Molecular Mechanism of Retinal Guanylate Cyclase Activation by GCAPs

描述（由申请人提供）：在感光细胞中，胞内cGMP水平的变化将视紫红质的光检测与膜超极化和突触信号传导联系起来[2]。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（PDE）水解cGMP，鸟苷酸环化酶（GC）合成cGMP。PDE被视紫红质的光检测激活，而GC被光检测事件后的低Ca 2+浓度激活。GC活性的调节需要豆蔻酰化的Ca 2+结合蛋白，称为鸟苷酸环化酶激活蛋白或GCAP [2]。这些GCAP经历Ca 2+诱导的构象变化，介导其在高Ca 2+浓度下抑制GC和在低Ca 2+浓度下激活GC的能力[4-6]。破坏GC功能或GCAP调节GC的能力的突变导致视网膜病变，导致部分或完全失明[8]。GCAP 1中的四个点突变（包括一个充分表征的Y 99 C突变）已被证明会导致人类视锥细胞或视锥-视杆细胞营养不良[9-12]。这些突变中的大多数似乎将GCAP 1锁定在GC活化构象中，从而失去了作为Ca 2+浓度的函数调节GC的能力。我们需要阐明野生型和突变型GCAP 1在Ca 2+结合和释放时所经历的构象变化以及GCAP 1与GC相互作用的分子基础。这些是理解GCAP-GC在光转导中的关键作用及其在视网膜营养不良病因学中的功能的关键步骤。作为初步的数据，我们已经解决了第一个结构的豆蔻酰化GCAP（myrGCAP 1）与钙离子结合。我们建议进行结构和生物化学实验，以探测钙离子诱导的构象变化，使GCAP调节活性的GC响应于钙离子。成功完成本提案中概述的目标将在我们对光转导调节的理解中开辟新的天地。它也将提供一个平台，以探讨潜在的人类视网膜营养不良的分子机制所造成的GCAP和GC的功能障碍。公共卫生相关性：鸟苷酸环化酶激活蛋白或GCAP是调节鸟苷酸环化酶（GC）活性的Ca 2+结合蛋白。这种调节活性对于视网膜中感光细胞的正常生理学是必需的，并且它们的功能障碍导致视网膜疾病和部分或全部失明。该提案的重点是了解GCAP对GCs的调控以及GCAP 1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.