Wang Q, Mullah B, Hansen C, Asundi J, Robishaw J D
Henry Hood MD Research Program, Pennsylvania State University, College of Medicine, Danville, Pennsylvania 17822, USA.
J Biol Chem. 1997 Oct 10;272(41):26040-8. doi: 10.1074/jbc.272.41.26040.
Human HEK 293 cells present a simple and tractable system to directly test the hypothesis that the G protein gamma subunits contribute to the specificity of receptor signaling pathways in vivo. To begin to elucidate the functions of the individual gamma subunits in these cells, a ribozyme strategy was used to specifically inactivate the mRNA encoding the gamma7 subunit. A phosphorothioated DNA-RNA chimeric hammerhead ribozyme was constructed and analyzed for specificity toward the targeted gamma7 subunit. In vitro cleavage analysis of this ribozyme revealed a highly efficient cleavage activity directed exclusively toward the gamma7 RNA transcript. In particular, this ribozyme did not result in cleavage of the gamma12 RNA transcript, which is 75% identical to the gamma7 RNA transcript. Using a transient transfection assay, in vivo analysis of this ribozyme showed a specific reduction in both the mRNA and protein expression of the gamma7 subunit in HEK 293 cells. Coincident with this loss in gamma7 subunit, there was a specific reduction in the protein expression of the beta1 subunit, suggesting that the beta1 and gamma7 subunits may functionally interact to form a betagamma dimer in vivo. Functional analysis of the consequences of ribozyme-mediated suppression of the gamma7 subunit expression indicated that it was associated with significant attenuation of isoproterenol-, but not prostaglandin E1-, stimulated adenylylcyclase activity. Suppression of the gamma7 subunit expression had no effect on carbachol- and ATP-mediated stimulation of phosphatidylinositol turnover. Taken together, these results not only indicate the feasibility of using the ribozyme technology to determine the roles of individual gamma subunits in receptor-G protein-effector pathways in vivo, but they point to a specific role of the gamma7 subunit in the regulation of adenylylcyclase activity in response to isoproterenol.
人HEK 293细胞提供了一个简单且易于操作的系统，可直接验证G蛋白γ亚基在体内有助于受体信号通路特异性的假说。为了开始阐明这些细胞中各个γ亚基的功能，采用了一种核酶策略来特异性地使编码γ7亚基的mRNA失活。构建了一种硫代磷酸化DNA-RNA嵌合锤头状核酶，并分析了其对靶向γ7亚基的特异性。对该核酶的体外切割分析显示，其具有高效的切割活性，且仅针对γ7 RNA转录本。特别地，该核酶不会导致与γ7 RNA转录本有75%同源性的γ12 RNA转录本的切割。使用瞬时转染试验，对该核酶的体内分析表明，HEK 293细胞中γ7亚基的mRNA和蛋白表达均有特异性降低。与γ7亚基的这种缺失同时发生的是，β1亚基的蛋白表达有特异性降低，这表明β1和γ7亚基在体内可能在功能上相互作用形成βγ二聚体。对核酶介导的γ7亚基表达抑制后果的功能分析表明，它与异丙肾上腺素刺激的腺苷酸环化酶活性的显著减弱有关，但与前列腺素E1刺激的腺苷酸环化酶活性无关。γ7亚基表达的抑制对卡巴胆碱和ATP介导的磷脂酰肌醇周转刺激没有影响。综上所述，这些结果不仅表明了使用核酶技术来确定各个γ亚基在体内受体-G蛋白-效应器途径中的作用的可行性，而且还指出了γ7亚基在响应异丙肾上腺素调节腺苷酸环化酶活性中的特定作用。
