RECOVERY OF PHOTOTRANSDUCTION

描述（由申请人提供）：我的长期目标是了解杆和锥光转导之间的根本差异的分子基础，并将知识应用于其他G蛋白信号系统。视杆和视锥光感受器使用类似的方案将光转换成神经元信号。视杆细胞比视锥细胞更敏感，但反应慢。光传导途径涉及不同但相关的基因。本申请的前两个目的利用视杆和视锥基因表达的这种差异来突出视杆恢复中的限速步骤。第三个目标是对GRK 7进行研究，它是G蛋白偶联受体激酶（GRK）家族的新成员，也是一种潜在的视锥细胞视蛋白激酶。目的1是通过分别产生RGS 9 - 1和GBeta 5-L转基因小鼠并进行杂交，使用4.4Kb小鼠视蛋白启动子在小鼠光感受器中过表达RGS 9 -1/GBeta 5-L GAP复合物。过表达对视杆光转导的影响将通过测量转导素GTP水解的速率和通过在昏暗闪光条件下的单杆抽吸记录来检查。视锥细胞衍生的视网膜电图（ERG）的“配对闪光”分析将用于评估RGS 9 -1/G β 5-L过表达对视锥细胞光转导的影响。目的2是使用如上所述的转基因在小鼠光感受器中过表达法尼基化（正常）GRK 1和香叶基香叶基化（突变）GRK 1。将通过使用常规生物化学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过表达的影响。单视杆记录和“成对闪光”视锥细胞衍生ERG分析将分别用于检查对视杆细胞和视锥细胞的生理效应。目的3：表达纯化的重组GRK 1和GRK 7，并比较其对激活的视紫红质的活性，以及在GRK 1空白背景下产生在小鼠光感受器中异位表达人GRK 7的转基因小鼠。hGRK 7转基因小鼠的表征将如目的2所述进行。从所提出的体外和体内实验中获得的结果确定GRK 7是否替代GRK 1，这是GRK 1缺陷的人类口病患者报告的正常明视视力的必然结果。将使用GRK 7作为诱饵在视网膜cDNA文库上进行酵母双杂交筛选，以鉴定视网膜中潜在的GRK 7底物和/或调节剂。这些方法将在信号转导领域产生有用的试剂，显着推进我们对恢复视杆细胞和视锥细胞光转导的知识，并提供有价值的洞察GRK 7的生理功能。

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.