VISUAL PIGMENT AND PHOTORECEPTOR ADAPTATION

总体目标是阐明手术，在完整的视网膜 色素上皮（RPE），类维生素A处理反应，介导11- 顺式视网膜形成并因此支持视杆中的视紫红质再生。 从蟾蜍（B. marinus）“RPE-眼杯”（后眼杯 缺乏神经视网膜）表明，光感受器间类维生素A结合 蛋白（IRBP），一种感光细胞间基质的可溶性糖蛋白， 显著刺激RPE从全反式合成11-顺式视黄醛 前体，以及释放这种顺式视网膜顶端 细胞外介质 这些实验旨在确定IRBP如何 对RPE类维生素A的加工产生这些影响， 退行性疾病影响加工反应的状态。 下 IRBP、细胞外类维生素A和 天然眼杯的照明预漂白），（3 H）类维生素A的命运 将在蟾蜍RPE-眼杯中检查掺入RPE的细胞。 具体 目的是确定11-顺式视黄酯作为前体的活性 11-顺式视黄醛或（通过反向异构化）反式酯的合成，以及这种合成是否 反应是IRBP依赖性的。 “VPP”转基因小鼠，一种经过充分研究的 将研究缓慢进展的感光细胞变性模型 缓慢进展的感光细胞变性模型，将用于 实验分析漂白诱导的维甲酸水平的变化， 视网膜和视网膜色素上皮细胞。 一个关键的目标是确定类维生素A 在这种视蛋白相关的细胞中， 退化性疾病 在使用蟾蜍RPE-眼杯的进一步实验中， 具有确定序列的重组IRBP多肽（由我们的 合作者）将测试在促进11-顺式视黄醇 从RPE释放和向RPE递送全反式视黄醇。 观察到的 测试多肽的类维生素A转移活性将解释为 与其（天然和突变体）氨基酸序列的关系，目的是 阐明了天然IRBP在RPE顶膜作用的基础。

The overall goal is to elucidate the operation, within intact retinal pigment epithelium (RPE), of retinoid processing reactions that mediate 11- cis retinal formation and thus support rhodopsin regeneration in the rods. Results obtained from the toad (B. marinus) "RPE-eyecup" (posterior eyecup devoid of neural retina) show that interphotoreceptor retinoid-binding protein (IRBP), a soluble glycoprotein of the interphotoreceptor matrix, markedly stimulates the RPE's synthesis of 11-cis retinal from all-trans precursor, as well as the release of this cis retinal to the apical extracellular medium. The experiments are aimed at determining how IRBP exerts these effects on RPE retinoid processing, and how progressive rod degenerative disease affects the status of the processing reactions. Under defined conditions of treatment with IRBP, extracellular retinoid, and illumination pre-bleaching of the native eyecup), the fate of (3H) retinoid incorporated into the RPE will be examined in toad RPE-eyecups. Specific aims are to determine the activities of 11-cis retinyl ester as a precursor of 11-cis retinal or (by back isomerization) trans ester, and whether such reactions are IRBP-dependent. The "VPP" transgenic mouse, a well-studied model of slowly progressing photoreceptor degeneration, will be studied model of slowly progressing photoreceptor degeneration, will be used in experiments to analyze bleach-induced changes in retinoid levels within the retina and RPE in vivo. A key aim is to determine whether retinoid processing becomes impaired at early times in this opsin-linked degenerative disease. In further experiments using toad RPE-eyecup, recombinant IRBP polypeptides of defined sequence (to be provided by our collaborators) will be tested for activity in promoting 11-cis retinal release from, and all-trans retinol delivery to, the RPE. The observed retinoid transfer activities of test polypeptides will be interpreted in relation to their (native and mutant) amino acid sequences, with the aim of elucidating the basis of native IRBP's action at the RPE apical membrane.