Molecular Mechanism of Retinal Guanylate Cyclase Activation by GCAPs

描述(申请人提供)：在光感受器细胞中，细胞内cGMP水平的变化将视紫红质与膜超极化和突触信号检测联系起来[2]。CGMP的周转严格受降解cGMP的磷酸二酯酶(PDE)和合成cGMP的鸟苷环化酶(GC)相互对立的活性控制。PDE是由于视紫红质的光检测而激活的，而GC是由光检测事件后的低钙浓度激活的。GC活性的调节需要肉豆蔻酸化的钙结合蛋白，称为鸟苷环化酶激活蛋白或GCAPs[2]。这些GCAP经历了钙离子诱导的构象变化，这些变化介导了它们在高钙浓度下抑制GC和在低钙浓度下激活GC的能力[4-6]。破坏GC功能或GCAP调节GC能力的突变会导致视网膜病变，导致部分或完全失明[8]。GCAP1中的四个点突变，包括一个特征良好的Y99C突变，已经被证明会导致人类的锥体或锥杆营养不良[9-12]。这些突变中的大多数似乎将GCAP1锁定在GC激活构象中，失去了作为钙离子浓度函数来调节GC的能力。我们需要阐明野生型和突变型GCAP1在钙结合和释放过程中的构象变化，以及GCAP1与GC相互作用的分子基础。这些都是了解GCAP-GC在光转导中的关键作用及其在视网膜营养不良病因中的作用的关键步骤。作为初步数据，我们已经解决了钙离子结合的肉豆蔻酰化GCAP(MyrGCAP1)的第一结构。我们建议进行结构和生化实验，以探索钙离子诱导的构象变化，使GCAP能够调节GC对钙离子的反应活性。成功完成这项提案中概述的目标将为我们对光转导调控的理解开辟新的天地。它还将为探索GCAP和GC故障导致的人类视网膜营养不良的分子机制提供一个平台。与公共健康相关：鸟苷环化酶激活蛋白或GCAP是一种钙结合蛋白，调节鸟苷环化酶(GC)的活性。这种调节活动对视网膜感光细胞的正常生理是必不可少的，它们的故障会导致视网膜疾病和部分或完全失明。这项建议的重点是了解GCAP对GCs的调控和GCAP1致病突变的分子机制。

DESCRIPTION (provided by applicant): In photoreceptor cells, changes in intracellular levels of cGMP link light detection by rhodopsin with membrane hyperpolarization and synaptic signaling [2]. The turnover of cGMP is strictly controlled by the opposing activities of a phosphodiesterase (PDE), which hydrolyzes cGMP, and guanylate cyclase (GC), that synthesizes cGMP. PDE is activated as a result of light detection by rhodopsin, while GC is activated by the low Ca2+ concentration that follows a light detection event. Regulation of the GC activity requires myristoylated Ca2+ binding proteins called Guanylate Cyclase Activating Proteins or GCAPs [2]. These GCAPs undergo Ca2+ induced conformational changes that mediate their ability to inhibit GC at high Ca2+ concentration and activate GC at low Ca2+ concentration [4-6]. Mutations disrupting GC function or the ability of GCAPs to regulate GC result in retinopathies leading to partial or complete blindness [8]. Four point mutations in GCAP1 including a well characterized Y99C mutation, have been shown to cause cone or cone-rod dystrophies in humans [9-12]. Most of these mutations appear to lock GCAP1 in the GC activating conformation losing the ability to regulate GC as a function of Ca2+ concentration. We need to elucidate the conformational changes undergone by wild type and mutant GCAP1 upon Ca2+ binding and release as well as the molecular basis of GCAP1 interaction with GC. These are crucial steps towards understanding the key role of GCAP-GC in phototransduction and their function in the etiology of retinal dystrophies. As preliminary data we have solved the first structure of a myristoylated GCAP (myrGCAP1) with Ca2+ bound. We propose to carry out structural and biochemical experimentation to probe the Ca2+ induced conformational change that allows GCAPs to modulate the activity of GCs in response to Ca2+. Successful completion of the goals outlined in this proposal will break new ground in our understanding of phototransduction regulation. It will also provide a platform to probe the molecular mechanisms underlying the human retinal dystrophies caused by malfunction of GCAP and GC. PUBLIC HEALTH RELEVANCE: Guanylate Cyclase Activating Proteins or GCAPs are Ca2+ binding proteins that regulate the activity of Guanylate Cyclases (GCs). This regulatory activity is essential for normal physiology of photoreceptor cells in the retina and their malfunction leads to retinal disease and partial or total blindness. This proposal is focused on understanding the GCAP regulation of GCs and the molecular mechanisms underlying disease-causing mutation in GCAP1.