INACTIVATION AND ADAPTATION OF THE ROD PHOTORESPONSE

在视杆细胞中，受体蛋白视紫红质的光吸收， 启动一种酶的级联反应，导致细胞内 CGMP水平。[cGMP]i的这种短暂下降导致关闭 杆状外节质膜上的阳离子通道和 视觉细胞的超极化(即光反应)。敏感度 和光响应在不同条件下的适应 光照在很大程度上取决于该酶级联的失活， CGMP水平的恢复。停用能力的损害 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Arrestin，它是一种 参与光转导酶级联失活的蛋白质。 拟议研究的目标是确定芳香化的机制 营业额，并研究这一机制与驱动力的关系 视杆内侧(IS)和视杆(IS)之间Arrestin的光相关运动背后 外部段(OS)。理性的是初步数据表明 紫罗兰素在光照下的降解。如果这样的退化 当芦荟素出现时，芦荟素在棒状体内的半衰期可能会比这短 以及一种降解机制--除了视盘脱落-- -可能有牵连。胞浆蛋白水解酶靶向arrestin(例如， Calain)被认为是一种可能的机制。一种新的分析方法 识别害虫区域的arrestin序列(即 胞浆蛋白水解酶发出的蛋白分解信号)与此一致 假设。在此基础上，研究了(I)芳香素的生物合成和 (2)作为arrestin“失活”机制的有限蛋白分解是 建议。使用生化、免疫细胞化学和 形态学方法已经计划好了。拟议研究的具体目标 包括：(目标1a)确定棒状物中芳香草素的周转率 光暴露的功能，以及(AIM 1b)检查可能的参与 微丝在芳香素在IS和OS之间的运输中的作用； 检测arrestin是否被ROS(胞浆)酶降解 (例如，钙蛋白酶和/或泛素)，以及(目标3)检查功能 Arrestin有限蛋白分解的后果。实验以确定 蛋白降解是否发生在arrestin序列的有害区域 也是有计划的。 持续更新--不仅是arrestin--而且是杆状体内的所有蛋白质， 对正常的视觉细胞功能至关重要。建议的长期目标 研究是确定更新过程中的缺陷是否可能 视网膜退行性疾病，如视网膜色素变性。

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.