CYCLIC GMP IN PHOTORECEPTORS

描述(改编自申请人的摘要)：建议的目标 研究旨在定义调控光信号转导的新机制和 视觉感受器的适应过程。主要的焦点是确定 一种新的钙结合蛋白p24(GCAP-2)的生理作用 两个光感受器谷氨酰环酶(GCs)合成cGMP，第二个 照片响应中的信使。这项建议是基于最近的 发现两种不同的膜GC，RetGC-1和RetGC-2，存在于 光感受器细胞和两种不同的钙结合蛋白调节 两种环化酶的活性均与游离钙离子浓度有关。这 研究结果显示了提供反馈的监管要素的多样性 环鸟苷酸水解后再合成的机制 光激发。这项研究的第一个目的是通过使用 定向突变，GCAP-2的哪些部分激活GC。GCAP-1和GCAP-2 是同源物，RetGC-1和RetGC-2也是同源物。因此， 这项建议的第二个目标是评估它们的本地化，如所确定的那样 通过免疫细胞化学分析。RetGC-1的相对效率和 GCAP-1或GCAP-2对RetGC-2的调节也将在体外进行测量。 这些实验将表明两个GCAP中的任何一个 与特定的环化酶相互作用，它们还将提供特征 这样的互动。第三个目标是评估其相对重要性 GCAP-1和-2以及RetGC-1和-2对cGMP的调节作用 在截断的外部节段中进行合成。这个项目的长期目标是 是确定GCAP-2参与GC的所有结构决定因素 监管。这里提出的实验与理解 复杂的反馈控制，调节光感受器的活动 可能患有几种遗传性视网膜疾病。

DESCRIPTION (Adapted from applicant's abstract): The goal of the proposed study is to define new mechanisms regulating the phototransduction and adaptation processes in visual receptors. The major focus is to determine the physiological role of a novel Ca2+-binding protein, p24 (GCAP-2), and two photoreceptor gluanylyl cyclase (GCs) in synthesis of cGMP, a second messenger in the photoresponse. This proposal is based on the recent finding that two different membrane GCs, RetGC-1 and RetGC-2, are present in photoreceptor cells and two different Ca2+-binding proteins can regulate activity of both cyclases as a function of free Ca2+ concentration. This finding demonstrates the diversity of regulatory elements providing feedback mechanisms for resynthesis of cGMP after its hydrolysis caused by photoexcitation. The first aim of this study is to define, by using directed mutagenesis, what parts of GCAP-2 activate GC. GCAP-1 and GCAP-2 are homologs and RetGC-1 and RetGC-2 are also homologs. Therefore, the second aim of this proposal is to assess their localization, as determined by immunocytochemical analysis. The relative efficiency of RetGC-1 and RetGC-2 regulation by GCAP-1 or GCAP-2 will also be measured in vitro. These experiments will indicate whether either of the two GCAPs specifically interacts with a certain cyclase and they will also provide characteristics of such interactions. The third aim is to evaluate the relative importance of GCAP-1 and -2 as well as RetGC-1 and -2 for the regulation of cGMP synthesis in truncated outer segments. The long-term goal of this project is to identify all structural determinants of GCAP-2 involved in GC regulation. The experiments proposed here are relevant to understanding the intricate feedback controls that regulate photoreceptor activity and that might be perturbed in several inherited retinal diseases.