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

通过识别 USH2A 和 ADGRV1 的相互作用蛋白来了解它们在光感受器中的功能

• 批准号: 9891346
• 负责人: Jun Yang
• 金额: $ 22.88万
• 依托单位: UNIVERSITY OF UTAH
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-02-01 至 2021-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9891346
• 关键词: Adhesions; Affect; Affinity Chromatography; Antibodies; Binding; Biochemical; Bioinformatics; Biological Assay; Blindness; Cattle; Cells; Complex; Coupled; Defect; Disease; Extracellular Matrix; G-Protein-Coupled Receptors; Generations; Genes; Immunoprecipitation; In Situ; Individual; Inherited; Integral Membrane Protein; Knowledge; Lead; Ligation; Liquid Chromatography; Mass Spectrum Analysis; Membrane; Missense Mutation; Monkeys; Mus; Mutation; Ontology; Pathogenesis; Pathogenicity; Patients; Peripheral; Photoreceptors; Play; Protein Region; Proteins; Recombinant Proteins; Research; Retina; Retinal Degeneration; Retinitis Pigmentosa; Role; Scaffolding Protein; Structural Protein; Techniques; Testing; USH2A gene; Usher Proteins; Usher Syndrome; V1 Receptors; VLGR1 gene; WHRN gene; base; deaf; early experience; extracellular; hearing impairment; insight; legally blind; molecular diagnostics; mutant; novel; programs; protein structure; screening; tandem mass spectrometry; therapeutic development

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.

项目概要 Usherin (USH2A) 和粘附 G 蛋白偶联受体 V1 (ADGRV1) 是两个大的跨膜 光感受器中纤毛周膜复合物（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色素性视网膜炎 (arRP) 和 Usher 综合症（USH）。 USH2A 基因是 arRP 和 USH 最常见的致病基因。后者是一个 arRP 病症与听力损失相结合，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，并变得完全或 最终在法律上失明。目前，arRP 或 USH 尚无治愈方法。在 PMC 的光感受器中，USH2A ADGRV1 通过其细胞内的 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 中的角色。然而，USH2A 和 ADGRV1 的功能仍然很大程度上未知，因为 这两种蛋白质的大部分区域位于光感受器外部，尚未得到充分研究。我们的 使用免疫沉淀和标记蛋白下拉分析与质量相结合的初步研究 光谱法在牛和小鼠中鉴定出几种 USH2A 和 ADGRV1 相互作用的候选蛋白 视网膜。其中一些相互作用已通过小鼠的免疫共沉淀和共定位得到证实 视网膜或通过使用重组蛋白的下拉测定。基于这些初步发现，我们建议 对 USH2A 和 ADGRV1 胞外域相互作用蛋白进行全面、无偏见的分析。在 目标 1，将从牛视网膜中鉴定出潜在的细胞外 USH2A 和 ADGRV1 相互作用蛋白 使用胞外域诱饵结合质谱法进行下拉测定来检测裂解物。在目标 2 中，确定 潜在的 USH2A 和 ADGRV1 胞外域相互作用蛋白将在小鼠和猴子中得到证实 标准生化结合测定和定位研究，以及已知 USH2A 和 ADGRV1 的影响 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 对光感受器中 PMC 功能和视网膜变性的发病机制的新见解 USH2A 和 ADGRV1 缺陷。