinase was synergistically stimulated by Gβγ and a tyrosine-phosphorylated peptide. 3. The small GTPase, Rab5, was identified as one of p110β-binding proteins in yeast two-hybrid system. In in vitro-binding experiments, p110β was indeed capable of associating with GTP-bound Rab5. However, such interaction was not observed with GDP-bound Rab5 or the various forms of Ras. 4. In cultured cells, insulin-induced activation of protein kinase B (Akt), which is the target enzyme of PI 3-kinase product, appeared to be simulated by active Rab5 and inhibited by the dominant-negative form. 5. A novel effector for Rab5, which contained SH2 domain, was also identified in yeast two-hybrid system, and the effector appeared to present in early endosomes. 6. Gab 2 was identified as one of adaptor molecules involved in signal transduction pathway from Fcγ receptors to PI 3-kinase, and the adaptor was phosphorylated in Tyr and Ser/Thr residues upon stimulation of Fcγ and chemotactic peptide (GPCR) receptors.
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