Regulation of Cyclic GMP Synthesis in Photoreceptors

描述（由申请方提供）：光转导信使cGMP介导视杆细胞和视锥细胞对光的反应。视网膜鸟苷酸环化酶（RetGC）合成cGMP，由钙通过鸟苷酸环化酶激活蛋白（GCAP）控制，是光反应恢复中最关键的步骤之一。其调节的缺陷导致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建立，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。寻求本提案中所述问题的答案符合NEI的使命，即“开展致盲性眼病、视觉功能机制和视力保护方面的研究并传播信息”。这一建议是基于对导致Leber先天性黑蒙（LCA）的RetGC新突变的表征和关于RetGC调节的新发现：1）致病突变使GCAP 1在体内优先靶向RetGC 1同工酶：2）GCAP 1作为“第一反应”Ca 2+传感器在光反应早期激活RetGC 1; 3）GCAP 1的N-脂肪酰化通过分子内的“拖拽”作用影响其作为RetGC 1的钙传感器的功能：4）GCAP 1分子中的两个区域可能作为环化酶结合界面出现; 5）光感受器蛋白RD 3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。我们提出了一个广泛的综合方法，以验证新的假设，并使用蛋白质生物化学，分子生物学和分子遗传学相结合的RetGC/GCAP调控途径的机制描绘。目的1将解决GCAP 1的分子结构，重点是建立GCAP 1的构象转变到RetGC激活状态的关键，阐明致病组成型活性GCAP 1突变体的蛋白质结构，并测试一个假设的“钙2 +-肉豆蔻酰拖船”-跨分子的N-脂肪酰基的行动，控制钙2+敏感性的GCAP 1。目的2将研究RetGC催化活性和调节的分子机制如何被新表征的突变改变，导致LCA 1失明。目的3将寻求RD 3作为一种新的生物学作用的理解，与LCA和锥杆变性，RetGC/GCAP通路的负调节剂有关。通过完成这些特定的目标，我们希望克服一些关键的障碍，在理解RetGC的调节及其在正常的感光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.