Deafness‐Associated ADGRV1 Mutation Impairs USH2A Stability through Improper Phosphorylation of WHRN and WDSUB1 Recruitment

Abstract The ankle‐link complex (ALC) consists of USH2A, WHRN, PDZD7, and ADGRV1 and plays an important role in hair cell development. At present, its architectural organization and signaling role remain unclear. By establishing Adgrv1 Y6236fsX1 mutant mice as a model of the deafness‐associated human Y6244fsX1 mutation, the authors show here that the Y6236fsX1 mutation disrupts the interaction between adhesion G protein‐coupled receptor V subfamily member 1 (ADGRV1) and other ALC components, resulting in stereocilia disorganization and mechanoelectrical transduction (MET) deficits. Importantly, ADGRV1 inhibits WHRN phosphorylation through regional cAMP‐PKA signaling, which in turn regulates the ubiquitination and stability of USH2A via local signaling compartmentalization, whereas ADGRV1 Y6236fsX1 does not. Yeast two‐hybrid screening identified the E3 ligase WDSUB1 that binds to WHRN and regulates the ubiquitination of USH2A in a WHRN phosphorylation‐dependent manner. Further FlAsH‐BRET assay, NMR spectrometry, and mutagenesis analysis provided insights into the architectural organization of ALC and interaction motifs at single‐residue resolution. In conclusion, the present data suggest that ALC organization and accompanying local signal transduction play important roles in regulating the stability of the ALC.