Regulation of Cyclic GMP Synthesis in Photoreceptors

描述（由申请人提供）：光导信使cGMP，介导杆状体和锥体对光的反应。视网膜鸟苷酸环化酶（RetGC）在钙的控制下通过鸟苷酸环化酶激活蛋白（GCAPs）合成cGMP，是光响应恢复过程中最关键的步骤之一。它的调节缺陷导致了多种形式的先天性人类失明。虽然RetGC调控的一般重要性和基本原理已经确立，但一些关键的机制方面仍然知之甚少，特别是GCAP和RetGC中的蛋白质-蛋白质相互作用如何导致环化酶激活和抑制或引发视网膜疾病。寻求本提案所述问题的答案符合NEI “就致盲眼病、视觉功能机制和视力保护进行研究和传播信息”的使命。本研究基于对引起Leber先天性黑朦（Leber congenital amaurosis， LCA）的RetGC新突变的表征和RetGC调控的新发现：1)GCAP1在体内被致病突变脱敏感性优先靶向RetGC1同工酶；2) GCAP1作为“第一反应”Ca2+传感器，在光响应早期激活RetGC1；3) GCAP1中的n -脂肪酰化通过分子内“拉扯”作用影响其作为RetGC1钙传感器的功能；4) GCAP1分子中的两个区域可能成为环化酶结合界面；5)光感受器蛋白RD3作为RetGC活性的有效抑制剂，但当受到lca相关突变的影响时，不能抑制环化酶。我们提出了一种广泛的综合方法来验证新的假设，并结合蛋白质生物化学、分子生物学和分子遗传学来描述RetGC/GCAP调控途径的机制。目的1将讨论GCAP1的分子结构，重点是建立GCAP1进入RetGC激活状态的构象转变的关键，阐明致病的组成型活性GCAP1突变体的蛋白质结构，并验证“Ca2+-肉豆芽酰基牵引”的假设-控制GCAP1 Ca2+敏感性的n -脂肪酰基的跨分子作用。目的2将研究导致LCA1失明的新特征突变如何改变RetGC催化活性和调控的分子机制。目的3将寻求理解RD3作为一种新的生物学作用，与LCA和锥杆变性有关，是RetGC/GCAP通路的负调节因子。通过完成这些具体目标，我们希望克服一些关键障碍，了解RetGC调控及其在正常光感受器生理和视网膜疾病中的作用。

DESCRIPTION (provided by applicant): The phototransduction messenger, cGMP, mediates rod and cone response to light. The synthesis of cGMP by retinal guanylyl (guanylate) cyclase (RetGC), controlled by calcium through guanylyl cyclase activating proteins (GCAPs), is one of the most critical steps in photoresponse recovery. The defects in its regulation cause multiple forms of congenital human blindness. While the general importance and basic principles of the RetGC regulation have been established, some of the key mechanistic aspects remain poorly understood, especially how protein-protein interactions in GCAP and RetGC result in the cyclase activation and inhibition or triggering retinal diseases. Seeking answers to the questions addressed in this proposal conforms to the NEI mission to "conduct research and disseminate information with respect to blinding eye diseases, mechanisms of visual function and preservation of sight". This proposal is based on characterization of new RetGC mutations causing Leber congenital amaurosis (LCA) and novel findings about the RetGC regulation: 1) that GCAP1 desensitized by disease-causing mutations preferentially targets RetGC1 isozyme in vivo; 2) that GCAP1 acts as the 'first-response' Ca2+ sensor activating RetGC1 early in photoresponse; 3) that N-fatty acylation in GCAP1 affects its function as a calcium sensor for RetGC1 via an intramolecular 'tug' action; 4) that two regions in GCAP1 molecule emerge as a likely cyclase-binding interface; 5) that a photoreceptor protein, RD3, acts as a potent inhibitor of RetGC activity, but fails to inhibit the cyclase when affected by LCA-related mutations. We propose a broad integrated approach to verify new hypotheses and delineate mechanisms in RetGC/GCAP regulatory pathways using a combination of protein biochemistry, molecular biology, and molecular genetics. Aim 1 will address the molecular structure of GCAP1 with the emphasis on establishing the key to the conformational transition of GCAP1 into its RetGC activator state, elucidating the protein architecture for the disease-causing constitutive active GCAP1 mutants, and testing a hypothesis of "Ca2+-myristoyl tug" - across-the-molecule action of the N-fatty acyl group that controls Ca2+ sensitivity of GCAP1. Aim 2 will investigate how the molecular mechanisms of RetGC catalytic activity and regulation become altered by newly characterized mutations causing LCA1 blindness. Aim 3 will seek understanding of biological role of RD3 as a novel, linked to LCA and cone-rod degeneration, negative regulator of the RetGC/GCAP pathway. By completing these specific aims, we expect to overcome some critical barriers in understanding of RetGC regulation and its role in normal photoreceptor physiology and in retinal diseases.