Molecular Mechanism of Retinal Guanylate Cyclase Activation by GCAPs

描述（由申请人提供）：在光感受器细胞中，胞内cGMP水平的变化通过具有膜超极化和突触信号[2]的视紫红质连接光检测。cGMP的周转受到磷酸二酯酶（PDE）和鸟苷酸环化酶（GC）的相反活性的严格控制。磷酸二酯酶水解cGMP，鸟苷酸环化酶合成cGMP。PDE是由于紫红质的光检测而激活的，而GC是由光检测事件后的低Ca2+浓度激活的。GC活性的调节需要肉豆酰化的Ca2+结合蛋白，称为鸟苷酸环化酶激活蛋白或GCAPs[2]。这些gcap经过Ca2+诱导的构象改变，介导了它们在高Ca2+浓度下抑制GC和在低Ca2+浓度下激活GC的能力[4-6]。破坏GC功能的突变或gcap调节GC的能力会导致视网膜病变，导致部分或完全失明。GCAP1的四个点突变，包括一个已被充分表征的Y99C突变，已被证明会导致人类锥体或锥杆营养不良[9-12]。大多数这些突变似乎将GCAP1锁定在GC激活构象中，失去了作为Ca2+浓度函数调节GC的能力。我们需要阐明野生型和突变型GCAP1在Ca2+结合和释放时发生的构象变化，以及GCAP1与GC相互作用的分子基础。这些都是理解GCAP-GC在光传导中的关键作用及其在视网膜营养不良病因学中的功能的关键步骤。作为初步数据，我们已经解决了与Ca2+结合的肉豆蔻酰化GCAP （myrGCAP1）的第一个结构。我们建议进行结构和生化实验来探索Ca2+诱导的构象变化，使GCAPs能够调节GCs响应Ca2+的活性。成功完成本提案中概述的目标将为我们对光传导调控的理解开辟新天地。这也将为探究由GCAP和GC功能障碍引起的人类视网膜营养不良的分子机制提供一个平台。公共卫生相关性：鸟苷酸环化酶激活蛋白（gcap）是调节鸟苷酸环化酶（GCs）活性的Ca2+结合蛋白。这种调节活动对视网膜感光细胞的正常生理至关重要，它们的功能障碍会导致视网膜疾病和部分或完全失明。本研究的重点是了解GCAP对GCs的调控以及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.