Regulation of Cyclic GMP Synthesis in Photoreceptors

描述(申请人提供)：光传导信使，cGMP，介导视杆和视锥对光的反应。视网膜鸟苷环化酶(RetGC)合成cGMP是光反应恢复过程中最关键的步骤之一，它受钙通过鸟苷酸环化酶激活蛋白(GCAPs)调控。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定，但一些关键的机制仍不清楚，特别是GCAP和RetGC中的蛋白质-蛋白质相互作用如何导致循环酶激活和抑制或引发视网膜疾病。寻求本提案中涉及的问题的答案符合NEI的使命，即“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”。这一建议是基于对导致Leber先天性黑色素(LCA)的新的RetGC突变的表征和关于RetGC调节的新发现：1)由致病突变脱敏的GCAP1优先针对活体中的RetGC1同工酶；2)GCAP1在光反应早期作为激活RetGC1的“第一反应”钙传感器；3)GCAP1中的N-脂肪酰化通过分子内的‘TUG’作用影响其作为RetGC1的钙传感器的功能；4)GCAP1分子中的两个区域作为可能的环化结合界面出现；5)光感受器蛋白RD3是RetGC活性的有效抑制剂，但当受到LCA相关突变的影响时，它不能抑制循环酶。我们提出了一种广泛的综合方法来验证新的假设和描述RetGC/GCAP调控通路中的机制，结合蛋白质生物化学、分子生物学和分子遗传学。目的1讨论GCAP1的分子结构，重点是确定GCAP1构象转变为其RetGC激活状态的关键，阐明致病的结构性活性GCAP1突变体的蛋白质结构，并检验一个假说，即控制GCAP1对钙的敏感性的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催化活性和调节的分子机制。目的3了解RD3作为一种新的与LCA和视锥-杆退行性变相关的生物学作用，视锥-杆变性是RetGC/GCAP途径的负调控因子。通过完成这些特定的目标，我们希望克服在理解RetGC调节及其在正常光感受器生理学和视网膜疾病中的作用方面的一些关键障碍。

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.