Identification of functional regions of guanylate cyclase-activating protein 1 (GCAP1) using GCAP1/GCIP chimeras
Identification of functional regions of guanylate cyclase-activating protein 1 (GCAP1) using GCAP1/GCIP chimeras
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DOI：
10.1515/bc.2001.148
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发表时间：
2001-08-01
影响因子：
3.7
通讯作者：
Baehr, W
中科院分区：
文献类型：
--
作者：
Li, N;Sokal, I;Baehr, W
鸟苷酸环化酶激活蛋白1（GCAP 1）和鸟苷酸环化酶抑制蛋白（GCIP）是在脊椎动物感光细胞中表达的钙调素相关的Ca 2+结合蛋白。GCAP 1在低游离[Ca 2 +]（< 50 nM，在光照下）激活光感受器鸟苷酸环化酶1（GC 1），但在生理高[Ca 2 +]（1 μ M，在黑暗中）抑制它。GCIP是一种来自蛙视网膜的Ca ~（2+）结合蛋白，在接近1 μ M [Ca ~（2+）]时抑制GC_1，但在低[Ca ~（2+）]时不能刺激环化酶。在这项研究中，我们通过产生GCAP 1/GCIP嵌合体来探索GCAP 1和GC 1之间的相互作用，并测试它们刺激GC 1的能力。我们制备了八对结构，其中GCIP和GCAP 1的N-末端部分分别被GCAP 1和GCIP的相应结构域相继取代。纯化表达的蛋白质，并测试在50 nm [Ca 2 +]下对GC 1的刺激，以及它们在高[Ca 2 +]下竞争性抑制Ca 2+不敏感的GCAP 1突变体GCAP 1-tm对GC 1刺激的能力。虽然所有的GCAP 1/GCIP嵌合体竞争性抑制GCAP 1-tm在高[Ca 2 +]下对GC 1的刺激，但几种GCIP/GCAP 1嵌合体没有作用。由GCIP的残基1 - 20和GCAP 1的残基21 - 205组成的嵌合体在低[Ca 2 +]下对GC 1没有影响，这表明与GCIP没有序列相似性的N-末端区域MGNIMDGKSVEELSSTEHQ是GC 1刺激所必需的关键组分之一。GCAP 1/GCIP嵌合体由GCAP 1的残基1 - 43（包括非功能性EF 1）和GCIP的残基56-206组成，在低[Ca 2 +]下刺激GC 1，在高[Ca 2 +]下抑制GC 1，表明将抑制蛋白转化为活化蛋白所需的必需组分包含在GCAP 1的N-末端区域（残基1 - 43）内。
uanylate cyclase-activating protein 1 (GCAP1) and guanylate cyclase-inhibitory protein (GCIP) are calmodulin-related Ca2+-binding proteins expressed in vertebrate photoreceptor cells. GCAP1 activates photoreceptor guanylate cyclase 1 (GC1) at low free [Ca2+] (< 50 nM, in the light), but inhibits it at physiological high [Ca2+] (1 muM, in the dark). GCIP, a Ca2+-binding protein from frog retina, inhibits GC1 at similar to1 muM [Ca2+], but is unable to stimulate cyclase at low [Ca2+]. In this study, we probed the interaction between GCAP1 and GC1 by producing GCAP1/GCIP chimeras and tested their capability to stimulate GC1. We prepared eight pairs of constructs in which the N-terminal portions of GCIP and GCAP1 were successively replaced by corresponding domains of GCAP1, and GCIP, respectively. The expressed proteins were purified and tested for stimulation of GC1 at 50 nm [Ca2+], and their ability to competitively inhibit GC1 stimulation by a Ca2+-insensitive GCAP1 mutant, GCAP1 -tm, at high [Ca2+]. While all GCAP1/GCIP chimeras competitively inhibited GC1 stimulation at high [Ca2+] by GCAP1 -tm, several of the GCIP/GCAP1 chimeras had no effect. A chimera consisting of residues 1 - 20 of GCIP and 21 - 205 of GCAP1 had no effect on GC1 at low [Ca2+], suggesting that the N-terminal region MGNIMDGKSVEELSSTECHQ, which has no sequence similarity to GCIP, is among the key components necessary for GC1 stimulation. A GCAP1/GCIP chimera consisting of residues 1 - 43 (including nonfunctional EF1) of GCAP1 and residues 56-206 of GCIP stimulated GC1 at low [Ca2+] and inhibited GC1 at high [Ca2+], suggesting that the essential components required to transform an inhibitory to an activating protein are contained within the N-terminal region of GCAP1 (residues 1 - 43).