Inactivation of EF-hands makes GCAP-2 (p24) a constitutive activator of photoreceptor guanylyl cyclase by preventing a Ca2+-induced ''activator-to-inhibitor'' transition
Inactivation of EF-hands makes GCAP-2 (p24) a constitutive activator of photoreceptor guanylyl cyclase by preventing a Ca2+-induced ''activator-to-inhibitor'' transition
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DOI：
10.1074/jbc.271.32.19346
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发表时间：
1996-08-09
影响因子：
4.8
通讯作者：
Hurley, JB
中科院分区：
文献类型：
--
作者：
Dizhoor, AM;Hurley, JB
鸟苷酸环化酶激活蛋白GCAP-1和GCAP-2（Dizhoor等，1995，Gorczyca等，1995）是最近鉴定的EF-手型Ca 2+结合蛋白亚类的成员，其对Ca 2+的反应不同于EF-手超家族的任何其它已知成员。GCAP仅在其无Ca 2+形式下获得活化构象。对应于暗适应脊椎动物光感受器水平的游离Ca 2+浓度抑制GCAP激活光感受器鸟苷酸环化酶（RetGCs）的能力。我们研究了阻断Ca 2+与GCAP-2的EF-手结合的突变的影响。与其他EF-手蛋白不同，当它们的EF-手被突变失活时，它们不能激活它们的靶标，具有任何单个EF-手失活的GCAP-2保持活性，并且对Ca 2+的抑制作用的敏感性低3-6倍。任何两个或所有三个EF-手的失活产生活性形式的GGAP-2，其对生理细胞内浓度的Ca 2+的抑制不敏感。出乎意料的是，我们还发现，通过Ca 2+不敏感突变体激活RetGCs被负载Ca 2+的野生型GCAP-2抑制。GCAP-2可以以两种极端的功能形式存在：激活RetGCs的apo形式和阻断RetGCs激活的Ca 2+负载形式。2）GCAP-2的所有三个EF-手都有助于Ca 2+的抑制作用。3)三个EF-手中的两个的失活足以将“激活剂-转运体”转变到细胞内游离Ca 2+的生理范围之外。
Guanylyl cyclase activator proteins GCAP-1 and GCAP-2 (Dizhoor et al, 1995, Gorczyca et al,, 1995) are members of a recently identified subclass of EF-hand type Ca2+-binding proteins that respond to Ca2+ differently than any other known members of the EF-hand superfamily. GCAPs acquire an activating conformation only in their Ca2+-free form. Free Ca2+ concentrations corresponding to levels in dark-adapted vertebrate photoreceptors inhibit the ability of GCAPs to activate photoreceptor guanylyl cyclases (RetGCs). We studied the effects of mutations that block binding of Ca2+ to the EF-hands of GCAP-2. Unlike other EF-hand proteins, which fail to activate their target when their EF-hands are inactivated by mutations, GCAP-2 with any single EF-hand inactivated remains active and is 3-6 times less sensitive to the inhibitory effect off Ca2+. Inactivation of any two or all three EF-hands produces active forms of GGAP-2 that are insensitive to inhibition by physiological intracellular concentrations of Ca2+. Unexpectedly we also found that activation of RetGCs by a Ca2+-insensitive mutant is inhibited by Ca2+-loaded wild type GCAP-2. We propose the following, 1) GCAP-2 can exist in two extreme functional forms: an apo form that activates RetGCs and a Ca2+-loaded form that blocks activation of RetGCs, 2) All three EF-hands of GCAP-2 contribute to the inhibitory effect of Ca2+. 3) Inactivation of two off three EF-hands is sufficient to shift the ''activator-inhibitor'' transition outside the physiological range of intracellular free Ca2+.