MOLECULAR GENETICS OF PHOTOTRANSDUCTION

描述：研究人员将利用果蝇的光传导作为一种 模型系统，以了解激活和协调 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，了解它是如何调节 光转导。研究人员已经证明，INAD与两个 参与视觉激活和去激活的组件 卡斯卡德。INAD与Trp相互作用，失去相互作用会导致缓慢的 视觉反应的恢复。Inad与效应器Norpa联系在一起 视觉瀑布。Norpa苍蝇缺乏任何光触发的感受器 潜力。获得的知识可能有助于识别INAD同源基因 脊椎动物。调查者建议：1)调查INAD-Trp 相互作用；2)调查INAD-Norpa关联；3)研究 体内INAD的结构与功能关系；4)产生空等位基因 (功能丧失)，并检查电生理缺陷。 变种人。 这项申请建议研究果蝇的视觉转导。这个 人们关注的是一种名为inad的基因。上世纪70年代的S、白比尔和他的同事们 分离出大量有缺陷的果蝇视觉突变体 在ERG中。Inad是这个集合的一部分；它不是 了不起的变种人。其基本的ERG缺陷被称为 失活--无后电势，因此得名。世界上的 自20世纪70年代以来，光传导发生了很大的变化。现在有了 对脊椎动物的视觉转导有相当扎实的描述 G蛋白偶联，cGMP介导的级联反应产生它。目前的形势 因为果蝇没有那么高级，但显然经历了一个 不同的途径，磷脂酶C(Norpa)是IP-3的主要参与者 和DAG(二酰甘油)。 最近，该领域一直在研究适应机制： 如何针对背景光适当地调制照片响应 条件。这显然是一个复杂的过程，涉及到对 沿着转导级联的许多水平。 脊椎动物的失活和适应：钙离子参与-调节 感光敏感度。CA(暗)通过cGMP门控通道进入 通过鸟苷环化酶激活蛋白(Ca)抑制鸟苷环化酶 结合蛋白)。第二条途径：基因扩增的调控 光激活的视黄素。CA抑制视蛋白的磷酸化。调解人 钙结合蛋白S-调制素(Recoverin)。CA调制cGMP门控通道 通过降低cGMP的亲和力。] 目前的主要问题是：光响应和适应是如何的 综合反应？；耦合机制是什么？；以及‘它们怎么样？ 控制？调查人员生成的初步工作表明 对于果蝇来说，inad可能是一个核心角色，因此，主要的智力 对本提案的兴趣。对INAD的研究是及时的和解决的 目前，该领域主要关注的是。调查员已经证明 Inad蛋白与另外两个蛋白相互作用：Trp和Norpa。 从表型来看，inad和trp突变体有些相似：它们是 光反应的失活和适应有缺陷。出现TRP 可能是一种钙通道，可能介导适应背后的离子反应。 最有趣的发现是与诺帕的互动。诺帕站在那里 因为没有感受器潜力。它是一种磷脂酶C，一直是 据信是激活级联中的主要参与者。因此， Inad在光导电级联中的放置非常有趣： 在激活级联和适应之间的早期链接 回应。 在这项提案中，调查者关注的是 消除INAD、Trp和Norpa之间的蛋白质-蛋白质相互作用。她 会产生突变，改变或破坏INAD之间的相互作用 和Trp，以及Inad和Norpa之间。适当的转基因技术将是 制作。然后调查者将检查电生理学 后果。表达将被评估为适当水平的控制 和亚细胞定位。

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.