Understanding the functions of USH2A and ADGRV1 in photoreceptors by identifying their interacting proteins

项目摘要 Usherin(USH2A)和黏附G蛋白偶联受体V1(ADGRV1)是两个大的跨膜蛋白 光感受器睫状膜复合体中的蛋白质。编码这些基因的突变 两种蛋白质破坏PMC并导致常染色体隐性视网膜色素变性(Arrp)和Usher 综合征(USH)。USH2A基因是arrp和USH最常见的致病基因。后者是一种 ARRP合并听力损失，是世界上遗传性聋盲的主要原因。 ARRP和USH患者都会经历早夜和周边视力丧失，并完全或 法律上最终是盲目的。目前，还没有治愈ARRP或USH的方法。在光感受器的PMC，USH2A 和ADGRV1通过细胞内的PDZ结合基序与支架蛋白Whirlin(WHRN)相互作用 C-末端。在这三种蛋白质中，我们之前的研究表明ADGRV1起着最重要的作用 在PMC中扮演的角色。然而，USH2A和ADGRV1的功能在很大程度上仍不清楚，因为 这两个蛋白质的大部分区域位于光感受器之外，目前还没有得到很好的研究。我们的 免疫沉淀法和标记蛋白下拉结合质谱法的初步研究 光谱分析在牛和小鼠体内鉴定出几种与USH2A和ADGRV1相互作用的候选蛋白 视网膜。其中一些相互作用被免疫共沉淀和小鼠的共定位所证实 视网膜或通过使用重组蛋白的下拉试验。根据这些初步调查结果，我们建议 对USH2A和ADGRV1胞外结构域相互作用蛋白进行全面、无偏见的分析。在……里面 目的1.从牛视网膜中鉴定潜在的胞外USH2A和ADGRV1相互作用蛋白 裂解物通过使用胞外诱饵的下拉分析，结合质谱分析。在目标2中，已确定的 将通过以下方法在小鼠和猴子中确认潜在的USH2A和ADGRV1胞外结构域相互作用蛋白 标准生化结合分析和定位研究以及已知的USH2A和ADGRV1的作用 这些相互作用上的致病错义突变将被检查。这项研究的结果将提供 光感受器中PMC功能与视网膜变性发病机制的新认识 USH2A和ADGRV1缺陷。

Project Summary Usherin (USH2A) and adhesion G protein-coupled receptor V1 (ADGRV1) are two large transmembrane proteins in the periciliary membrane complex (PMC) in photoreceptors. Mutations in the genes encoding these two proteins disrupt the PMC and lead to autosomal recessive retinitis pigmentosa (arRP) and Usher syndrome (USH). The USH2A gene is the most common causative gene for arRP and USH. The latter is a condition of arRP combined with hearing loss and is the leading cause of inherited deaf-blindness in the world. Both arRP and USH patients experience early night and peripheral vision loss and become completely or legally blind eventually. Currently, no cure is available for arRP or USH. At the PMC in photoreceptors, USH2A and ADGRV1 interact with a scaffold protein whirlin (WHRN) through their PDZ-binding motif at the intracellular C-terminus. Among the three proteins, our previous studies suggest that ADGRV1 plays the most important role in the PMC. However, the functions of USH2A and ADGRV1 remain largely unknown, because the majority region of these two proteins is located outside photoreceptors and has not been well studied. Our preliminary studies using immunoprecipitation and tagged protein pull-down assays coupled with mass spectrometry identified several USH2A- and ADGRV1-interacting candidate proteins in bovine and mouse retinas. Some of these interactions were confirmed by coimmunoprecipitation and colocalization in mouse retinas or by pull-down assays using recombinant proteins. Based on these initial findings, we propose to conduct a comprehensive, non-biased analysis of USH2A and ADGRV1 ectodomain-interacting proteins. In Aim 1, potential extracellular USH2A- and ADGRV1-interacting proteins will be identified from bovine retinal lysates by pull-down assays using ectodomain baits, coupled with mass spectrometry. In Aim 2, the identified potential USH2A and ADGRV1 ectodomain-interacting proteins will be confirmed in mice and monkeys by standard biochemical binding assays and localization studies, and the effect of known USH2A and ADGRV1 pathogenic missense mutations on these interactions will be examined. The findings of this study will provide novel insights into PMC function in photoreceptors and the pathogenesis of retinal degeneration caused by USH2A and ADGRV1 defects.