RECOVERY OF PHOTOTRANSDUCTION

描述(由申请者提供)：我的长期目标是了解视杆细胞和视锥细胞光转导之间根本差异的分子基础，并将所学知识应用于其他G蛋白信号系统。视杆感光器和视锥感光器使用类似的方案将光转化为神经元信号。杆状物比锥状物更敏感，但反应速度较慢。光传导途径涉及不同但相关的基因。这项应用的前两个目的是利用视杆和视锥基因表达的这种差异来突出视杆回收中的限速步骤。第三个目的是研究G蛋白偶联受体激酶(GRK)家族的新成员GRK7，它是一种潜在的人类视锥蛋白激酶。目的1利用4.4kb的小鼠视蛋白启动子，通过建立RGS9-L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复合体。在昏暗的闪光条件下，将通过测量转导蛋白GTP的水解率和单杆吸力记录来检验过度表达对杆状细胞光转导的影响。我们将利用视锥细胞视网膜电图的“双闪”分析来评估RGS9-1/GBeta5-L过表达对视锥细胞光信号转导的影响。目的2利用上述转基因技术在小鼠光感受器中过表达法尼化(正常)GRK 1和香叶基香叶化(突变)GRK 1。过度表达的影响将通过使用常规生化技术和先进的质谱仪测量视紫红质磷酸化的速度和位置来测试。单视杆记录和“对闪光”视锥ERG分析将被用来分别检测视杆和视锥的生理效应。目的3表达和比较纯化的重组GRK1和GRK7对激活的视紫红质的活性，并在GRK1缺失的背景下获得异位表达人GRK7的转基因小鼠。HGRK7转基因小鼠的特征将按照AIM 2的描述进行。从拟议的体外和体内实验获得的结果确定GRK7是否替代了GRK1，GRK1缺陷的人类Oguchi病患者报告的正常明视视力的必然结果。使用GRK7作为诱饵的酵母双杂交筛选将在视网膜cDNA文库上进行，以确定视网膜中潜在的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，显著提高我们对视杆和视锥细胞光转导恢复的了解，并为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.