Mochizuki N, Cho G, Wen B, Insel P A
Department of Pharmacology, University of California, San Diego, La Jolla 92093-0636, USA.
Gene. 1996 Nov 28;181(1-2):39-43. doi: 10.1016/s0378-1119(96)00456-8.
We have used the yeast two-hybrid system to identify proteins that interact with the alpha-subunit of the heterotrimeric GTP-binding protein, Gi2. We screened a human B cell cDNA library with full-length G alpha i2 and isolated four positive colonies, one of which expressed the 44-kDa COOH terminus of a previously unrecognized 677-amino acid (aa) protein. A full-length clone was isolated from a HeLa cell cDNA library. The deduced protein contains 10 Leu-Gly-Asn repeats, and thus we named it LGN. Computer analysis indicates that LGN is a mosaic protein with seven repeated sequences of about 40 aa in length at its N-terminal end, and four repeated sequences of about 34 aa at its C-terminal end. Each of the two repeat regions shows substantial similarity to proteins found in other organisms. RT-PCR analysis of human tissues showed that the mRNA of LGN was ubiquitously expressed. The specificity of interaction between G alpha i2 and LGN was confirmed by an in vitro binding assay using recombinant proteins. These data indicate that the yeast two-hybrid system can identify novel proteins, such as LGN, that interact with G alpha proteins. As a mosaic protein, LGN shows similarity with portions of proteins from many species and thus may define a new protein family.
我们利用酵母双杂交系统来鉴定与异源三聚体GTP结合蛋白Gi2的α亚基相互作用的蛋白质。我们用全长Gαi2筛选了一个人B细胞cDNA文库，分离出4个阳性菌落，其中一个表达了一种先前未被识别的677个氨基酸（aa）蛋白质的44-kDa羧基末端。从HeLa细胞cDNA文库中分离出一个全长克隆。推导的蛋白质含有10个亮氨酸-甘氨酸-天冬酰胺重复序列，因此我们将其命名为LGN。计算机分析表明，LGN是一种镶嵌蛋白，其N末端有7个长度约为40 aa的重复序列，C末端有4个长度约为34 aa的重复序列。两个重复区域中的每一个都与其他生物体中发现的蛋白质有显著的相似性。对人体组织的RT-PCR分析表明，LGN的mRNA普遍表达。使用重组蛋白的体外结合试验证实了Gαi2与LGN之间相互作用的特异性。这些数据表明，酵母双杂交系统可以鉴定与Gα蛋白相互作用的新蛋白质，如LGN。作为一种镶嵌蛋白，LGN与许多物种的蛋白质部分具有相似性，因此可能定义一个新的蛋白质家族。
