Department of Cell Biology, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma 73104, USA.
Invest Ophthalmol Vis Sci. 2011 Aug 11;52(9):6355-62. doi: 10.1167/iovs.10-7138.
Phosphoinositide 3-kinase (PI3K) consists of a p110 catalytic protein and a p85α regulatory protein, required for the stabilization and localization of p110-PI3K activity. The biological significance of PI3K was investigated in vertebrate rod photoreceptors by deleting its regulatory p85α protein and examining its role in photoreceptor structure, function, and protein trafficking.
Mice that expressed Cre recombinase in rods were bred to mice with a floxed p85α (pik3r1) regulatory subunit of PI3K to generate a conditional deletion of pik3r1 in rods. Functional and structural changes were determined by ERG and morphometric analysis, respectively. PI3K activity was measured in retinal homogenates immunoprecipitated with an anti-PY antibody. Akt activation was determined by Western blot analysis with a pAkt antibody.
Light-induced stress increased PI3K activity in retinal immunoprecipitates and phosphorylation of Akt. There was no effect of pik3r1 deletion on retinal structure. However, twin flash electroretinography revealed a slight delay in recovery kinetics in pik3r1 knockout (KO) mice compared with wild-type controls. The movement of arrestin in the pik3r1 KO mice was slower than that in the wild-type mouse retinas at 5 minutes of exposure to light. At 10 minutes of exposure, the ROS localization of arrestin was almost identical between the wild-type and pik3r1 KO mice.
The results provide the first direct evidence that rods use PI3K-generated phosphoinositides for photoreceptor function. The lack of phenotype in pik3r1 KO rod photoreceptors suggests a redundant role in controlling PIP(3) synthesis.
磷酸肌醇 3-激酶（PI3K）由 p110 催化蛋白和 p85α 调节蛋白组成，对于 p110-PI3K 活性的稳定和定位是必需的。通过删除其调节 p85α 蛋白并研究其在光感受器结构、功能和蛋白运输中的作用，研究了 PI3K 在脊椎动物杆状光感受器中的生物学意义。
在杆状细胞中表达 Cre 重组酶的小鼠与 floxed p85α（pik3r1）PI3K 调节亚基的小鼠交配，以在杆状细胞中产生 pik3r1 的条件性缺失。通过 ERG 和形态计量分析分别确定功能和结构变化。用抗-PY 抗体免疫沉淀视网膜匀浆测量 PI3K 活性。用 pAkt 抗体通过 Western blot 分析确定 Akt 激活。
光诱导的应激增加了视网膜免疫沉淀物中的 PI3K 活性和 Akt 的磷酸化。pik3r1 缺失对视网膜结构没有影响。然而，双闪光视网膜电图显示，与野生型对照相比，pik3r1 敲除（KO）小鼠的恢复动力学略有延迟。与野生型小鼠视网膜相比，pik3r1 KO 小鼠中视紫红质的运动在暴露于光 5 分钟时较慢。在暴露 10 分钟时，野生型和 pik3r1 KO 小鼠之间的 arrestin 的 ROS 定位几乎相同。
这些结果首次直接证明了 rods 使用 PI3K 产生的磷酸肌醇来发挥光感受器功能。pik3r1 KO 杆状光感受器中缺乏表型表明在控制 PIP（3）合成中存在冗余作用。
