The WD40-repeat Proteins WDR-20 and WDR-48 Bind and Activate the Deubiquitinating Enzyme USP-46 to Promote the Abundance of the Glutamate Receptor GLR-1 in the Ventral Nerve Cord of Caenorhabditis elegans

Background: USP-46 deubiquitinates the C. elegans glutamate receptor GLR-1. Results: WDR-20 and WDR-48 bind and activate USP-46 in vitro and increase the abundance of GLR-1 in neurons. Conclusion: WD40-repeat proteins stimulate USP-46 activity, resulting in increased GLR-1 stability in neurons and alterations in glutamate-dependent behavior. Significance: WD40-repeat protein regulation of DUB activity is important for glutamate receptor trafficking in vivo. Ubiquitin-mediated endocytosis and degradation of glutamate receptors controls their synaptic abundance and is implicated in modulating synaptic strength. The deubiquitinating enzymes (DUBs) that function in the nervous system are beginning to be defined, but the mechanisms that control DUB activity in vivo are understood poorly. We found previously that the DUB USP-46 deubiquitinates the Caenorhabditis elegans glutamate receptor GLR-1 and prevents its degradation in the lysosome. The WD40-repeat (WDR) proteins WDR20 and WDR48/UAF1 have been shown to bind to USP46 and stimulate its catalytic activity in other systems. Here we identify the C. elegans homologs of these WDR proteins and show that C. elegans WDR-20 and WDR-48 can bind and stimulate USP-46 catalytic activity in vitro. Overexpression of these activator proteins in vivo increases the abundance of GLR-1 in the ventral nerve cord, and this effect is further enhanced by coexpression of USP-46. Biochemical characterization indicates that this increase in GLR-1 abundance correlates with decreased levels of ubiquitin-GLR-1 conjugates, suggesting that WDR-20, WDR-48, and USP-46 function together to deubiquitinate and stabilize GLR-1 in neurons. Overexpression of WDR-20 and WDR-48 results in alterations in locomotion behavior consistent with increased glutamatergic signaling, and this effect is blocked in usp-46 loss-of-function mutants. Conversely, wdr-20 and wdr-48 loss-of-function mutants exhibit changes in locomotion behavior that are consistent with decreased glutamatergic signaling. We propose that WDR-20 and WDR-48 form a complex with USP-46 and stimulate the DUB to deubiquitinate and stabilize GLR-1 in vivo.