Receptor levels determine binding affinity of WNT-3A to Frizzled 7 in a colorectal cancer model

Abstract: WNT binding to Frizzleds (FZD) is a crucial step that leads to the initiation of signalling cascades governing multiple processes during embryonic development, stem cell regulation and adult tissue homeostasis. Recent efforts have enabled us to shed light on WNT-FZD pharmacology in overexpressed HEK293 cell systems. However, it is important to assess ligand binding at endogenous receptor levels as there might be differential binding behaviour in a native environment. Here, we focus on one FZD paralogue: FZD₇, and study its interactions with WNT-3A in a CRISPR-Cas9-edited SW480 colorectal cancer model. SW480 cells were CRISPR-Cas9-edited to insert a HiBiT-tag on the N-terminus of FZD₇, preserving the native signal peptide. Subsequently, these cells were used to study eGFP-WNT-3A association to endogenous and overexpressed HiBiT-FZD₇ using NanoBiT/BRET to measure ligand binding and quantification of NanoBiT-emitted luminescence to assess receptor internalization. eGFP-WNT-3A bound to endogenous HiBiT-FZD7 with significantly higher k_on and with lower K_d than to overexpressed receptors. Importantly, as the fluorescent probe is an agonist, experiments performed in cell lysates demonstrated that eGFP-WNT-3A/HiBiT-FZD₇ binding assessment is not altered by receptor internalization. In conclusion, binding affinities of eGFP-WNT-3A to HiBiT-FZD₇ decreased with increasing receptor concentrations suggesting that HiBiT-FZD₇ overexpression fails to recapitulate ligand binding behaviour in a (patho-)physiologically relevant context where endogenous receptor expression levels are lower.