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

项目摘要 Usherin（USH2A）和粘附G蛋白偶联受体V1（ADGRV 1）是两个大的跨膜受体， 光感受器中的睫状体膜复合体（PMC）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 arRP合并听力损失，是世界上遗传性盲的主要原因。 arRP和USH患者都经历早期夜间和周边视力丧失，并完全或完全丧失。 最终失明。目前，arRP或USH没有治愈方法。在PMC的光感受器，USH2A 和ADGRV 1通过其PDZ结合基序在细胞内与支架蛋白whirlin（WHRN）相互作用， C端在这三种蛋白中，我们以前的研究表明ADGRV 1起着最重要的作用， 在PMC中的角色。然而，USH2A和ADGRV 1的功能在很大程度上仍然是未知的，因为 这两种蛋白质的大部分区域位于光感受器之外，尚未得到很好的研究。我们 使用免疫沉淀和标记蛋白下拉测定结合质谱的初步研究 光谱法在牛和小鼠中鉴定了几种USH2A和ADGRV 1相互作用的候选蛋白质 视网膜这些相互作用中的一些已经通过免疫共沉淀和小鼠体内的共定位得到证实 视网膜或通过使用重组蛋白的下拉测定。根据这些初步研究结果，我们建议 对USH2A和ADGRV 1胞外结构域相互作用蛋白进行全面、无偏倚的分析。在 目的1、从牛视网膜中鉴定潜在的细胞外USH2A和ADGRV 1相互作用蛋白 通过使用胞外域诱饵的下拉测定法结合质谱法测定裂解物。在目标2中， 将通过以下方法在小鼠和猴中证实潜在的USH2A和ADGRV 1胞外结构域相互作用蛋白： 标准生物化学结合测定和定位研究，以及已知USH2A和ADGRV 1的作用 将检查这些相互作用的致病性错义突变。这项研究的结果将提供 对PMC在光感受器中的功能和由PMC引起的视网膜变性的发病机制的新见解 USH2A和ADGRV 1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.