MOLECULAR GENETICS OF PHOTOTRANSDUCTION

光传导的分子遗传学

• 批准号: 2684555
• 负责人: BIH-HWA SHIEH
• 金额: $ 25.97万
• 依托单位: VANDERBILT UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1993
• 资助国家: 美国
• 起止时间: 1993-04-01 至 2001-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2684555
• 关键词: Drosophilidae; G protein; animal genetic material tag; diacylglycerols; electrophysiology; enzyme activity; gene mutation; genetically modified animals; inositol phosphates; intermolecular interaction; molecular genetics; phospholipase C; photoactivation; visual photoreceptor; visual phototransduction

DESCRIPTION: The investigator will use phototransduction in Drosophila as a model system to understand the coordination of the activation and deactivation processes of signal transduction. The experimental strategy is to study a protein named INAD, to understand how it regulates phototransduction. The investigator has shown that INAD interacts with two components involved in the activation and deactivation of the visual cascade. INAD interacts with TRP and a loss of interaction leads to a slow recovery of the visual response. INAD associates with NORPA, the effector of the visual cascade. NORPA flies lack any light-triggered receptor potential. Knowledge gained may help identify INAD homologues in vertebrates. The investigator proposes to : 1) investigate the INAD-TRP interaction; 2) investigate the INAD-NORPA association; 3) study the structure-function relationship of INAD in vivo; 4) generate null alleles (loss-of-function) of InaD and examine the electrophysiological defects of the mutants. This application proposes to study visual transduction in Drosophila. The focus is on a gene called InaD. In the 1970's, Bill Pak and his colleagues isolated a large collection of Drosophila visual mutants that were defective in the ERG. InaD was part of this collection; it was not one of the remarkable mutants. Its basic ERG defect was called inactivation-no-afterpotential, hence its name. The world of phototransduction has changed considerably since the 1970's. there is now have a fairly solid description of vertebrate visual transduction and the G-protein coupled, cGMP-mediated cascade that generates it. The situation for Drosophila is somewhat less advanced, but clearly goes through a different pathway with Phospholipase C (NORPA) being a major player via IP-3 and DAG (diacylglycerol). More recently, the field has been investigating mechanisms of adaptation: how photo responses are modulated appropriately for background light conditions. This is clearly a complicated process and involves control at many levels along the transduction cascade. [Vertebrate inactivation and adaptation: Ca involved - regulates photoreceptor sensitivity. Ca (dark) enters via cGMP-gated channels inhibits guanylate cyclase via guanylate cyclase activating protein (Ca binding protein). Second pathway: regulation of amplification by photoactivated opsins. Ca inhibits phosphorylation of opsin. Mediated by Ca binding protein S-modulin (recoverin). Ca modulates cGMP-gated channel by decreasing affinity of cGMP.] Presently, the main issues are: how are the photoresponse and adaptation responses integrated?; what are the coupling mechanisms?; and' how are they controlled? Preliminary work generated by the investigator indicates that for Drosophila, INAD may be a central player, hence, the main intellectual interest in the present proposal. The study of INAD is timely and addresses the main concerns of the field, at present. The investigator has shown that INAD protein interacts physically with 2 others: TRP and NORPA. Phenotypically, InaD and trp mutants are somewhat similar: they are defective in deactivation and adaptation of the photoresponse. TRP appears to be a Ca channel that may mediate ionic responses underlying adaptation. The most interesting finding is the interaction with NORPA. NORPA stands for no receptor potential. It is a Phospholipase C that has always been believed to be the major player in the activation cascade. Thus, the placement of InaD in the phototransduction cascade is very intriguing: early in the link between the activation cascade and the adaptation response. In this proposal the investigator focuses on the functional consequences of eliminating protein-protein interactions between INAD, TRP, and NORPA. She will generate mutations that alter or destroy the interaction between INAD and TRP, and between INAD and NORPA. The appropriate transgenics will be produced. The investigator will then examine electrophysiological consequences. Expression will assessed to control for appropriate levels and subcellular localization.

描述：研究人员将使用果蝇中的光转导作为一种 模型系统，以了解激活和 信号转导的失活过程。 实验策略是 研究一种名为INAD的蛋白质， 光传导 研究人员已经表明，INAD与两个 参与视觉的激活和去激活的组件 级联。 INAD与TRP相互作用，失去相互作用会导致缓慢的 恢复视觉反应。 INAD与NORPA相关，NORPA是效应器， 视觉级联的结果。 NORPA果蝇缺乏任何光触发受体 潜力 获得的知识可能有助于确定INAD同系物， 脊椎动物 研究者建议：1）研究INAD-TRP 交互作用; 2）研究INAD-NORPA关联; 3）研究 INAD在体内结构-功能关系; 4）产生无效等位基因 （功能丧失）的InaD和检查电生理缺陷， 变种人 本申请旨在研究果蝇的视觉转导。 的 重点是一个叫做InaD的基因。 20世纪70年代，比尔·帕克和他的同事 分离出大量果蝇视觉突变体， 在ERG。 InaD是这个系列的一部分;它不是其中之一。 非凡的突变体 它的基本ERG缺陷被称为 失活无后电位，因此得名。 的世界 自20世纪70年代以来，光传导已经发生了相当大的变化。 现在有 有一个相当坚实的描述脊椎动物的视觉转导和 G蛋白偶联、cGMP介导的级联反应产生了它。情况 对于果蝇来说，它的进化程度稍低，但很明显， 不同的途径，磷脂酶C（NORPA）是通过IP-3的主要参与者 和DAG（二酰基甘油）。 最近，该领域一直在研究适应机制： 如何适当地调制背景光的光响应 条件 这显然是一个复杂的过程， 沿着转导级联的许多水平。 [脊椎动物失活和适应：钙参与-调节 光感受器敏感性 Ca（暗）通过cGMP门控通道进入 通过鸟苷酸环化酶激活蛋白（Ca）抑制鸟苷酸环化酶 结合蛋白）。 第二条途径：调节扩增 光活化视蛋白 Ca抑制视蛋白的磷酸化。 介导 钙结合蛋白S-调制蛋白（恢复蛋白）。 Ca调节cGMP门控通道 降低cGMP的亲和力。 目前主要的问题是：光反应和适应性如何 综合反应？耦合机制是什么？他们怎么样 控制？ 调查员的初步工作表明， 对于果蝇来说，INAD可能是一个核心角色，因此，主要的智力 对目前的提案感兴趣。 国家农业发展研究所的研究是及时的， 目前该领域的主要担忧。 调查人员表明， INAD蛋白与另外两种蛋白质相互作用：TRP和NORPA。 表型上，InaD和trp突变体有些相似：它们是 在光响应的失活和适应方面有缺陷。 TRP出现 是一个钙通道，可能介导的离子反应的基础适应。 最有趣的发现是与NORPA的互动。 NORPA支架 没有受体电位。 它是一种磷脂酶C， 被认为是激活级联反应的主要参与者。 因此 InaD在光转导级联中的位置非常有趣： 在激活级联和适应之间的早期联系中， 反应 在本提案中，研究者重点关注以下功能性后果： 消除INAD、TRP和NORPA之间的蛋白质-蛋白质相互作用。 她 将产生突变，改变或破坏INAD之间的相互作用， 和TRP，以及INAD和NORPA之间。 合适的转基因将是 制作。 然后，研究者将检查电生理学 后果 将评估表达以控制适当水平 和亚细胞定位。