GUANYLATE CYCLASE TRANSDUCTION SYSTEMS IN RETINA

视网膜中的鸟苷酸环化酶转导系统

• 批准号: 6125118
• 负责人: RAMESHWAR K SHARMA
• 金额: $ 35.42万
• 依托单位: UNIV OF MED/DENT NJ-SCH OSTEOPATHIC MED
• 依托单位国家: 美国
• 财政年份: 1994
• 资助国家: 美国
• 起止时间: 1994-08-01 至 2002-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6125118
• 关键词: SDS polyacrylamide gel electrophoresis; adenosine triphosphate; affinity chromatography; animal tissue; atrial natriuretic peptide; autocrine; biological signal transduction; computer simulation; enzyme activity; guanylate cyclase; laboratory rabbit; molecular cloning; neural transmission; paracrine; receptor binding; receptor expression; retina; retinal adaptation; rod cell; tissue /cell culture; visual phototransduction

The ultimate aim of this research proposal is to decode the molecular and biochemical definition of the recovery-adaptation process of phototransduction and of a new process probably linked with the synaptic transmission of the retina. The foundation of the project is the cloning the native rod outer segment membrane guanylate cyclase (ROS-GC), the enzyme pivotal to both the transduction processes of vision and also to the retinal neuronal synaptic activity. Recent evidence indicates that the enzyme exhibits these two activities by acting as a double-sensor of Ca/2+. It has two switches. One switch inhabits and the other switch stimulates the enzymes at micromolar concentrations of Ca/2+. The inhibitory switch is linked to phototransduction and the stimulatory switch to retinal synaptic activity. The major objective is to elucidate the most basic molecular mechanisms involved in the inhibitory and stimulatory switching components of ROS-GC. Recent evidences indicates that the intracellular segment of ROS-GC is modular in nature and the modules are specific for diverse Ca/2+ signals linked to the two processes. Signals related to both processes are mediated by the Ca/2+ binding proteins: GCAP1 and GCAP2 in phototransduction and S100beta in synaptic transmission. Domains of three distinct modules of ROS-GC involved in GCAPs and S100beta stimulation of ROS-GC have been localized and termed Ca/2+-regulated modules (CRMs): CRM1 is linked to GCAP1 signaling. CRM2 to S100beta and CRM3 to GCAP2. A second form of ROS-GC, ROS-GC2, has been cloned from the retina, and is also Ca/2+ modulated, suggesting its linkage to phototransduction. Neither its linkage to synaptic transduction nor its modular nature has been established. Specific Aims (1-5) as stated below are to determine: (1) sequence motifs of CRM1, CRM2 and CRM3 which act as "turn on" switches for GCAP1, GCAP2 and S100beta; (2) the Ca/2+-induced folding changes in the GCAPs and S100beta sequence motifs critical in ROS-GC; (3) sequence motifs of GCAP1, GCAP2 and S100beta needed to interact with CRM1, CRM2 and CRM3; (4) the mechanisms of ROS-GC2 activation by GCAPs, 1 and 2; and (5) if ROS-GC2 is also modulated by Ca/2+ in a stimulatory fashion. If it is, determine its mechanism of modulation. The combined tools of biochemistry, genetic remodeling, computer simulation-molecular modeling, and immunology will be used to accomplish these most basic goals, which have direct ramifications in understanding the molecular processes related to vision and retinal neurological disorders.

这项研究计划的最终目的是解码分子和 恢复适应过程的生化定义 光转导和可能与突触相关的新过程 视网膜的传输。该项目的基础是克隆 天然杆外节膜鸟苷酸环化酶（ROS-GC）， 酶对于视觉转导过程和 视网膜神经元突触活动。最近的证据表明 酶通过充当双传感器来表现出这两种活性 钙/2+。它有两个开关。一个开关处于工作状态，另一个开关处于工作状态 在微摩尔浓度的 Ca/2+ 下刺激酶。这 抑制开关与光转导和刺激有关 切换到视网膜突触活动。主要目标是阐明 参与抑制和抑制的最基本的分子机制 ROS-GC 的刺激开关组件。最近的证据表明 ROS-GC 的细胞内片段本质上是模块化的，并且 模块特定于连接到两个的不同 Ca/2+ 信号 流程。与这两个过程相关的信号均由 Ca/2+ 介导 结合蛋白：光转导中的 GCAP1 和 GCAP2 以及光转导中的 S100beta 突触传递。 ROS-GC 三个不同模块的域 参与 ROS-GC 的 GCAP 和 S100beta 刺激已被定位 并称为 Ca/2+ 调节模块 (CRM)：CRM1 与 GCAP1 相连 发信号。 CRM2 到 S100beta 以及 CRM3 到 GCAP2。 ROS-GC的第二种形式， ROS-GC2，已从视网膜克隆，并且也是 Ca/2+ 调制的， 表明其与光转导的联系。它的链接也不是 突触转导及其模块化性质尚未确定。 下面所述的具体目标 (1-5) 是确定： (1) 序列基序 CRM1、CRM2 和 CRM3 充当 GCAP1、GCAP2 的“打开”开关 和S100beta； (2) Ca/2+诱导的GCAPs折叠变化和 S100beta 序列基序在 ROS-GC 中至关重要； (3) GCAP1的序列基序， GCAP2和S100beta需要与CRM1、CRM2和CRM3交互； （4） GCAP 1 和 2 激活 ROS-GC2 的机制； (5) 如果 ROS-GC2 是 也通过 Ca/2+ 以刺激方式调节。如果是，则确定其 调制机制。生物化学、遗传学的综合工具 重塑、计算机模拟分子建模和免疫学将 用于实现这些最基本的目标，这些目标有直接的影响 了解与视觉和视网膜相关的分子过程 神经系统疾病。