Mochizuki N, Hibi M, Kanai Y, Insel P A
Department of Pharmacology, University of California San Diego, La Jolla 92093-0636, USA.
FEBS Lett. 1995 Oct 9;373(2):155-8. doi: 10.1016/0014-5793(95)01031-9.
The heterotrimeric G protein, G alpha i2, transduces signals from seven membrane spanning receptors to effectors such as adenylyl cyclase and ion channels. The purpose of this study was to identify these or other cellular proteins that interact with G alpha i2 by use of the yeast two-hybrid system. A human B cell cDNA library was screened by this system using full length G alpha i2. Four positive colonies were obtained. Two of the four were identified as nucleobindin, a calcium binding protein and a putative antigen to which anti-nuclear antibodies are generated in mice with a disorder that resembles systemic lupus erythematosus. Nucleobindin has a leucine zipper, EF hands, and a signal peptide sequence and is thought to localize to the nucleus as well as being secreted. The specificity of intehraction between G alpha i2 and nucleobindin was confirmed by an in vitro binding assay using recombinant proteins. Transfection of G alpha i2 and nucleobindin in COS cells increased G alpha i2 expression relative to cells transfected with G alpha i2 and mock vector. Our results indicate that the yeast two-hybrid system provides a means to identify novel proteins that interact with G alpha proteins. Nucleobindin appears to represent one of those proteins.
异源三聚体G蛋白Gαi2将信号从七跨膜受体传导至腺苷酸环化酶和离子通道等效应器。本研究的目的是利用酵母双杂交系统鉴定与Gαi2相互作用的这些或其他细胞蛋白。使用全长Gαi2通过该系统筛选人B细胞cDNA文库。获得了四个阳性菌落。四个中的两个被鉴定为核结合蛋白，一种钙结合蛋白，也是在患有类似系统性红斑狼疮疾病的小鼠中产生抗核抗体的假定抗原。核结合蛋白具有亮氨酸拉链、EF手结构和信号肽序列，被认为定位于细胞核以及分泌到细胞外。使用重组蛋白的体外结合试验证实了Gαi2与核结合蛋白之间相互作用的特异性。与用Gαi2和空载体转染的细胞相比，在COS细胞中转染Gαi2和核结合蛋白增加了Gαi2的表达。我们的结果表明，酵母双杂交系统提供了一种鉴定与Gα蛋白相互作用的新蛋白的方法。核结合蛋白似乎代表了其中一种蛋白。
