WNT Receptor Requirements for Dishevelled Phosphorylation

Abstract

The Dishevelled (DVL) protein is a key component of WNT signaling that relays signals from receptors to downstream effectors. It has been shown that following WNT ligand binding to Frizzled (FZD) receptors, DVL is recruited to the membrane and is phosphorylated. Downstream signals of WNT require the presence of DVL. Since FZD receptors are the known receptor for WNT ligands, it has been assumed, but not shown that Wnt‐stimulated DVL phosphorylation is dependent on a FZD receptor. Since there are 19 WNTs, 10 FZDs, 3 DVLs and co‐receptors, such as LRP5/6 and ROR1/2, that play a role in Wnt signaling, it is suspected that the specific combination that forms the signaling complex determines the downstream signaling output. Most WNTs have been shown to activate canonical, β‐catenin dependent signaling, whereas WNT5a can stimulate multiple non‐canonical effector pathways such as planar cell polarity and calcium signaling. We hypothesize that the specific receptors, whether it be ROR1/2 or a particular FZD, that bind to WNT5a determine the non‐canonical pathway that is activated. In order to look further downstream, our primary objective is to determine which receptors are required in response to WNT5a to activate DVL phosphorylation.By using cellular models missing key proteins in the WNT signaling complex, we can determine their individual functions and redundancies. To study WNT‐FZD‐DVL interactions, we are utilizing cell lines which lack all ten FZDs (FZDless), ROR1/2 (Rorless), or DVL1/2/3 (DVLless) and assessing relative expression and phosphorylation of DVL. DVL expression is decreased in RORless cells and ROR1 and ROR2 expression are decreased in DVLless cells. LGK974 Porcupine inhibitor treatment to inhibit endogenous WNT secretion also reduces DVL expression in wildtype cells. In comparison, overexpression of the FZD2 receptor, results in increased ROR2 expression. FZDless cells show no phosphorylation of DVL under endogenous WNT stimulation or in the presence of WNT conditioned media (CM). In contrast, RORless cells show DVL phosphorylation under endogenous WNT expression, but not with LGK974 and subsequent WNT5a CM treatment.In conclusion, expression and phosphorylation of DVL are dependent on the expression of specific WNT receptors. WNT‐induced DVL phosphorylation is dependent on the presence of FZD receptors but expression of ROR receptors is not necessary. However, these requirements are dependent on the particular WNT ligand in question. WNT5a not only requires FZD receptors to stimulate DVL phosphorylation, but also requires ROR receptors. Overall, these findings demonstrate the intricacies of WNT signaling and lends itself to further investigation of these receptor requirements.Support or Funding InformationVan Andel Institute Graduate School Fellowship