WNT-3A-induced β-catenin signaling does not require signaling through heterotrimeric G proteins

The network of Wingless/Int-1 (WNT)-induced signaling pathways includes beta-catenin-dependent and -independent pathways. beta-Catenin regulates T cell factor/lymphoid enhancer-binding factor (TCF/LEF)-mediated gene transcription, and in response to WNTs, beta-catenin signaling is initiated through engagement of a Frizzled (FZD)/LDL receptor-related protein 5/6 (LRP5/6) receptor complex. FZDs are G protein-coupled receptors, but the question of whether heterotrimeric G proteins are involved in WNT/beta-catenin signaling remains unanswered. Here, we investigate whether acute activation of WNT/beta-catenin signaling by purified WNT-3A requires functional signaling through heterotrimeric G proteins. Using genome editing, we ablated expression of G(s)/G(olf)/G(q)/G(11)/G(12)/G(13)/G(z) in HEK293 (Delta G7) cells, leaving the expression of pertussis toxin (PTX)-sensitive G(i/o) proteins unchanged, to assess whether WNT-3A activates WNT/beta-catenin signaling in WT and Delta G7 cells devoid of functional G protein signaling. We monitored WNT-3A-induced activation by detection of phosphorylation of LDL receptor-related protein 6 (LRP6), electrophoretic mobility shift of the phosphoprotein Dishevelled (DVL), beta-catenin stabilization and dephosphorylation, and TCF-dependent transcription. We found that purified, recombinant WNT-3A efficiently induces WNT/beta-catenin signaling in Delta G7 cells in both the absence and presence of G(i/o)-blocking PTX. Furthermore, cells completely devoid of G protein expression, so called G alpha-depleted HEK293 cells, maintain responsiveness to WNT-3A with regard to the hallmarks of WNT/beta-catenin signaling. These findings corroborate the concept that heterotrimeric G proteins are not required for this FZD- and DVL-mediated signaling branch. Our observations agree with previous results arguing for FZD conformation-dependent functional selectivity between DVL and heterotrimeric G proteins. In conclusion, WNT/beta-catenin signaling through FZDs does not require the involvement of heterotrimeric G proteins.