Immunocytochemical Analysis of Endogenous Frizzled-(Co-)Receptor Interactions and Rapid Wnt Pathway Activation in Mammalian Cells

Abstract

The differential activation of Wnt pathways (canonical: Wnt/β-catenin; non-canonical: planar cell polarity (PCP), Wnt/Ca2+) depends on the cell-specific availability and regulation of Wnt receptors, called Frizzled (FZD). FZDs selectively recruit co-receptors to activate various downstream effectors. We established a proximity ligation assay (PLA) for the detection of endogenous FZD–co-receptor interactions and analyzed time-dependent Wnt pathway activation in cultured cells. Prostate cancer cells (PC-3) stimulated by Wnt ligands (Wnt5A, Wnt10B) were analyzed by Cy3-PLA for the co-localization of FZD6 and co-receptors (canonical: LRP6, non-canonical: ROR1) at the single-cell level. Downstream effector activation was assayed by immunocytochemistry. PLA allowed the specific (siRNA-verified) detection of FZD6–LRP6 and FZD6–ROR1 complexes as highly fluorescent spots. Incubation with Wnt10B led to increased FZD6–LRP6 interactions after 2 to 4 min and resulted in nuclear accumulation of β-catenin within 5 min. Wnt5A stimulation resulted in a higher number of FZD6–ROR1 complexes after 2 min. Elevated levels of phosphorylated myosin phosphatase target 1 suggested subsequent Wnt/PCP activation in PC-3. This is the first study demonstrating time-dependent interactions of endogenous Wnt (co-)receptors followed by rapid Wnt/β-catenin and Wnt/PCP activation in PC-3. In conclusion, the PLA could uncover novel signatures of Wnt receptor activation in mammalian cells and may provide new insights into involved signaling routes.

The role of WNT10B in physiology and disease: A 10-year update

“…WNT10B has been shown to interact with both LRP5 and LRP6 in different tissue-specific contexts. In the prostate cancer cell line PC3, WNT10B was shown by proximal ligation assay (PLA) to co-localize with FZD6 and LRP6 and then induce nuclear β-catenin in 5 min ( Neuhaus et al, 2021 ). In osteoblasts, WNT10B upregulates the transcription of fatty acid metabolism genes via LRP5 (but not LRP6) ( Frey et al, 2015 ).…”

The Possible Role of Electrical Stimulation in Osteoporosis: A Narrative Review

“…The WNT pathway is an evolutionarily conserved signaling pathway that is involved in various biological activities. It can be divided into a canonical Wnt/β-catenin signaling pathway, planar cell polarity pathway, Wnt/Ca+ pathway, and spindle regulation pathway [ 46 , 47 , 48 , 49 , 50 ]. The WNT signaling pathway plays an important role in promoting osteogenesis and inhibiting osteoporosis as well [ 51 , 52 , 53 ].…”