CYCLIC GMP IN PHOTORECEPTORS

描述（改编自申请人的摘要）：拟议的目标 研究的目的是确定调节光转导的新机制， 视觉感受器的适应过程 主要重点是确定 一种新的钙离子结合蛋白p24（GCAP-2）的生理作用， 两种感光体谷氨酰环化酶（GCs）在cGMP的合成，第二种 光反应中的信使 这一建议是根据最近 发现两种不同的膜GC，RetGC-1和RetGC-2，存在于 感光细胞和两种不同的Ca 2+结合蛋白可以调节 两种环化酶的活性作为游离Ca 2+浓度的函数。 这 这一发现表明了提供反馈的调节元件的多样性 cGMP水解后再合成的机制 光激发 本研究的第一个目的是通过使用 定向诱变，GCAP-2的哪些部分激活GC。 GCAP-1和GCAP-2 RetGC-1和RetGC-2也是同源物。 因此 这项建议的第二个目的是评估它们的本地化， 免疫细胞化学分析。 RetGC-1和 还将在体外测量GCAP-1或GCAP-2对RetGC-2的调节。 这些实验将表明，这两种GCAP中的任何一种， 与某种环化酶相互作用，它们也会提供 这样的互动。 第三个目的是评估相对重要性 GCAP-1和GCAP-2以及RetGC-1和RetGC-2用于调节cGMP 在截短的外节中合成。 这个项目的长期目标是 目的是确定GC中GCAP-2的所有结构决定因素 调控 这里提出的实验与理解 复杂的反馈控制，调节感光细胞的活动， 可能在几种遗传性视网膜疾病中受到干扰。

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.