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Abstract
The network of Wingless/Int-1 (WNT)-induced signaling pathways includes β-catenin-dependent and -independent pathways. β-Catenin regulates T cell factor/lymphoid enhancer-binding factor (TCF/LEF)-mediated gene transcription, and in response to WNTs, β-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/β-catenin signaling remains unanswered. Here, we investigate whether acute activation of WNT/β-catenin signaling by purified WNT-3A requires functional signaling through heterotrimeric G proteins. Using genome editing, we ablated expression of Gs/Golf/Gq/G11/G12/G13/Gz in HEK293 (ΔG7) cells, leaving the expression of pertussis toxin (PTX)-sensitive Gi/o proteins unchanged, to assess whether WNT-3A activates WNT/β-catenin signaling in WT and ΔG7 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), β-catenin stabilization and dephosphorylation, and TCF-dependent transcription. We found that purified, recombinant WNT-3A efficiently induces WNT/β-catenin signaling in ΔG7 cells in both the absence and presence of Gi/o-blocking PTX. Furthermore, cells completely devoid of G protein expression, so called Gα-depleted HEK293 cells, maintain responsiveness to WNT-3A with regard to the hallmarks of WNT/β-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/β-catenin signaling through FZDs does not require the involvement of heterotrimeric G proteins.
Highlights
The network of Wingless/Int-1 (WNT)-induced signaling pathways includes -catenin– dependent and –independent pathways. -Catenin regulates T cell factor/lymphoid enhancer– binding factor (TCF/LEF)-mediated gene transcription, and in response to WNTs, -catenin signaling is initiated through engagement of a Frizzled (FZD)/LDL receptor–related protein 5/6 (LRP5/6) receptor complex
We ablated expression of Gs/Golf/Gq/G11/G12/G13/Gz in HEK293 (⌬G7) cells, leaving the expression of pertussis toxin (PTX)-sensitive Gi/o proteins unchanged, to assess whether WNT-3A activates WNT/-catenin signaling in WT and ⌬G7 cells devoid of functional G protein signaling
Proteins in receptor-mediated cell responses [16]. This methodological advance has allowed us to conclude that heterotrimeric G proteins are not necessary to mediate major hallmarks of WNT/-catenin signaling and TCF/LEF transcriptional activity in response to recombinant and purified WNT-3A
Summary
WNT/-catenin signaling through FZDs does not require the involvement of heterotrimeric G proteins. WNT stimulation of the pathway leads to FZD/LRP5/6 signalosome formation, which includes phosphorylation of LRP5/6 and recruitment of the scaffold protein disheveled (DVL). Formation of the WNT/ FZD/LRP5/6/DVL complex at the plasma membrane results in the inactivation of the -catenin destruction complex, manifesting in reduced glycogen synthase kinase 3 phosphorylation of -catenin, increasing its cytosolic stabilization and nuclear translocation. Because FZDs are heptahelical receptors, which function as G protein– coupled receptors [4], the question of the involvement of heterotrimeric G proteins in the WNT/-catenin pathway was raised many years ago and has been a question of intense debate ever since [5, 6]. Experimental studies suggest that heterotrimeric G proteins participate in WNT-induced signal transduction to -catenin–mediated transcriptional reg-
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