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

由agonists induces the dissociation of the signal coupling proteins into GTP-bound α和βγ subunits （Gβγ）的Stimulation of G蛋白偶联受体（GPCRs）。在添加到α亚单元中， Gβγ最近被显示为调节效应酶或离子通道的活动。在目前的研究中，我们通过调节效应器活动的βγ子单位的分子机制来研究。Moreover，我们研究了对p110β催化剂和p85调节子单位的磷脂酰肌醇3-OH激酶（PI 3-激酶）的控制，这些酶是由GPCRs和酪氨酸激酶受体的刺激而协同激活的。1.第一个WD motif of G_在对比下，Gβγ与Raf-1激酶在大约两个混合系统中相互作用的第三和第六个动力2.脂质激酶在p110β/p85 PI 3中的活性-激酶被Gβγ和酪氨酸磷酸化肽同步刺激。3. Rab 5是一种小GTPase，在两混合系统中被识别为一种p110β-绑定蛋白。在体外绑定实验中，第110页被认为能够与GTP绑定Rab 5关联。However，这样的交互并没有被GDP绑定Rab 5或各种形式的Ras所观察。4.在文化细胞中，蛋白激酶B （Akt）的胰岛素诱导激活是PI 3-激酶产品的目标酶，似乎是由活性Rab 5模拟的，并由主导阴性形式抑制。5.一种适合Rab 5的新型效应器，其中包含SH 2域，也在两个混合系统中被识别，而且效应器出现在早期的内体中。6. Gab 2被识别为一种适配器分子从Fcγ受体向PI 3-激酶的信号转化途径，该适配器在Tyr和Ser/Thr溶液刺激Fcγ和化学肽（GPCR）受体中磷酸化。

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.