Ogihara Hideki, Shima Fumi, Naito Kana, Asato Tsuyoshi, Kariya Ken-ichi, Kataoka Tohru
Division of Molecular Biology, Department of Molecular and Cellular Biology, Kobe University Graduate School of Medicine, Kobe, Japan.
Kobe J Med Sci. 2004;50(3-4):111-21.
Genetic studies on Schizosaccharomyces pombe adenylyl cyclase (cyr1) have shown that its activity is positively regulated by a heterotrimetric G protein a subunit gpa2 and that the resulting increase in intracellular cAMP concentration causes inhibition of sexual development including mating and meiosis. However, molecular mechanism underlying this gpa2-dependent regulation of cyr1 remains to be clarified. Here, we show that gpa2 exhibits a direct and GTP-dependent binding to the Ras-associating domain (RAD) of cyr1, which is identified by a computer algorithm-based search of the cyr1 amino acid sequence. Overexpression of this RAD results in acceleration of the sexual development of fission yeast cells presumably by competitive sequestration of gpa2. Furthermore, cyr1 is activated in vitro by the addition of purified gpa2, which is converted to the active state by treatment with AlF4-. These results indicate a crucial role of the RAD as a direct binding site of gpa2 in activation of cyr1. Thus, RADs, which have been defined as a conserved motif shared among the Ras-family small G protein-associating domains, are for the first time shown to exhibit a functional association with a member of the heterotrimeric G proteins.
对粟酒裂殖酵母腺苷酸环化酶（cyr1）的遗传学研究表明，其活性受到异源三聚体G蛋白α亚基gpa2的正向调节，并且由此导致的细胞内cAMP浓度升高会抑制包括交配和减数分裂在内的有性发育。然而，cyr1这种依赖gpa2的调节背后的分子机制仍有待阐明。在这里，我们表明gpa2表现出与cyr1的Ras结合结构域（RAD）直接且依赖GTP的结合，这是通过基于计算机算法对cyr1氨基酸序列进行搜索而确定的。该RAD的过表达可能通过竞争性隔离gpa2导致裂殖酵母细胞有性发育加速。此外，通过添加纯化的gpa2可在体外激活cyr1，gpa2经AlF4-处理后转变为活性状态。这些结果表明RAD作为gpa2在激活cyr1中的直接结合位点起着关键作用。因此，RADs（已被定义为Ras家族小G蛋白结合结构域中共享的保守基序）首次被证明与异源三聚体G蛋白的一个成员存在功能关联。
