Mutations in WHRN lead to
Usher syndrome type 2d or to non-syndromic
hearing impairment. The WHRN-encoded gene product whirlin directly interacts with the intracellular regions of the other two
Usher syndrome type 2-associated
proteins, usherin and ADGRV1. In photoreceptor cells, this
protein complex constitutes fibrous links between the periciliary membrane and the connecting cilium. However, the molecular mechanism(s) of
retinal degeneration due to compromised formation and function of the USH2-associated
protein complex remains elusive. To unravel this pathogenic mechanism, we isolated and characterized whirlin-associated
protein complexes from zebrafish photoreceptor cells. We generated transgenic zebrafish that express Strep/FLAG-tagged Whrna, a zebrafish ortholog of human whirlin, under the control of a photoreceptor-specific promoter. Affinity purification of Strep/FLAG-tagged Whrna and associated
proteins from adult transgenic zebrafish retinas followed by mass spectrometry identified 19 novel candidate associated
proteins. Pull down experiments and dedicated yeast two-hybrid assays confirmed the association of Whrna with 7 of the co-purified
proteins. Several of the co-purified
proteins are part of the synaptic
proteome, which indicates a role for whirlin in the photoreceptor synapse. Future studies will elucidate which of the newly identified
protein-
protein interactions contribute to the development of the
retinal phenotype observed in USH2d patients. SIGNIFICANCE: Since
protein-
protein interactions identified using targeted in vitro studies do not always recapitulate interactions that are functionally relevant in vivo, we established a transgenic zebrafish line that stably expresses a Strep/FLAG-tagged ortholog of human whirlin (SF-Whrna) in photoreceptor cells. Affinity purification of in vivo-assembled SF-Whrna-associated
protein complexes from
retinal lysates followed by mass spectrometry, identified 19 novel candidate interaction partners, many of which are enriched in the synaptic
proteome. Two human orthologs of the identified candidate interaction partners, FRMPD4 and Kir2.3, were validated as direct interaction partners of human whirlin using a yeast two-hybrid assay. The strong connection of whirlin with
postsynaptic density proteins was not identified in previous in vitro
protein-
protein interaction assays, presumably due to the absence of a biologically relevant context. Isolation and identification of in vivo-assembled whirlin-associated
protein complexes from the tissue of interest is therefore a powerful methodology to obtain novel insight into tissue specific
protein-
protein interactions and has the potential to improve significantly our understanding of the function of whirlin and the molecular pathogenesis underlying
Usher syndrome type 2.