Nucleobindin 1 Is a Calcium-regulated Guanine Nucleotide Dissociation Inhibitor of Gαi1
Nucleobindin 1 Is a Calcium-regulated Guanine Nucleotide Dissociation Inhibitor of Gαi1
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DOI：
10.1074/jbc.m110.148429
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发表时间：
2010-10-08
影响因子：
4.8
通讯作者：
Sakmar, Thomas P.
中科院分区：
文献类型：
--
作者：
Kapoor, Neeraj;Gupta, Ruchi;Sakmar, Thomas P.
核结合蛋白1（NUCB 1）是一种广泛表达的多结构域钙结合蛋白，其确切的生理和生化功能尚不清楚。我们设计并异源表达了可溶性NUCB 1（sNUCB 1），并表征了其生物物理和生化特性。我们表明，sNUCB 1存在于溶液中的二聚体，每个单体结合两个二价钙阳离子。钙离子结合引起sNUCB 1的构象变化，通过圆二色谱和荧光光谱实验判断。早期的报道表明NUCB 1可能与异源三聚体G蛋白α亚基相互作用。我们发现，二聚体无钙sNUCB 1结合表达G α（i1）和钙结合抑制相互作用。sNUCB 1与G α（i1）的结合抑制了GDP释放的基础速率，并减缓了GTP γ S摄取的速率和程度。此外，我们的组织培养实验表明，sNUCB 1阻止受体介导的G α（i）依赖性抑制腺苷酸环化酶。因此，我们得出结论，sNUCB 1是一种钙依赖性G α（i1）鸟嘌呤核苷酸解离抑制剂（GDI）。据我们所知，sNUCB 1是异源三聚体G蛋白钙依赖性GDI的第一个例子。我们还表明，sNUCB 1的GDI活性的机制是独特的，并没有出现在RGS蛋白中发现的共识GoLoco基序。我们建议，细胞质NUCB 1可能的功能，以调节异源三聚体G蛋白的运输和G蛋白偶联受体介导的信号转导途径。
Nucleobindin 1 (NUCB1) is a widely expressed multidomain calcium-binding protein whose precise physiological and biochemical functions are not well understood. We engineered and heterologously expressed a soluble form of NUCB1 (sNUCB1) and characterized its biophysical and biochemical properties. We show that sNUCB1 exists as a dimer in solution and that each monomer binds two divalent calcium cations. Calcium binding causes conformational changes in sNUCB1 as judged by circular dichroism and fluorescence spectroscopy experiments. Earlier reports suggested that NUCB1 might interact with heterotrimeric G protein alpha subunits. We show that dimeric calcium-free sNUCB1 binds to expressed G alpha(i1) and that calcium binding inhibits the interaction. The binding of sNUCB1 to G alpha(i1) inhibits its basal rate of GDP release and slows its rate and extent of GTP gamma S uptake. Additionally, our tissue culture experiments show that sNUCB1 prevents receptor-mediated G alpha(i)-dependent inhibition of adenylyl cyclase. Thus, we conclude that sNUCB1 is a calcium-dependent guanine nucleotide dissociation inhibitor (GDI) for G alpha(i1). To our knowledge, sNUCB1 is the first example of a calcium-dependent GDI for heterotrimeric G proteins. We also show that the mechanism of GDI activity of sNUCB1 is unique and does not arise from the consensus GoLoco motif found in RGS proteins. We propose that cytoplasmic NUCB1 might function to regulate heterotrimeric G protein trafficking and G protein-coupled receptor-mediated signal transduction pathways.