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（GPCR）诱导信号偶联蛋白在GTP结合的α和βγ亚基（Gβγ）中解离。除α亚基外，最近已显示Gβγ调节效应酶或离子通道的活性。在本研究中，我们研究了βγ亚基调节效应子活性的分子机制。此外，我们研究了由p110β催化和p85调节亚基组成的磷脂酰肌醇3-OH激酶（PI 3-激酶），该亚基通过刺激GPCR和酪氨酸激酶受体协同激活。 1。Gβγ的第一个WD基序似乎参与了刺激GβγD2依赖性2型腺苷酸环化酶的激活。相反，Gβγ的第三和第六基序与酵母两杂化系统中的RAF-1激酶相互作用。 2。p110β/p85 Pi 3-激酶的脂质激酶活性通过Gβγ和酪氨酸磷酸化肽协同刺激。 3。小GTPase Rab5被确定为酵母两杂化系统中的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RAB5关联。但是，与结合GDP的RAB5或各种形式的RAS未观察到这种相互作用。 4。在培养的细胞中，胰岛素诱导的蛋白激酶B（AKT）的激活是PI 3-激酶产物的靶酶，似乎是由活性RAB5模拟的，并被显性阴性形式抑制。 5。在酵母两杂交系统中也发现了一个包含SH2结构域的Rab5的新型效应子，并且效应子似乎在早期内体中存在。 6. GAB 2被确定为参与从Fcγ受体到PI 3-激酶的信号转导途径的适配器分子之一，并且在刺激Fcγ和趋化性胡椒（GPCR）受体的情况下将适配器磷酸化。