Daple, a Wnt Signaling Regulator, is a Non‐Receptor GEF for Trimeric G Proteins

Wnt signaling is essential for development and tissue homeostasis, and its dysregulation causes multiple diseases. Wnt ligands activate Frizzled receptors (FZDRs), which belong to the GPCR superfamily. Although trimeric G protein activation has been shown to mediate Wnt signaling, definitive evidence for the direct activation of G proteins by FZDRs remains elusive. This raises the possibility that FZDRs activate G proteins indirectly via intermediate proteins. Recent discoveries have led to the identification of non‐receptor G protein activators that may fulfil this role. Using bioinformatics we identified a putative Guanine nucleotide Exchange Factor (GEF) motif in a protein called Daple. Daple is a cytoplasmic protein previously described as regulator of Wnt signaling that directly binds to the scaffold Dishevelled (Dvl). By using homology modeling, site‐directed mutagenesis, in vitro biochemistry and yeast‐based assays, we demonstrate that Daple is a bona fide GEF with all the biochemical features previously reported for members of the same G protein regulator class (e.g., GIV or Calnuc). Daple binds with submicromolar affinity to Gαi subunits when the G protein is in the inactive but not active conformation and accelerates the rate of nucleotide exchange 2.5 to 3‐fold. Daple also activates Gαi3 in functional assay in yeast that monitors G protein activity. All these features are disrupted upon mutation of the GEF motif. Taken together these results demonstrate that Daple contains a functional GEF motif that binds and activates Gα subunits of the Gi subfamily. Future efforts will focus on testing the hypothesis that Daple regulates Wnt signaling by linking the core Wnt scaffold Dvl to G protein activation. Support: American Cancer society (RSG‐13‐362‐01‐TBE) and NIH (R01GM108733).