Translocation of soluble proteins in rod photoreceptors

视杆光感受器中可溶性蛋白质的易位

• 批准号: 7233141
• 负责人: Edward N Pugh
• 金额: $ 38.48万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-06-01 至 2009-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7233141
• 关键词: Affect; Affinity; African; Antigens; Arrestin; Arrestins; Binding; Binding Sites; Biosensor; Blood Vessels; Calcium; Carrier Proteins; Cells; Chimeric Proteins; Cilia; Condition; Confocal Microscopy; Cytoplasm; Cytosol; Dark Adaptation; Drosophila genus; Epitopes; Exhibits; Eye; G-Protein-Coupled Receptors; Generations; In Vitro; Inflammation; Laboratories; Life; Light; Lighting; Membrane; Metabolism; Methods; Molecular; Movement; Mus; Names; Nature; Numbers; Opsin; Phosphatidylinositol 4,5-Diphosphate; Phosphatidylinositols; Phosphorylation; Photons; Photoreceptors; Phototransduction; Physiological; Play; Process; Proteins; Proteolysis; Research Personnel; Retina; Retinal; Retinal Pigments; Rhodopsin; Role; Site; Solutions; Testing; Time; Tracer; Work; Xenopus; Xenopus laevis; Xenopus sp.; autoimmune uveitis; cell type; in vivo; migration; neuronal cell body; prevent; programs; recoverin protein; research study; response; retinal rods; rhodopsin kinase; sensor

DESCRIPTION (provided by applicant): Rod photoreceptors outnumber by 20:1 all other cell types in most vertebrate retinas. Arrestin is a 48 KDa protein expressed specifically in rod photoreceptor cells. Arrestin is the second-most abundant protein in the retina, estimated to be present at 1:10 to 1:2 the quantity of the most abundant retinal protein, rhodopsin, the visual pigment and G-protein coupled receptor that captures light and initiates the rod's light response. Arrestin plays an essential role in the inactivation of light-activated rhodopsin, capping the latter once it is phosphorylated. Arrestin was originally discovered and named retinal S-antigen, because it can evoke uveitis, an autoimmune inflammation of the blood-vessel rich layers of the eye. Arrestin undergoes a remarkable light-dependent migration between the two major compartments of the rod cell: in the dark-adapted eye it is concentrated largely in the inner segment region, while under light-adapted conditions it translocates almost completely to the outer segment, where rhodopsin resides. The proposed work investigates the molecular mechanisms underlying the light-dependent translocation of arrestin in living rod cells of the African clawed frog, Xenopus laevis, using arrestin-EGFP fusion proteins as tracers/biosensors, and 2-photon, confocal microscopy to quantify the protein and its movement. Work by several laboratories has shown such fusion proteins faithfully track the movement of native arrestin, and work from this laboratory has established that the dark-adapted distribution of arrestin is in disequilibrium with the cytoplasm. The proposed work will test several hypothesis about the nature of the arrestin dark disequilibrium and light-driven movement, including that (a) the disequilbrium is effected by a molecular "gate" in the connecting cilium; (b) the light-driven movement occurs purely by passive (diffusional) redistribution upon massive binding epitope creation by rhodopsin activation; (c) the redistribution is a light-activated process involving intraflagellar transport proteins, which serve to carry proteins from their origin to site of use in polarized, ciliated cells.

描述（由申请人提供）：在大多数脊椎动物视网膜中，所有其他细胞类型都超过20：1。逮捕蛋白是一种在杆感光细胞中特别表达的48 kDa蛋白。逮捕素是视网膜中第二多数蛋白质，估计存在于1:10至1：2最丰富的视网膜蛋白，视紫红质，视觉颜料和G蛋白偶联受体的数量，可捕获光并启动杆的光反应。逮捕素在光激活的视紫红素的灭活中起着至关重要的作用，一旦将其磷酸化就可以限制后者。最初被逮捕蛋白被发现并命名为视网膜S抗原，因为它可以唤起葡萄膜炎，这是眼睛富含血管的自身免疫性炎症。逮捕蛋白在杆细胞的两个主要隔室之间经历了显着的光依赖性迁移：在黑暗的眼睛中，它主要集中在内部段区域，而在光适应的条件下，它几乎完全转移到了若愿蛋白居住的外部段。拟议的工作调查了使用抑制素-EGFP融合蛋白作为示踪剂/生物传感器和2光角色，共摄影显微镜来量化蛋白质及其运动的非洲爪叶诺植物Laevis的光依赖性易位易位的分子机制。几个实验室的工作表明，这种融合蛋白忠实地跟踪了本地骤停的运动，该实验室的工作已经确定，抑制素的黑暗适应分布与细胞质不平衡。拟议的工作将检验一些关于阻止的黑暗不平衡和轻驱动运动的性质的假设，包括（a）不平衡由连接纤毛中的分子“栅极”实现； （b）轻度驱动的运动纯粹是通过被动（扩散）重新分布而发生的，该重新分布在大量结合表位通过视紫红质激活而产生； （c）重新分布是一个涉及flagellar转运蛋白的光激活过程，该过程可将蛋白从其起源携带到极化的纤毛纤毛细胞中的使用位置。