Interaction of GCAP1 with retinal guanylyl cyclase and calcium: sensitivity to fatty acylation.
Interaction of GCAP1 with retinal guanylyl cyclase and calcium: sensitivity to fatty acylation.
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DOI：
10.3389/fnmol.2012.00019
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发表时间：
2012
影响因子：
4.8
通讯作者：
Dizhoor AM
中科院分区：
文献类型：
--
作者：
Peshenko IV;Olshevskaya EV;Dizhoor AM
关酰环化酶激活蛋白（GCAPs）是EF-hand蛋白超家族中神经元钙传感器蛋白组（NCS）中的钙/镁结合蛋白。gcap激活脊椎动物光感受器中的视网膜观酰基环化酶（RetGC），以响应细胞内游离Ca2+浓度的光依赖性下降。gcap由四个EF-hand结构域组成，并含有n端脂肪酰化甘氨酸，在GCAP1中，这是RetGC正常激活所必需的。我们分析了在HEK293细胞中异种表达重组GCAP1时，禁止n -肉豆肉酰化（Gly2→Ala）取代对重组GCAP1与其靶酶共定位能力的影响。我们还比较了纯化的非酰化G2A突变体和C14:0酰化GCAP1在体外的Ca2+结合和retgc激活特性。用c端GFP标签表达的G2A GCAP1能够与环化酶共定位，尽管效率低于野生型，但在体外刺激环化酶活性的效率要低得多。G2A GCAP1的Ca2+结合等温线略微向更高的游离Ca2+浓度移动，因此用G2A突变体重建的RetGC的Ca2+敏感性也有所改变。同时，肉豆蔻酰化对GCAP1三维结构中肉豆蔻酰残基近端EF-hand的高亲和力Ca2+结合影响不大。这些数据表明，n端脂肪酰基可能通过目前未知的分子内机制改变GCAP1分子远端ef -hand的活性。
Guanylyl cyclase activating proteins (GCAPs) are calcium/magnesium binding proteins within neuronal calcium sensor proteins group (NCS) of the EF-hand proteins superfamily. GCAPs activate retinal guanylyl cyclase (RetGC) in vertebrate photoreceptors in response to light-dependent fall of the intracellular free Ca2+ concentrations. GCAPs consist of four EF-hand domains and contain N-terminal fatty acylated glycine, which in GCAP1 is required for the normal activation of RetGC. We analyzed the effects of a substitution prohibiting N-myristoylation (Gly2 → Ala) on the ability of the recombinant GCAP1 to co-localize with its target enzyme when heterologously expressed in HEK293 cells. We also compared Ca2+ binding and RetGC-activating properties of the purified non-acylated G2A mutant and C14:0 acylated GCAP1 in vitro. The G2A GCAP1 expressed with a C-terminal GFP tag was able to co-localize with the cyclase, albeit less efficiently than the wild type, but much less effectively stimulated cyclase activity in vitro. Ca2+ binding isotherm of the G2A GCAP1 was slightly shifted toward higher free Ca2+ concentrations and so was Ca2+ sensitivity of RetGC reconstituted with the G2A mutant. At the same time, myristoylation had little effect on the high-affinity Ca2+-binding in the EF-hand proximal to the myristoyl residue in three-dimensional GCAP1 structure. These data indicate that the N-terminal fatty acyl group may alter the activity of EF-hands in the distal portion of the GCAP1 molecule via presently unknown intramolecular mechanism.
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影响因子：
4.3
作者：
Dizhoor, Alexander M.;Olshevskaya, Elena V.;Peshenko, Igor V.
通讯作者：
Peshenko, Igor V.
影响因子：
4.8
作者：
Dizhoor, AM;Hurley, JB
通讯作者：
Hurley, JB
影响因子：
34.7
作者：
Burgoyne, Robert D.
通讯作者：
Burgoyne, Robert D.
影响因子：
4.8
作者：
Ames, JB;Tanaka, T;Stryer, L
通讯作者：
Stryer, L
影响因子：
2.9
作者：
Hwang, JY;Koch, KW
通讯作者：
Koch, KW