Abstract
TRPV4 is a cell surface-expressed calcium-permeable cation channel that mediates cell-specific effects on cellular morphology and function. Dominant missense mutations of TRPV4 cause distinct, tissue-specific diseases, but the pathogenic mechanisms are unknown. Mutations causing peripheral neuropathy localize to the intracellular N-terminal domain whereas skeletal dysplasia mutations are in multiple domains. Using an unbiased screen, we identified the cytoskeletal remodeling GTPase RhoA as a TRPV4 interactor. TRPV4-RhoA binding occurs via the TRPV4 N-terminal domain, resulting in suppression of TRPV4 channel activity, inhibition of RhoA activation, and extension of neurites in vitro. Neuropathy but not skeletal dysplasia mutations disrupt TRPV4-RhoA binding and cytoskeletal outgrowth. However, inhibition of RhoA restores neurite length in vitro and in a fly model of TRPV4 neuropathy. Together these results identify RhoA as a critical mediator of TRPV4-induced cell structure changes and suggest that disruption of TRPV4-RhoA binding may contribute to tissue-specific toxicity of TRPV4 neuropathy mutations.
Highlights
Transient receptor potential vanilloid 4 (TRPV4) is a cell surface-expressed calcium-permeable cation channel that mediates cellspecific effects on cellular morphology and function
In ongoing efforts to identify novel TRPV4 protein–protein interactions that are relevant to TRPV4-related neuropathy, we used an unbiased proteomics approach in which we expressed C-terminal FLAGtagged wild type (WT) or neuropathy mutant (R237L) TRPV4 in HEK293T cells followed by immunoprecipitation and liquid chromatography-mass spectrometry
Given that overexpression of RhoA leads to actomyosin contraction, formation of stress fibers, and a rounded cellular morphology[32], we investigated whether these morphological changes on their own could be responsible for the reduced TRPV4 response to hypotonicity, or whether TRPV4–RhoA interaction was necessary for the inhibitory effect
Summary
TRPV4 is a cell surface-expressed calcium-permeable cation channel that mediates cellspecific effects on cellular morphology and function. Inhibition of RhoA restores neurite length in vitro and in a fly model of TRPV4 neuropathy Together these results identify RhoA as a critical mediator of TRPV4-induced cell structure changes and suggest that disruption of TRPV4-RhoA binding may contribute to tissue-specific toxicity of TRPV4 neuropathy mutations. Studies of TRPV4 disease mutants in heterologous systems have demonstrated that both neuropathy and skeletal dysplasia mutations lead to gain of ion channel function, increased basal and stimulated TRPV4-mediated calcium influx, and cytotoxicity that can be rescued with channel antagonists[1,2,7,16,24,25]. Our results demonstrate that neuropathy mutations within the TRPV4 ARD disrupt the ion channel-independent scaffolding function of TRPV4, which may be an important pathological event contributing to neuron-specific disease
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