Mammalian Phototransduction: Role of cGMP and Calcium

说明(申请人提供)：光感受器鸟苷环化酶(GC1和GC2)产生光转导的内部信使cGMP。阳离子由被称为鸟苷环化酶激活蛋白(GCAPs)的钙结合蛋白介导，调节它们的活性。常染色体显性遗传性视锥细胞营养不良(AdCD)和常染色体显性遗传性视锥-视杆细胞营养不良(AdCORD)与GCAP1基因(GUCA1A)中的钙结合突变有关。生化缺陷表现为在黑暗中对Gc1的主要、持续的刺激[钙]。在特定的目标1中，我们将描述基于GCAP1突变的adCD和adCORD的动物模型，并开发一种RNA干扰策略来击倒突变和正常的GCAP1基因。部分或完全敲除将延迟或治愈视锥细胞营养不良，因为GCAP基因缺失的小鼠只有很小的表型(暗适应延迟)，并且没有视网膜退化。这些实验是最终人类基因治疗的前奏。在特定的目的2中，我们建议通过在GCAPS基因敲除背景上转基因表达GCAP2来产生GCAP1基因敲除小鼠，以研究在GCAP1缺失的情况下恢复的动力学。此外，我们将首先在体外，最后在转基因实验中，探索与显性RP相关的GCAP2(G157R)突变的生化后果。在特定目标3中，我们建议在GCAPs基因敲除背景下建立仅表达GC1或GC2的小鼠，以分析在没有钙刺激的情况下每种酶对暗电流的贡献。我们进一步建议使用锥体特异性启动子在小鼠锥体中产生表达GC2的转基因小鼠。目的是研究表达GC2但不表达GC1的视锥细胞的生理特性，以及在GCAP1(在体外对GC2刺激很差)存在时GC2对钙的敏感性。在分析小鼠GC1和GC2基因敲除的表型时，我们发现这两个GCs作为完整的膜蛋白，在膜相关蛋白(PDE、GRK1、转导蛋白)的外段运输中起着关键作用。因此，我们建议使用表达绿色荧光蛋白的构建体、缺少靶向序列VXPX的视紫红质C-末端片段和含有所提出的靶向基序的C-末端GC片段(S)来鉴定转基因非洲爪哇中的靶向序列。这项研究将有助于我们理解光感受器中的cGMP/钙反馈系统，以及基于GCAP突变导致视网膜疾病的机制。

DESCRIPTION (provided by applicant): Photoreceptor guanylate cyclases (GC1 and GC2) produce cGMP, the internal messenger of phototransduction. Cations, mediated by Ca2+-binding proteins termed guanylate cyclase activating proteins (GCAPs), regulate their activities. Mutations affecting Ca2+-binding in the GCAP1 gene (GUCA1A) have been linked to autosomal dominant cone dystrophy (adCD) and autosomal dominant cone-rod dystrophy (adCORD). The biochemical defects present as a dominant, persistent stimulation of GC1 at dark [Ca2+]. In specific aim 1, we will characterize animal models of adCD and adCORD based on GCAP1 mutations, and develop an RNA interference strategy to knock down both the mutant and the normal GCAP1 genes. A partial or complete knockdown will delay onset or cure the cone dystrophy since GCAP null mice have only a minor phenotype (delay in dark adaptation) and no retinal degeneration. These experiments are a prelude to eventual human gene therapy. In specific aim 2, we propose to generate a GCAP1 knockout mouse by transgenic expression of GCAP2 on a GCAPs knockout background to investigate the kinetics of recovery in the absence of GCAP1. Further, we will explore the biochemical consequence of a GCAP2(G157R) mutation linked to dominant RP, first in-vitro and finally in a transgenic experiment. In specific aim 3, we propose to generate mice expressing GC1 only or GC2 only on a GCAPs knockout background to dissect the contribution of each enzyme to the dark current in the absence of Ca2+ stimulation. We further propose to generate transgenic mice expressing GC2 in mouse cones using a cone specific promoter. The purpose is to characterize the physiology of cones expressing GC2 but not GC1, and the Ca2+ sensitivity of GC2 in the presence of GCAP1 (which poorly stimulates GC2 in vitro). While analyzing the phenotypes of GC1 and GC2 knockouts in mouse, we discovered that both GCs, as integral membrane proteins, have key roles in transport of membrane-associated proteins (PDE, GRK1, Transducin) to the outer segments. We therefore propose to identify the targeting sequences in transgenic Xenopus using constructs expressing EGFP, a rhodopsin C-terminal segment lacking the targeting sequence VXPX, and C-terminal GC fragments containing the proposed targeting motif(s). The proposed research will contribute to our understanding of the cGMP/Ca2+ feed back system in photoreceptors, as well mechanisms leading to retina disease based on GCAP mutations.