Guanine nucleotide dissociation inhibitor activity of the triple GoLoco motif protein G18: alanine-to-aspartate mutation restores function to an inactive second GoLoco motif
Guanine nucleotide dissociation inhibitor activity of the triple GoLoco motif protein G18: alanine-to-aspartate mutation restores function to an inactive second GoLoco motif
复制标题
DOI：
10.1042/bj20031686
复制
发表时间：
2004-03-15
影响因子：
4.1
通讯作者：
Siderovski, DP
中科院分区：
文献类型：
--
作者：
Kimple, RJ;Willard, FS;Siderovski, DP
GoLoco（'Ga（i/o）-Loco'相互作用）基序蛋白最近被鉴定为异源三聚体G蛋白α亚基的新型GDI（鸟嘌呤核苷酸解离抑制剂）。G18是哺乳动物GoLoco-motif基因家族的成员，通过分析人类和小鼠基因组的匿名开放阅读框发现。预测编码的G18多肽含有三个19个氨基酸的GoLoco基序，其已在其他蛋白质中显示出结合Ga亚基并抑制自发核苷酸释放。然而，G18蛋白迄今尚未进行生物化学表征。在这里，我们克隆并表达了G18蛋白，并评估了其作为GDI的能力。G18能够同时结合一个以上的Ga（II）亚基。在与不可水解的GTP类似物鸟苷5 '-[γ-硫代]三磷酸的结合测定中，G18表现出GDI活性，减慢。盖尔用GDP换GTP只有G18内的第一个和第三个GoLoco基序能够与Ga亚基相互作用，并且这些基序仅以低微摩尔亲和力结合GDP结合形式的Gail，而不结合Ga。，Ga、Gas或Ga α（12）。在G18的失活的第二GoLoco基序中将Ala-121突变为天冬氨酸，以恢复与Ga及其结合的核苷酸形成关键接触的标志性酸性-谷氨酰胺-精氨酸三肽[Kimple，Kimple，Betts，Sondek和Siderovski（2002）Nature（伦敦）416，878-881]，导致Ga结合和GDI活性的功能获得。
GoLoco ('Galpha(i/o)-Loco' interaction) motif proteins have recently been identified as novel GDIs (guanine nucleotide dissociation inhibitors) for heterotrimeric G-protein alpha subunits. G18 is a member of the mammalian GoLoco-motif gene family and was uncovered by analyses of human and mouse genomes for anonymous open-reading frames. The encoded G18 polypeptide is predicted to contain three 19-amino-acid GoLoco motifs, which have been shown in other proteins to bind Galpha subunits and inhibit spontaneous nucleotide release. However, the G18 protein has thus far not been characterized biochemically. Here, we have cloned and expressed the G18 protein and assessed its ability to act as a GDI. G18 is capable of simultaneously binding more than one Galpha(il) subunit. In binding assays with the non-hydrolysable GTP analogue guanosine 5'-[gamma-thio]triphosphate, G18 exhibits GDI activity, slowing. the exchange of GDP for GTP by Gail. Only the first and third GoLoco motifs within G18 are capable of interacting with Ga subunits, and these bind with low micromolar affinity only to Gail in the GDP-bound form, and not to Ga., Ga, Gas or Galpha(12). Mutation of Ala-121 to aspartate in the inactive second GoLoco motif of G18, to restore the signature acidic-glutamine-arginine tripeptide that forms critical contacts with Ga and its bound nucleotide [Kimple, Kimple, Betts, Sondek and Siderovski (2002) Nature (London) 416, 878-881], results in gain-of-function with respect to Ga binding and GDI activity.