REGULATION OF CYCLIC GMP SYNTHESIS IN PHOTORECEPTORS

本研究的目的是确定视觉受体中调节光传导和适应的新过程。长期目标是确定通过视网膜膜观基环化酶调节光转导第二信使环GMP合成的分子机制。该建议是基于光感受器特异性钙结合蛋白（GCAPs）的发现，在光感受器恢复和光适应过程中，细胞内游离Ca2+浓度的变化对刺激cGMP合成至关重要。最近的证据表明，gcap刺激环化酶的能力是由它们的Ca2+结合域和它们的初级结构的三个其他片段决定的。因此，本研究的第一个目的是通过定向诱变确定GCAP中特定的氨基酸残基，这些氨基酸残基构成了GCAP激活剂和抑制剂状态之间的切换，并确定GCAP激活环化酶的能力。GCAPs通过环化酶胞内结构域起作用，最近有证据表明GCAPs的二聚化控制环化酶亚基之间的相互作用。因此，本课题的第二个目的是确定环化酶被GCAP激活后的结构变化，并利用蛋白质工程和高分辨率色谱法揭示环化酶/GCAP复合物的结构。影响gcap调控retGC的突变最近与人类视网膜变性有关。本研究的第三个目的是通过体外生化分析和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锥体变性相关的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，反馈机制调节光感受器的活动，当受到干扰时，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.