Profiling the TRPV4 ankyrin repeat domain interactome and its disruption by neuromuscular disease-causing mutations

The ankyrin repeat is one of the most abundant protein-protein interaction motifs in eukaryotes yet occurs in only a small number of ion channels. These channels are all members of the transient receptor potential (TRP) superfamily and contain prominent ankyrin repeat domains (ARDs) in their cytoplasmic N termini. In transient receptor potential vanilloid 4 (TRPV4), the importance of this domain has been highlighted by the finding that gain-of-function neuromuscular disease-causing missense mutations cluster on the ARD surface. Little is known currently about the extent of the TRPV4-ARD interactome, nor how it may be altered by disease-causing mutations. Here, we utilized a human proteome microarray to profile the ARD interactomes of WT and mutant TRPV4. Probing of the microarray with TRPV4^WT-ARD revealed 78 interactors, including proteins related to ubiquitination and small GTPase signaling, such as the ubiquitin ligase NEDD4L and the RhoGEF ARHGEF10. In parallel experiments, we also identified the deubiquitinase OTUB2 as an interactor of the proximal N terminus. Comparison of the ARD interactomes of WT and mutant TRPV4 revealed 21 interactions affected by disease-causing mutations. Strikingly, one of these interactors, ARHGEF10, is also mutated in neuromuscular disease. Cell-based studies confirmed that ARHGEF10 exhibits a reduced capacity to coimmunoprecipitate with mutant TRPV4. Furthermore, calcium imaging studies demonstrated that ARHGEF10 overexpression suppressed TRPV4^WT channel activity, but that this inhibition is abrogated by disease-causing mutations. Together, these findings provide insights into the functional roles of an ion channel ARD, as well as their disruption in disease, and offer a resource for future cell-based studies.