RECOVERY OF PHOTOTRANSDUCTION

描述（由申请人提供）：我的长期目标是了解杆状和锥状光导之间基本差异的分子基础，并将这些知识应用于其他g蛋白信号系统。杆状光感受器和锥状光感受器使用类似的方案将光转换为神经元信号。视杆细胞比视锥细胞更敏感，但反应更慢。光传导途径涉及不同但相关的基因。该应用程序的前两个目的是利用杆状和锥状基因表达的差异来突出杆状采油中的限速步骤。GRK7是g蛋白偶联受体激酶（GRK）家族的新成员，也是人类视锥蛋白激酶的潜在成员。目的1：利用4.4 Kb的小鼠视蛋白启动子，分别生成RGS9-1和GBeta5-L转基因小鼠并进行杂交，在小鼠光感受器中过表达RGS9-1/GBeta5-L GAP复合物。过表达对光导杆的影响将通过测量转导蛋白GTP水解率和在微弱闪光条件下的单杆吸吸记录来检查。视锥衍生视网膜电图（ERG）“配对闪光”分析将用于评估RGS9-1/GBeta5-L过表达对视锥光导的影响。目的2是利用上述转基因技术在小鼠光感受器中过表达法酰化（正常）grk1和香叶酰化（突变）grk1。过度表达的影响将通过使用常规生化技术和先进的质谱法测量视紫红质磷酸化的速率和位点来测试。单杆记录和“对闪”锥衍生的ERG分析将分别用于检查对杆和锥的生理影响。目的3是表达和比较纯化的重组GRK1和GRK7在活化视紫质上的活性，并在GRK1缺失的背景下在小鼠光感受器中产生异位表达人GRK7的转基因小鼠。hGRK7转基因小鼠的表征将按照目的2的描述进行。从体外和体内实验中获得的结果确定了GRK7是否替代GRK1，这是人类口口病患者报告的GRK1缺陷的正常光性视力的必然结果。以GRK7为诱饵对视网膜cDNA文库进行酵母双杂交筛选，以鉴定视网膜中潜在的GRK7底物和/或调节因子。这些方法将在信号转导领域产生有用的试剂，显著提高我们对杆状和锥状光导恢复的认识，并为了解GRK7的生理功能提供有价值的见解。

DESCRIPTION (provided by applicant): My long-term goal is to understand the molecular basis underlying the fundamental differences between rod and cone phototransduction and to apply the knowledge to other G-protein signaling systems. Rod and cone photoreceptors use similar schemes to convert light into neuronal signals. Rods are more sensitive but respond slower than cones. The phototransduction pathways involve different but related genes. The first two aims of this application exploit such differences in rod and cone gene expression to highlight the rate-limiting step in rod recovery. The third aim focuses on the characterization of GRK7, a novel member of the G-protein coupled receptor kinase (GRK) family and a potential cone opsin kinase in human. Aim 1 is to over-express the RGS9-1/GBeta5-L GAP complex in mouse photoreceptor using a 4.4 Kb mouse opsin promoter by generating RGS9-l and GBeta5-L transgenic mice respectively and by crossbreeding them. The effects of over-expression on rod phototransduction will be examined by measuring the rate of transducin GTP hydrolysis and by single rod suction recordings under dim flash conditions. "Paired-flash" analyses of cone-derived electroretinography (ERG) will be used to assess the effect of RGS9-1/GBeta5-L over-expression on cone phototransduction. Aim 2 is to over-express farnesylated (normal) GRK 1 and geranylgeranylated (mutant) GRK 1 in mouse photoreceptors using transgenesis as described above. The effects of over-expression will be tested by measuring the rate and the sites of rhodopsin phosphorylation using conventional biochemical techniques and advanced mass spectrometry. Single rod recordings and "Pair-flash" cone derived ERG analyses will be used to examine the physiological effects on rods and cones, respectively. Aim 3 is to express and compare the activities of purified recombinant GRK1 and GRK7 on activated rhodopsin and to generate transgenic mice ectopically expressing human GRK7 in mouse photoreceptors on a GRK1 null background. Characterizations of hGRK7 transgenic mice will be performed as described for aim 2. Results obtained from the proposed in vitro and in vivo experiments determine whether or not GRK7 substitutes for GRK1, a corollary for the normal photopic vision reported by human oguchi disease patients with defective GRK1. Yeast two-hybrid screen using GRK7 as a bait on a retinal cDNA library will be performed to identify potential GRK7 substrates and/or regulators in the retina. These approaches shall generate useful reagents in the field of signal transduction, significantly advance our knowledge on the recovery of rod and cone phototransduction and provide valuable insight into the physiological functions of GRK7.