Regulation of Cyclic GMP Synthesis in Photoreceptors

光感受器中环状 GMP 合成的调控

• 批准号: 9107875
• 负责人: ALEXANDER M DIZHOOR
• 金额: $ 39.04万
• 依托单位: SALUS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1996
• 资助国家: 美国
• 起止时间: 1996-08-01 至 2017-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9107875
• 关键词: Address; Affect; Amino Acids; Architecture; Binding; Binding Proteins; Biological; Biological Preservation; Biological Process; Biology; Blindness; Calcium; Cell Death; Cone; Cyclic GMP; Defect; Dimerization; Disease; Electrons; Eye diseases; Genes; Goals; Guanylate Cyclase; Health; Human; Isoenzymes; Knowledge; Leber' s amaurosis; Light; Link; Maps; Mediating; Membrane; Missense Mutation; Mission; Molecular; Molecular Biology; Molecular Genetics; Molecular Structure; Mutate; Mutation; Pathway interactions; Patients; Photoreceptors; Phototransduction; Physiology; Point Mutation; Process; Protein Biochemistry; Proteins; Recovery; Regulation; Regulatory Pathway; Reporting; Research; Retinal; Retinal Degeneration; Retinal Diseases; Retinitis Pigmentosa; Role; Signal Transduction; Site-Directed Mutagenesis; Spectrum Analysis; Structure; Testing; Therapeutic; Transgenic Animals; Transgenic Model; Translating; Vertebrate Photoreceptors; Vision; acyl group; base; cGMP production; cone-rod degeneration; conformational conversion; design; disease-causing mutation; fatty acylation; gene therapy; guanylate cyclase activating protein; in vitro Model; in vivo; inhibitor/antagonist; link protein; mouse model; mutant; novel; prevent; protein complex; protein protein interaction; research study; response; sensor; temporal measurement; treatment strategy

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.

描述（由申请人提供）：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，并通过鸟苷酸环化酶激活蛋白 (GCAP) 受钙控制，是光响应恢复中最关键的步骤之一。其调节缺陷导致多种形式的人类先天性失明。虽然 RetGC 调节的一般重要性和基本原理已经确定，但一些关键机制方面仍然知之甚少，特别是 GCAP 和 RetGC 中蛋白质-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。寻求本提案中提出的问题的答案符合 NEI 的使命，即“开展有关致盲眼病、视觉功能机制和视力保护的研究并传播信息”。该提案基于导致 Leber 先天性黑蒙 (LCA) 的新 RetGC 突变的特征以及有关 RetGC 调节的新发现：1) 因致病突变而脱敏的 GCAP1 在体内优先靶向 RetGC1 同工酶； 2) GCAP1 充当“第一响应”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“拖拉”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作为环化酶结合界面出现； 5) 光感受器蛋白 RD3 是 RetGC 活性的有效抑制剂，但在受到 LCA 相关突变影响时无法抑制环化酶。我们提出了一种广泛的综合方法，结合蛋白质生物化学、分子生物学和分子遗传学来验证新假设并描述 RetGC/GCAP 调控途径的机制。目标 1 将解决 GCAP1 的分子结构，重点是确定 GCAP1 构象转变为其 RetGC 激活剂状态的关键，阐明致病的组成型活性 GCAP1 突变体的蛋白质结构，并测试“Ca2+-肉豆蔻酰牵引”的假设 - 控制 Ca2+ 敏感性的 N-脂肪酰基的跨分子作用 GCAP1 的。目标 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。目标 3 将寻求了解 RD3 作为与 LCA 和视锥杆变性相关的新型 RetGC/GCAP 途径负调节因子的生物学作用。通过完成这些具体目标，我们期望克服理解 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的一些关键障碍。