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Analysis of the functions of G protein βγ-Subunit and application to drug design

G蛋白βγ亚基的功能分析及其在药物设计中的应用

基本信息

批准号：
10557220
负责人：
KATADA Toshiaki
金额：
$ 8.19万
依托单位：
The University of Tokyo
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
1998
资助国家：
日本
起止时间：
1998 至 1999
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-10557220/
关键词：
G protein βγ subunits membrane receptor tyrosine kinase lipid kinase Rab5 アデニル酸シクラーゼ

项目摘要

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.

激动剂刺激G蛋白偶联受体（GPCRs）可诱导信号偶联蛋白解离为GTP结合的α和βγ亚基（Gβγ）。除α亚基外，Gβγ最近被证明可调节效应酶或离子通道的活性。在本研究中，我们研究了βγ亚基调节效应器活性的分子机制。此外，我们还研究了由p110β催化亚基和p85调节亚基组成的磷脂酰肌醇3-OH激酶（PI 3-kinase），它可通过GPCR和酪氨酸激酶受体的刺激而协同激活。1. Gβγ的第一个WD基序似乎参与刺激G β D2依赖的2型腺苷酸环化酶的激活。而在酵母双杂交系统中，Gβγ的第3和第6基序与Raf-1激酶相互作用。2. Gβγ和酪氨酸磷酸化肽协同刺激p110β/p85 PI 3-激酶的脂激酶活性。3. Rab 5是酵母双杂交系统中鉴定的p110β结合蛋白之一。在体外结合实验中，p110β确实能够与GTP结合的Rab 5结合。然而，这种相互作用没有观察到GDP结合Rab 5或各种形式的Ras。4.在培养的细胞中，胰岛素诱导的PI 3-激酶产物的靶酶蛋白激酶B（Akt）的活化似乎被活性Rab 5所模拟，并被显性负性形式所抑制。5.在酵母双杂交系统中还发现了一个新的Rab 5效应子，该效应子含有SH 2结构域，并且似乎存在于早期的内体中。6. Gab 2是参与Fcγ受体到PI 3-激酶信号转导途径的接头分子之一，在Fcγ和趋化肽（GPCR）受体的刺激下，其Tyr和Ser/Thr残基发生磷酸化。