Regulation of Cyclic GMP Synthesis in Photoreceptors

描述（由申请人提供）：拟议研究的目的是定义视觉光感受器中调节光传导和适应的新过程。长期目标是确定通过视网膜膜观酰基环化酶（retGC）调节光转导第二信使环GMP （cGMP）合成的分子机制。该建议是基于发现光感受器特异性钙传感器蛋白，GCAPs，调节retGC响应细胞内游离钙浓度的变化，是正常的光反应和光感受器细胞存活所必需的。最近的证据支持了我们的假设，即两个EF-hand结构域的镁结合控制了GCAP与环化酶在光线下的对接。因此，本研究的第一个目标是通过定向诱变确定retGC和gcap的对接位点，并在体内和体外测试它们的功能。在脊椎动物中有两种普遍存在的gcap: GCAP1和GCAP2。最近使用GCAP1和GCAP2基因敲除小鼠模型的研究结果提出了一种假设，即cGMP合成响应光的激活是一个两步过程，其中GCAP1和GCAP2根据其不同的钙敏感性依次刺激cGMP的产生。还有两个retGC同工酶，retGC1和retGC2，是棒恢复所必需的。因此，第二个具体目标是确定小鼠棒中GCAP1和GCAP2如何调节retGC的两种不同同工酶。我们计划建立单个GCAP1或GCAP2对单个retgc的参数和生化调控，并确定它们对杆生理和GCAP1突变引起的光感受器细胞死亡进程的贡献。视锥细胞对光的反应和恢复比杆状细胞快，适应光的速度也快，因此有一种假设认为，视锥细胞的cGMP合成比杆状细胞的更快。因此，本提案的第三个具体目的是确定锥体中retGC的生化特性以及单个gcap在调节锥体retGC活性中的作用。提出的实验是相关的机制，控制光感受器活性和导致遗传性视网膜疾病的人类患者的理解。

DESCRIPTION (provided by applicant): The purpose of the proposed study is to define new processes that regulate phototransduction and adaptation in visual photoreceptors. The long-term goal is to determine the molecular mechanisms that regulate synthesis of a second messenger of phototransduction, cyclic GMP (cGMP), by retinal membrane guanylyl cyclases (retGC). The proposal is based on the finding that photoreceptor-specific calcium sensor proteins, GCAPs, that regulate retGC in response to the change in intracellular free calcium concentrations are essential for normal photoresponses and survival of photoreceptor cells. Recent evidence supports our hypothesis that magnesium binding in two EF-hand domains controls GCAP docking with the cyclase in the light. Therefore, the first aim of the proposal is to identify, by using directed mutagenesis, the docking sites in retGC and GCAPs and to test their function in vitro and in vivo. There are two ubiquitous GCAPs in vertebrates, GCAP1 and GCAP2. Recent findings using GCAP1 and GCAP2 gene knockout mouse models have lead to a hypothesis that activation of cGMP synthesis in response to light is a two-step process, in which GCAP1 and GCAP2 stimulate cGMP production sequentially, according to their different calcium sensitivity. There are also two isozymes of retGC, retGC1 and retGC2, required for rod recovery. Therefore, the second specific aim is to establish how the two different isozymes of retGC are regulated by GCAP1 and GCAP2 in mouse rods. We plan to establish parameters and biochemical regulation of individual retGCs by individual GCAP1 or GCAP2 and determine their contribution to the rod physiology and to the progression of photoreceptor cell death caused by mutations in GCAP1. Cone cells respond to light and recover faster than rods and adapt to light very rapidly, thus leading to a hypothesis that cGMP synthesis is regulated faster in cones than in rods. Therefore, the third specific aim of this proposal is to determine the biochemical properties of retGC in cones and the role of individual GCAPs in regulation of cone retGC activity. The proposed experiments are relevant to the understanding of the mechanisms that control photoreceptor activity and cause inherited retinal diseases in human patients. PUBLIC HEALTH RELEVANCE: The proposed study has relevance to public health, because its completion will provide better understanding of the processes important for both normal vision and visual disorders. The main goal for this research is to understand how retinal guanylyl cyclases and calcium-binding proteins control electrical responses of the retina and why the mutations that affect these proteins lead to congenital blindness. The specific purpose of this study is to elucidate molecular mechanisms of their interactions and their role in retinal physiology and retinal degeneration.