Kubo Hiroshi, Hazeki Kaoru, Takasuga Shunsuke, Hazeki Osamu
Division of Molecular Medical Science, Graduate School of Biomedical Sciences, Hiroshima University, Hiroshima 734-8551, Japan.
Biochem J. 2005 Dec 15;392(Pt 3):607-14. doi: 10.1042/BJ20050671.
We prepared CHO (Chinese hamster ovary) cells expressing both IR (insulin receptor) and A1R (A1 adenosine receptor). Treatment of the cells with insulin or PIA [N6-(2-phenylisopropyl)adenosine], a specific A(1)R agonist increased Akt activity in the cells in a PI3K- (phosphoinositide 3-kinase) dependent manner. Transfection of p110beta into the cells augmented the action of PIA with little effect on insulin. Introduction of a pH1 vector producing shRNA (short hairpin RNA) that targets p110beta abolished PIA-induced Akt activation. By contrast, an shRNA probe targeting p110alpha did not impair the effects of PIA. The effect of PIA in p110alpha-deficient cells was attenuated effectively by both Deltap85 and betaARK-CT (beta-adrenergic receptor kinase-C-terminal peptide). A Deltap85-derived protein possessing point mutations in its two SH2 domains did not impair PIA action. These results suggest that tyrosine-phosphorylated proteins and Gbetagamma (betagamma subunits of GTP-binding protein) are necessary for the specific function of p110beta in intact cells. The p110beta-middle (middle part of p110beta) may play an important role in signal reception from GPCRs (GTP-binding-protein-coupled receptor), because transfection of the middle part impaired PIA sensitivity.
我们制备了同时表达胰岛素受体（IR）和A1腺苷受体（A1R）的中国仓鼠卵巢（CHO）细胞。用胰岛素或PIA [N6-(2-苯异丙基)腺苷，一种特异性A(1)R激动剂]处理细胞，以磷脂酰肌醇3激酶（PI3K）依赖的方式增加了细胞中Akt的活性。将p110β转染到细胞中增强了PIA的作用，而对胰岛素的作用影响很小。导入一种产生靶向p110β的短发夹RNA（shRNA）的pH1载体，消除了PIA诱导的Akt激活。相比之下，靶向p110α的shRNA探针并未削弱PIA的作用。在p110α缺陷细胞中，PIA的作用被δp85和β肾上腺素能受体激酶C末端肽（βARK-CT）有效减弱。一种在其两个SH2结构域中具有点突变的δp85衍生蛋白并未削弱PIA的作用。这些结果表明，酪氨酸磷酸化蛋白和Gβγ（GTP结合蛋白的βγ亚基）对于完整细胞中p110β的特定功能是必需的。p110β中间部分（p110β的中间部分）可能在G蛋白偶联受体（GPCR）的信号接收中起重要作用，因为转染中间部分会损害PIA敏感性。
