The differential activation of Wnt pathways (canonical: Wnt/-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–coreceptor
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 -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/-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
| Identifer | oai:union.ndltd.org:DRESDEN/oai:qucosa:de:qucosa:89084 |
| Date | 17 January 2024 |
| Creators | Neuhaus, Jochen, Weimann, Annett, Berndt-Paetz, Mandy |
| Publisher | MDPI |
| Source Sets | Hochschulschriftenserver (HSSS) der SLUB Dresden |
| Language | English |
| Detected Language | English |
| Type | info:eu-repo/semantics/publishedVersion, doc-type:article, info:eu-repo/semantics/article, doc-type:Text |
| Rights | info:eu-repo/semantics/openAccess |
| Relation | 12057 |
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