cGMP and Photoreceptor Function

描述（由申请人提供）：虽然通常已知视杆细胞中视觉转导的基本特征，但对在光感受器磷酸二酯酶（PDE 6）激活和失活期间发生的蛋白质-蛋白质相互作用和变构调节知之甚少。此外，哺乳动物光反应的光适应的生化机制还没有得到很好的描述，但有强有力的证据表明，PDE 6调节的其他机制在光反应脱敏中发挥作用。视觉信号分子机制知识的这些严重空白需要弥补，以充分了解PDE 6或其结合伴侣的改变如何导致视觉功能障碍和视网膜疾病。长期目标是阐明在视网膜光感受器的光反应的激发、恢复和适应过程中控制PDE 6活性的多种机制。本申请的总体目标是定义生物化学和结构进展，其开始于通过转导蛋白光诱导的PDE 6全酶活化，随后是负责PDE 6恢复至暗适应状态及其在光适应期间脱敏的事件。统一的假设是，抑制？- PDE 6亚基（P？）负责介导PDE 6催化亚基和转导素以及与其他调节蛋白之间发生的变构相互作用。这一假设的实验基础依赖于最近的工作表明，P？与几个结构上相互作用 和PDE 6催化亚基的功能上不同的结构域。此外，线性扩展构象的P？与PDE 6催化二聚体结合提供了与活化的转导蛋白以及与另一种PDE 6结合配偶体GARP 2相互作用的多个位点。在目标1中，将定义导致转导素结合后PDE 6全酶瞬时激活的步骤顺序。目的2的目标是确定控制PDE 6活性寿命的变构机制。目的3将定义已知的PDE 6相互作用蛋白的拓扑关系和它们调节PDE 6活性的机制。在光反应过程中，生物化学途径信息与PDE 6信号复合物结构改变的创新整合将显着增加我们对视杆细胞和视锥细胞中光转导途径的理解。PDE 6及其相互作用伴侣的大分子复合物的活化、失活和适应步骤顺序的新知识是预测和治疗视网膜变性疾病和视觉障碍的先决条件，这些疾病是由光感受器细胞中PDE 6信号传导复合物功能障碍引起的。

DESCRIPTION (provided by applicant): While the basic features of visual transduction in rod photoreceptors are generally known, the protein-protein interactions and allosteric regulation that occur during activation and deactivation of the photoreceptor phosphodiesterase (PDE6) are poorly understood. Further, the biochemical mechanisms underlying light adaptation of the mammalian photoresponse are not well delineated, but there is strong evidence that additional mechanisms of PDE6 regulation play a role in photoresponse desensitization. These serious gaps in knowledge of the molecular mechanism of visual signaling need to be remedied to fully understand how alterations in PDE6 or its binding partners can lead to visual dysfunction and retinal disease. The long-term goal is to elucidate the multiple mechanisms controlling PDE6 activity during excitation, recovery, and adaptation of the photoresponse in retinal photoreceptors. The overall objective of this application is to define the biochemical and structural progression that starts with light-induced activation of PDE6 holoenzyme by transducin, followed by the events responsible for PDE6 recovery to the dark-adapted state and its desensitization during light adaptation. The unifying hypothesis is that the inhibitory ?-subunt of PDE6 (P?) is responsible for mediating allosteric interactions that occur between the PDE6 catalytic subunits and transducin as well as with other regulatory proteins. The experimental basis for this hypothesis relies on recent work showing that P? interacts with several structurally and functionally distinct domains of the PDE6 catalytic subunits. Furthermore, the linearly extended conformation of P? bound to the PDE6 catalytic dimer provides multiple sites of interaction with activated transducin, as well as with another PDE6 binding partner, GARP2. In Aim 1, the sequence of steps leading to transient activation of PDE6 holoenzyme subsequent to binding of transducin will be defined. The goal of Aim 2 is to determine the allosteric mechanisms by which the active lifetime of PDE6 is controlled. Aim 3 will define the topological relationship of known PDE6-interacting proteins and the mechanism(s) by which they modulate PDE6 activity. This innovative integration of biochemical pathway information with the structural alterations in the PDE6 signaling complex during the photoresponse will significantly increase our understanding of the phototransduction pathway in rods and cones. This new knowledge of the sequence of steps in the activation, inactivation, and adaptation of the macromolecular complex of PDE6 and its interacting partners is a prerequisite for predicting and treating retinal degenerative diseases and visual disorders that result from dysfunction of the PDE6 signaling complex in photoreceptor cells.