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

Jochen Neuhaus,Annett Weimann,Mandy Berndt-Paetz

doi:10.3390/ijms222112057

Jochen Neuhaus, Annett Weimann + Show 1 more

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https://doi.org/10.3390/ijms222112057

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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.

Highlights

IntroductionWnt (Wingless and Int1) signaling pathways play essential roles in embryonic development as well as in the maintenance of tissue homeostasis in adults by regulating proliferation, migration, and differentiation
We established a proximity ligation assay (PLA) to detect and quantify the colocalization of FZD6 and co-receptors in PC-3 cells, which indicates an interaction between these molecules
Since LRP6 was reported to be the major co-receptor in Wnt/β-catenin signaling, FZD6 interactions with LRP6 were analyzed to study the canonical Wnt route

Summary

Introduction

Wnt (Wingless and Int1) signaling pathways play essential roles in embryonic development as well as in the maintenance of tissue homeostasis in adults by regulating proliferation, migration, and differentiation. Wnt proteins bind to seven-transmembrane receptors, the so-called Frizzled (FZD) receptors [4]. FZDs can act together with a variety of co-receptors including low-density lipoprotein receptor (LRP) 5 and LRP6, inactive tyrosine-protein kinase transmembrane receptor (ROR) 1 and ROR2, and tyrosine-protein kinase RYK to activate canonical (β-catenin-dependent) and non-canonical (β-cateninindependent) signaling pathways [3,5]. The canonical Wnt/β-catenin pathway is initiated by the formation of a complex between Wnt, FZD, and LRP5/6, resulting in subsequent stabilization and nuclear translocation of the transcriptional regulator β-catenin [6].

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