# Characterization of Usher Syndrome Type 2-Associated Proteins in the Retina via Affinity Purification-Mass Spectrometry

## TL;DR

This study identifies proteins interacting with Usher syndrome type 2 proteins in the retina, revealing their roles in cell structure and signaling.

## Contribution

Systematic identification of USH2 protein interactors in the retina using affinity purification and mass spectrometry.

## Key findings

- USH2 proteins connect the extracellular matrix to the actin network and signal via Gαi/Gαq.
- ADGRV1 interacts with complexes involved in cell projections and TGFβ signaling.
- Usherin and ADGRV1 interact with proteins related to ECM remodeling and ciliary function.

## Abstract

Usher syndrome is the leading cause of inherited deaf-blindness, with type 2 (Usher syndrome type 2, USH2) being the most common form. USH2A, ADGRV1, and WHRN are the three known USH2 causative genes, which are also linked to isolated retinal degeneration and hearing loss. These genes encode usherin, ADGRV1, and whirlin, respectively, collectively called USH2 proteins. These proteins form a multiprotein complex (USH2 complex) at the periciliary membrane in retinal photoreceptors and at the stereociliary ankle link in inner ear hair cells. The molecular function of the USH2 complex and its disease mechanisms are poorly understood. Currently, there is no cure for diseases caused by mutations in the three USH2 genes. In this study, we employed multiple affinity purification methods combined with mass spectrometry to systematically identify the interaction partners of USH2 proteins in the retina. The ADGRV1 intracellular bait pulled down proteins involved in actin-based cell projections, the chaperone-containing TCP-1 complex, and the Bardet-Biedl syndrome complex. The extracellular domains of ADGRV1 and usherin pulled down proteins related to peptidase regulation, collagen biosynthesis and modification, and elastic fiber formation. The EAR/EPTP repeats of ADGRV1 specifically pulled down TGFβ signaling proteins. Further immunoprecipitation experiments identified, with high confidence, Gαi and Gαq as ADGRV1-interacting proteins, and retinal degeneration and ciliary proteins as interaction partners of USH2 proteins. We also demonstrated that the usherin extracellular domains interact with each other and with ADGRV1. Overall, these findings suggest that the USH2 complex connects the extracellular matrix (ECM) to the intracellular actin network, signals through Gαi and Gαq, and participates in ECM remodeling, TGFβ signaling, cell adhesion, and ciliary function in photoreceptors.

•Affinity purification approaches to systematically explore USH2-associated proteins.•The USH2 complex links to collagen/elastic fiber homeostasis and TGFβ activation.•ADGRV1 interacts with BBS/CCT complex components and actin-associated proteins.•ADGRV1 may signal via Gαi3/Gα11 and PI3K/PKB/mTOR pathways in the retina.•ADGRV1 interacts with usherin and EMILIN3; usherin interacts with EPHA3 and MMP19.

Affinity purification approaches to systematically explore USH2-associated proteins.

The USH2 complex links to collagen/elastic fiber homeostasis and TGFβ activation.

ADGRV1 interacts with BBS/CCT complex components and actin-associated proteins.

ADGRV1 may signal via Gαi3/Gα11 and PI3K/PKB/mTOR pathways in the retina.

ADGRV1 interacts with usherin and EMILIN3; usherin interacts with EPHA3 and MMP19.

Usher syndrome type 2 (USH2) is the leading cause of inherited deaf-blindness and is incurable. The mechanism underlying USH2, especially retinal degeneration, is unclear. To address this, we employed pull-down and immunoprecipitation approaches, coupled with LC-MS/MS, to explore the interactome of USH2 proteins. Our findings suggest that the USH2 protein complex connects extracellular matrix (ECM) constituents with the intracellular actin network, transduces signals via Gαi/Gαq, and participates in ECM remodeling, TGFβ signaling, cell adhesion, and ciliary function in photoreceptors.