INACTIVATION AND ADAPTATION OF THE ROD PHOTORESPONSE

视杆细胞感光反应的失活和适应

• 批准号: 3264617
• 负责人: NANCY J MANGINI
• 金额: $ 15.44万
• 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/3264617
• 关键词: Amphibia; animal tissue; autoradiography; binding proteins; chemical binding; cytochalasins; intracellular transport; laboratory rat; light adaptations; microfilaments; phosphodiesterases; phospholipase C; phosphorylation; photoactivation; photobiology; protein purification; protein sequence; 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， 启动酶级联反应，导致细胞内 cGMP水平。 这种[cGMP]i的短暂降低导致 阳离子通道的质膜上的杆外节和一个 视觉细胞的超极化（即，光响应）。 灵敏度 以及在不同条件下光响应的适应， 照明关键取决于这种酶级联的失活， cGMP水平的恢复。 一种丧失了 光诱导信号的潜在过程将导致异常 cGMP水平，并可导致视力下降，甚至感光细胞 死亡 本申请的重点是抑制蛋白，其是本发明的一个方面。 参与使光传导的酶级联失活的蛋白质。 这些研究的目的是确定抑制蛋白的机制， 营业额，并研究这一机制如何与驱动力 在视杆内（IS）和 外部段（OS）。 理由是初步数据表明， 光暴露后抑制蛋白的降解。 如果这种退化 抑制蛋白发生，那么抑制蛋白在杆中的半衰期可能比这短。 视蛋白和一种降解机制--除了椎间盘脱落-- - 可能有牵连。 通过胞质蛋白酶（例如， 钙蛋白酶）被假设为一种可能的机制。 一个新的分析， 识别PEST区域的抑制蛋白序列（即，条件性位点 胞质蛋白酶的蛋白水解信号）与此一致 假说. 在此基础上，研究（i）抑制素生物合成和 (ii)作为抑制蛋白“失活”机制的有限蛋白水解是 提出了 采用生物化学、免疫细胞化学和 规划了形态学方法。 拟议研究的具体目标 包括：（AIM 1a）确定视杆细胞中抑制蛋白的周转， 光暴露的功能，以及（AIM 1b）检查可能的参与 微丝在IS和OS之间转运arrestin;（AIM 2） 检查抑制蛋白是否被ROS（胞质）蛋白酶降解 (e.g.,钙蛋白酶和/或泛素），和（AIM 3）检查功能性 抑制蛋白的有限蛋白水解的结果。 实验以确定 蛋白水解是否发生在抑制蛋白序列的PEST区域， 也计划。 持续更新--不仅是抑制蛋白--还有杆内的所有蛋白质， 对正常的视觉细胞功能至关重要 提出的长期目标 研究的目的是确定这种更新过程中的缺陷是否可能 视网膜变性疾病如色素性视网膜炎的基础。