INACTIVATION AND ADAPTATION OF THE ROD PHOTORESPONSE

视杆细胞感光反应的失活和适应

• 批准号: 3264614
• 负责人: NANCY J MANGINI
• 金额: $ 17.44万
• 依托单位: UNIVERSITY OF ILLINOIS AT CHICAGO
• 依托单位国家: 美国
• 财政年份: 1988
• 资助国家: 美国
• 起止时间: 1988-03-01 至 1995-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3264614
• 关键词: Amphibia; animal tissue; autoradiography; binding proteins; chemical binding; cytochalasins; intracellular transport; laboratory rat; light adaptations; membrane reconstitution /synthesis; microfilaments; phosphodiesterases; phospholipase C; phosphorylation; photoactivation; photobiology; protein purification; protein structure function; protein transport; proteolysis; rhodopsin; rod cell; ubiquitin; visual photoreceptor; visual phototransduction; western blottings

In rod visual cells, light absorption by the receptor protein, rhodopsin, initiates an enzyme cascade that leads to a decrease in intracellular levels of cGMP. This transient decrease in [cGMP]i causes closure of cation channels on the plasma membrane of the rod outer segment and a hyperpolarization of the visual cell (i.e., a photoresponse). Sensitivity and adaptation of the photoresponse under varying conditions of illumination depend critically on the deactivation of this enzyme cascade, and the recovery of cGMP level. An impairment in the ability to deactivate processes underlying the light-induced signal will result in abnormal levels of cGMP, and can lead to poor vision or even photoreceptor cell death. The emphasis of the present application is on arrestin, one of the proteins involved in deactivating the enzyme cascade of phototransduction. The goal of proposed studies is to determine the mechanism of arrestin turnover, and to examine how this mechanism relates to driving forces behind light-dependent movements of arrestin between rod inner- (IS) and outer segments (OS). The rational is preliminary data suggesting enhanced degradation of arrestin upon light exposure. If such degradation of arrestin occurs, then arrestin's half-life in rods may be shorter than that of opsin, and a mechanism for degradation -- in addition to disk shedding - - might be involved. Targeting of arrestin by cytosolic proteases (e.g., calpain) is hypothesized as one possible mechanism. A novel analysis of the arrestin sequence identifying PEST regions (i.e., conditional sites of proteolysis signalling by cytosolic proteases) is consistent with this hypothesis. On this basis, studies examining (i) arrestin biosynthesis and (ii) limited proteolysis as a mechanism for arrestin "deactivation" are proposed. Experiments employing biochemical, immunocytochemical and morphological methods are planned. Specific aims of the proposed studies include: (AIM 1a) determining the turnover of arrestin in rods as a function of light exposure, and (AIM 1b) examining the possible involvement of microfilaments in the transport of arrestin between IS and OS; (AIM 2) examining whether arrestin is degraded by an ROS (cytosolic) protease (e.g., calpain, and/or ubiquitin), and (AIM 3) examining the functional consequences of limited proteolysis of arrestin. Experiments to determine whether proteolysis occurs at a PEST region of the arrestin sequence are also planned. Continued renewal -- not only of arrestin -- but all proteins within rods, is critical for normal visual cell function. A long-term goal of proposed studies is to determine whether defects in this renewal process may underlie retinal degenerative diseases such as Retinitis Pigmentosa.

在视杆细胞中，受体蛋白视紫红质吸收光， 启动酶级联反应，导致细胞内 cGMP 水平。 [cGMP]i 的这种短暂减少会导致 杆外节质膜上的阳离子通道和 视觉细胞的超极化（即光反应）。 灵敏度 以及不同条件下光响应的适应 照明主要取决于该酶级联的失活， 以及cGMP水平的恢复。 停用能力受损 光感应信号背后的过程将导致异常 cGMP水平，会导致视力不佳甚至感光细胞 死亡。 本申请的重点是视紫红质抑制蛋白，它是 参与使光转导酶级联失活的蛋白质。 拟议研究的目标是确定抑制蛋白的机制 营业额，并研究该机制与驱动力的关系 视杆内部（IS）和视杆细胞之间视紫红质抑制蛋白的光依赖性运动背后 外部段（OS）。 理由是初步数据表明增强 光暴露后抑制蛋白降解。 如果这种退化 如果出现视紫红质抑制蛋白，则视紫红质抑制蛋白在视杆细胞中的半衰期可能会比此短 视蛋白，以及降解机制——除了磁盘脱落之外—— - 可能涉及。 通过胞质蛋白酶靶向抑制蛋白（例如， calpain）被假设为一种可能的机制。 新颖的分析 抑制蛋白序列识别 PEST 区域（即条件位点） 胞质蛋白酶的蛋白水解信号传导）与此一致 假设。 在此基础上，研究检查了（i）视紫红质抑制蛋白的生物合成和 (ii) 有限的蛋白水解作为抑制蛋白“失活”的机制 建议的。 采用生物化学、免疫细胞化学和 形态学方法已规划。 拟议研究的具体目标 包括：（目标 1a）确定视杆细胞中视紫红质抑制蛋白的周转率 光暴露的功能，以及（AIM 1b）检查可能的参与 IS 和 OS 之间运输抑制蛋白的微丝； （目标2） 检查视紫红质抑制蛋白是否被 ROS（胞质）蛋白酶降解 （例如，钙蛋白酶和/或泛素），以及（AIM 3）检查功能 视紫红质抑制蛋白有限蛋白水解的后果。 实验确定 蛋白水解是否发生在视紫红质抑制蛋白序列的 PEST 区域 也有计划。 持续更新——不仅是视紫红质抑制蛋白——还有视杆细胞内的所有蛋白质， 对于正常的视觉细胞功能至关重要。 拟议的长期目标 研究的目的是确定更新过程中的缺陷是否可能 是视网膜退行性疾病的基础，例如色素性视网膜炎。