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

• DOI: 10.1111/bph.16090
• Publication Date: 2023-04-14T02:44:36Z
• Authors: Lukas Grätz; Joanna J. Sajkows...; Janine Wesslowski; Julia Kinsolving; Lloyd J. Bridge; Katja Petzold; Gary Davidson; Gunnar Schulte; Paweł Kozielewicz
• Keywords: info:eu-repo/classification/ddc/570; 570; biology; 610; Life sciences; Frizzled Receptors; Kinetics; HEK293 Cells; Wnt3A Protein; Cell Line, Tumor; Humans; ddc:570; Colorectal Neoplasms; Protein Binding

Background and Purpose Wnt binding to Frizzleds (FZD) is a crucial step that leads the initiation of signalling cascades governing multiple processes during embryonic development, stem cell regulation adult tissue homeostasis. Recent efforts have enabled us shed light on Wnt–FZD pharmacology using overexpressed HEK293 cells. However, assessing ligand at endogenous receptor expression levels important due differential behaviour in native environment. Here, we study FZD paralogue, 7 , analyse its interactions with Wnt‐3a live CRISPR‐Cas9‐edited SW480 cells typifying colorectal cancer. Experimental Approach were insert HiBiT tag N‐terminus preserving signal peptide. These used eGFP‐Wnt‐3a association HiBiT‐FZD NanoBiT/bioluminescence resonance energy transfer (BRET) NanoBiT measure internalization. Key Results With this new assay was compared receptors. Receptor overexpression results increased membrane dynamics, leading an apparent decrease on‐rate consequently higher, up 10 times, calculated K d . Thus, measurements affinities obtained are suboptimal from endogenously expressing Conclusions Implications Binding affinity overexpressing fail replicate assessed (patho)physiologically relevant context where lower. Therefore, future studies should be performed receptors expressed under promotion.