Zimmermann G, Zhou D, Taussig R
Department of Biological Chemistry, University of Michigan Medical School, Ann Arbor, Michigan 48109-0636, USA.
J Biol Chem. 1998 Mar 20;273(12):6968-75. doi: 10.1074/jbc.273.12.6968.
We describe the development of a genetic system allowing for the isolation of mutant mammalian adenylyl cyclases defective in their responses to G protein subunits, thus allowing for the identification of structural elements within the cyclase that are responsible for the recognition of these regulators. Expression of mammalian type V adenylyl cyclase in a cyclase-deleted yeast strain can conditionally complement the lethal phenotype of this strain. Type V adenylyl cyclase-expressing yeast grow only when the cyclase is activated by coexpression of Gsalpha or addition of forskolin to the medium; however, growth arrest is observed in the presence of both activators or under basal conditions. Utilizing this genetic system, we have isolated 25 adenylyl cyclase mutants defective in their response to Gsalpha. Sequence analysis and biochemical characterization of these mutants have identified residues in both cytoplasmic domains of the cyclase that are involved in the specific binding of and regulation by Gsalpha.
我们描述了一种遗传系统的开发，该系统可用于分离对G蛋白亚基反应存在缺陷的突变型哺乳动物腺苷酸环化酶，从而能够鉴定环化酶中负责识别这些调节因子的结构元件。在缺失环化酶的酵母菌株中表达哺乳动物V型腺苷酸环化酶可有条件地补充该菌株的致死表型。表达V型腺苷酸环化酶的酵母仅在环化酶通过共表达Gsα或向培养基中添加福斯可林而被激活时才能生长；然而，在同时存在两种激活剂或基础条件下会观察到生长停滞。利用这个遗传系统，我们分离出了25个对Gsα反应存在缺陷的腺苷酸环化酶突变体。对这些突变体的序列分析和生化特性鉴定确定了环化酶两个胞质结构域中参与Gsα特异性结合和调节的残基。
