NanoBiT- and NanoBiT/BRET-based assays allow the analysis of binding kinetics of WNT-3A to endogenous 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 cells. However, assessing ligand binding at endogenous receptor expression levels is important as there might be differential binding behaviour in a native environment. Here, we study one FZD paralogue: FZD7 , and analyze its interactions with WNT-3A in live CRISPR-Cas9-edited SW480 cells typifying colorectal cancer. 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 with endogenous and overexpressed HiBiT-FZD7 using NanoBiT/BRET and NanoBiT to measure ligand binding and receptor internalization. Using the newly developed assay, we detect the binding of eGFP-WNT-3A to endogenous HiBiT-FZD7 and compare it with overexpressed receptors. We show that receptor overexpression results in increased membrane dynamics, leading to an apparent decrease in binding on-rate and consequently in higher, up to 10 times, calculated Kd . Thus, measurements of binding affinities of WNT-3A to FZD7 obtained in overexpressed cells are suboptimal compared to the measurements from endogenously-expressing cells. Binding affinity measurements in the overexpressing cells fail to recapitulate ligand binding affinities assessed in a (patho-)physiologically relevant context where receptor expression is lower. Therefore, future studies on WNT-FZD7 binding should be performed using receptors expressed under endogenous promotion.