Calcium- and myristoyl-dependent properties of guanylate cyclase-activating protein-1 and protein-2
Calcium- and myristoyl-dependent properties of guanylate cyclase-activating protein-1 and protein-2
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DOI：
10.1021/bi026618y
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发表时间：
2002-10-29
期刊：
影响因子：
2.9
通讯作者：
Koch, KW
中科院分区：
文献类型：
--
作者：
Hwang, JY;Koch, KW
在视觉传导中，鸟苷酸环化酶激活蛋白（GCAP）在细胞质[Ca 2 +]（游离）低的条件下激活膜结合的鸟苷酸环化酶I（ROS-GC 1）合成cGMP。GCAP是一种神经元钙离子结合蛋白，具有三个功能性EF-手和一个N-末端豆蔻酰化的共有位点。GCAP-1和GCAP-2对ROS-GC 1活性的调节作用不同。GCAP-I中的肉豆蔻酰基对ROS-GC 1的Ca 2+依赖性调节具有强烈影响（IC 50偏移）。相比之下，GCAP-2的肉豆蔻酰化不改变环化酶活化特征（IC 50无变化）。因此，肉豆蔻酰基团控制GCAP-1的Ca 2+敏感性，但不控制GCAP-2的Ca 2+敏感性。肉豆蔻酰基限制了GCAP-1和GCAP-2中一个半胱氨酸的可及性，通过测量半胱氨酸的时间依赖性巯基反应性观察到。当EGTA缓冲Ca ~（2+）时，这种屏蔽效应并没有减轻。我们应用表面等离子体共振（SPR）光谱监测钙依赖性结合豆蔻酰化和nonmyristoylated GCAP-1和GCAP-2固定磷脂膜。没有GCAP表现出Ca 2 +-肉豆蔻酰转换，如观察到的恢复。因此，肉豆蔻酰基通过变构机制控制GCAP-1的Ca 2+敏感性（而不是GCAP-2的Ca 2+敏感性），但该控制步骤不涉及肉豆蔻酰基开关。
In visual transduction, guanylate cyclase-activating proteins (GCAPs) activate the membrane-bound guanylate cyclase I (ROS-GC1) to synthesize cGMP under conditions of low cytoplasmic [Ca2+](free). GCAPs are neuronal Ca2+-binding proteins with three functional EF-hands and a consensus site for N-terminal myristoylation. GCAP-1 and GCAP-2 regulated ROS-GC1 activities differently. The myristoyl group in GCAP-I had a strong influence on the Ca2+-dependent regulation of ROS-GC1 (shift in IC50). In contrast, myristoylation of GCAP-2 did not change the cyclase activation profile (no shift in IC50)Thus, the myristoyl group controlled the Ca2+-sensitivity of GCAP-1, but not that of GCAP-2. The myristoyl group restricted the accessibility of one cysteine in GCAP-I and GCAP-2 observed by measuring the time-dependent thiol reactivity of cysteines. This shielding effect was not relieved when Ca2+ was buffered by EGTA. We applied surface plasmon resonance (SPR) spectroscopy to monitor the Ca2+-dependent binding of myristoylated and nonmyristoylated GCAP-1 and GCAP-2 to immobilized phospholipid membranes. None of the GCAPs exhibited a Ca2+-myristoyl switch as observed for recoverin. Thus, the myristoyl group controls the Ca2+-sensitivity of GCAP-1 (not that of GCAP-2) by an allosteric mechanism, but this control step does not involve a myristoyl switch.