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.

Public Health Relevance

USH2A is the most common causative protein for Usher syndrome and autosomal recessive retinitis pigmentosa. While both diseases are incurable, Usher syndrome is also the leading cause of inherited deaf-blindness. In this proposal, we will identify proteins that interact with USH2A and its partner ADGRV1 in the retina in order to understand retinal degeneration associated with USH2A and ADGRV1 defects.

National Institute of Health (NIH)
National Eye Institute (NEI)
Exploratory/Developmental Grants (R21)
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Biology of the Visual System Study Section (BVS)
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Shen, Grace L
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University of Utah
Schools of Medicine
Salt Lake City
United States
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