REGULATION OF CYCLIC GMP SYNTHESIS IN PHOTORECEPTORS

这项拟议的研究的目的是定义调节视觉感受器中光传导和适应的新过程。长期目标是确定调节视网膜膜鸟苷酸环化酶合成环GMP的分子机制，环GMP是光传导的第二信使。这一建议是基于这样的发现，即光感受器特异的钙结合蛋白(GCAPs)对于刺激cGMP合成是必不可少的，以响应光感受器恢复和光适应过程中细胞内游离钙浓度的变化。最近的证据表明，GCAP刺激环化酶的能力是由它们的钙结合结构域和其他三个一级结构片段决定的。因此，本研究的第一个目的是利用定向突变技术确定GCAP中的特定氨基酸残基，这些氨基酸残基构成了GCAP激活和抑制状态之间的转换，并确定了GCAP激活环化酶的能力。GCAPs通过环化酶胞内结构域发挥作用，最近的证据表明GCAPs的二聚化控制着环化酶亚单位之间的相互作用。因此，本方案的第二个目的是利用蛋白质工程和高分辨层析技术，确定环化酶被GCAP激活后的结构变化，并揭示环化酶/GCAP复合体的结构。影响GCAP调节retGC的突变最近被认为与人类视网膜变性有关。这项建议的第三个目的是通过对GCAP突变的体外活性进行生化检测，并通过研究转基因视网膜的形态和功能变化，来表征与人类常染色体显性视锥变性相关的GCAP突变的生化和生物学效应。这里提出的实验与理解复杂的反馈机制有关，这些反馈机制调节光感受器的活动，当受到干扰时，会导致遗传性视网膜疾病。

The purpose of the proposed study is to define new processes that regulate phototransduction and adaptation in visual receptors. The long-term goal is to determine the molecular mechanisms that regulate synthesis of cyclic GMP, the second messenger of phototransduction, by retinal membrane guanylyl cyclases. The proposal is based on the findings that photoreceptor-specific calcium binding proteins, GCAPs, are essential for stimulation of cGMP synthesis in response to the change in the intracellular free Ca2+ concentrations during recovery and light adaptation of photoreceptors. Recent evidence demonstrate that the ability of GCAPs to stimulate the cyclase is determined by their Ca2+ binding domains and three other fragments of their primary structure. Therefore, the first aim of this study is to identify by using directed mutagenesis the specific amino acid residues in GCAPs that: constitute a switch between the GCAP activator and inhibitor states and determine the ability of GCAPs to activate the cyclase. GCAPs act via the cyclase intracellular domain, and there are recent evidence that dimerization of GCAPs control the interaction between the cyclase subunits. Therefore, the second aim of this proposal is to determine the structural changes in the cyclase upon its activation by GCAP and to reveal the structure of the cyclase/GCAP complexes by using protein engineering and high resolution chromatography. Mutations that affect regulation of retGC by GCAPs have been recently linked to human retinal degeneration. The third aim of this proposal is to characterize biochemical and biological effects of a GCAP mutation associated with a human autosomal dominant cone degeneration by using biochemical assays for its activity in vitro and by studying the morphological and the functional changes in transgenic retinas. The experiments proposed here are relevant to understanding of the intricate feedback mechanisms that regulate photoreceptor activity and, when perturbed, cause inherited retinal diseases.