PHOTOTRANSDUCTION--METABOLISM OF CGMP AND CALCIUM

描述(摘自申请者的摘要)：两种关键酶 参与cGMP调节的是光感受器特异性的磷酸二酯酶 (PDE)和鸟苷环化酶(GC)。PDE是一种多亚单位酶(推定为 化学计量比PDE-α/β/Gamma2/Delta2)和外围 膜相关，而GC是单亚基完整的膜蛋白。 Gc1受[Ca~(2+)]通过一个或两个Ca~(2+)结合调节剂(鸟苷)调节 环化酶激活蛋白GCAP1和GCAP2)。此应用程序被定向 利用分子技术对这些组分进行表征 生物学。在具体目标1中，将共表达PDE亚基以建立 生物活性的基本成分。光感受器PDE是 只有尚未在单细胞中功能性表达的哺乳动物PDE 系统。作为一个子目标，将测试新的PDE亚基PDE-Delta 利用基因表达替换技术在小鼠体内发挥功能 野生型PDE-Delta基因已被靶向中断(PDE-Delta“基因 在特定目标2中，GCAP1的表达和诱变研究 将寻求确定与Gc1交互的域。这里的子目标，作为一个 合作的努力，就是敲除编码其中一种或两者的基因 GCAP。具体目标3针对gc1的表达和突变。这个 应聘者对RD鸡特别感兴趣，这是一种 早发型隐性常染色体视网膜色素变性(Leber‘s先天性 黑猩猩)。初步数据表明，Rd中编码Gc1的基因 鸡视网膜没有表达，这项提议旨在识别 特定的基因缺陷。总体而言，这项研究将加强基础 对参与的部件的结构/功能关系的知识 CGMP和钙离子这两个重要的第二信使的调节 光转导。

DESCRIPTION (Adapted from applicant's abstract): The two key enzymes involved in regulation of cGMP are photoreceptor-specific phosphodiesterase (PDE) and guanylate cyclase (GC). PDE is a multisubunit enzyme (putative stoichiometry PDE-alpha/beta/gamma2/delta2) and peripherally membrane-associated, while GC is a single subunit integral membrane protein. GC1 is regulated by [Ca2+] via one or two Ca2+-binding regulators (guanylate cyclase activating proteins, GCAP1 and GCAP2). This application is directed at the characterization of these components by techniques of molecular biology. In specific aim 1, PDE subunits will be co-expressed to establish the essential components for biological activity. Photoreceptor PDE is the only mammalian PDE that has not been functionally expressed in unicellular systems. As a subaim, the novel PDE subunit PDE-delta will be tested for function using gene expression replacement techniques in mice whose wild-type PDE-delta gene has been targeted for disruption (PDE-delta "gene knock-out"). In specific aim 2, GCAP1 expression and mutagenesis studies will seek to identify domains interacting with GC1. A subaim here, as a collaborative effort, is to knock out the genes encoding either or both GCAPs. Specific aim 3 addresses expression and mutagenesis of GC1. The applicant is particularly interested in the rd chicken, an animal model for early onset recessive autosomal retinitis pigmentosa (Leber's congenital amaurosis). Preliminary data indicate that the gene encoding GC1 in the rd chicken retina is not expressed, and this proposal aims to identify the specific gene defect. Collectively, this research will enhance fundamental knowledge of structure/function relationships of components participating in the regulation of cGMP and Ca2+, two important second messengers of phototransduction.