NanoBiT‐ and NanoBiT/BRET‐based assays allow the analysis of binding kinetics of Wnt‐3a to endogenous Frizzled 7 in a colorectal cancer model

Grätz, Lukas; Sajkowska-Kozielewicz, Joanna J.; Wesslowski, Janine; Kinsolving, Julia; Bridge, Lloyd J.; Petzold, Katja; Davidson, Gary; Schulte, Gunnar; Kozielewicz, Paweł

Abstract

Background and Purpose: 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 using overexpressed HEK293 cells. However, assessing ligand binding at endogenous receptor expression levels is important due to differential binding behaviour in a native environment. Here, we study FZD paralogue, FZD7, and analyse its interactions with Wnt-3a in live CRISPR-Cas9-edited SW480 cells typifying colorectal cancer. Experimental Approach: SW480 cells were CRISPR-Cas9-edited to insert a HiBiT tag on the N-terminus of FZD7, preserving the native signal peptide. These cells were used to study eGFP-Wnt-3a association with endogenous and overexpressed HiBiT-FZD7 using NanoBiT/bioluminescence resonance energy transfer (BRET) and NanoBiT to measure ligand binding and receptor internalization. Key Results: With this new assay the binding of eGFP-Wnt-3a to endogenous HiBiT-FZD7 was compared with overexpressed receptors. 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 to FZD7 obtained in overexpressed cells are suboptimal compared with the measurements from endogenously expressing cells. Conclusions and Implications: Binding affinity measurements in the overexpressing cells fail to replicate 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.

• Journal Article Type: Article
• Acceptance Date: Apr 5, 2023
• Online Publication Date: Apr 13, 2023
• Publication Date: Oct 31, 2024
• Deposit Date: May 9, 2023
• Publicly Available Date: Nov 1, 2024
• Journal: British Journal of Pharmacology
• Print ISSN: 0007-1188
• Electronic ISSN: 1476-5381
• Publisher: Wiley
• Peer Reviewed: Peer Reviewed
• Volume: 81
• Issue: 20
• DOI: https://doi.org/10.1111/bph.16090
• Keywords: Pharmacology; binding affinity shift; CRISPR-Cas9; FZD7; NanoBiT/BRET binding; SW480 cells; Wnt
• Public URL: https://uwe-repository.worktribe.com/output/10762252
• Publisher URL: https://bpspubs.onlinelibrary.wiley.com/doi/10.1111/bph.16090