NanoBiT- and NanoBiT/BRET-based assays allow the analysis of binding kinetics of Wnt-3a to endogenous Frizzled 7 in a colorectal cancer model.
CRISPR-Cas9
FZD7
NanoBiT/BRET binding
SW480 cells
Wnt
binding affinity shift
Journal
British journal of pharmacology
ISSN: 1476-5381
Titre abrégé: Br J Pharmacol
Pays: England
ID NLM: 7502536
Informations de publication
Date de publication:
13 Apr 2023
13 Apr 2023
Historique:
revised:
06
03
2023
received:
15
11
2022
accepted:
05
04
2023
pubmed:
14
4
2023
medline:
14
4
2023
entrez:
13
4
2023
Statut:
aheadofprint
Résumé
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, FZD SW480 cells were CRISPR-Cas9-edited to insert a HiBiT tag on the N-terminus of FZD With this new assay the binding of eGFP-Wnt-3a to endogenous HiBiT-FZD 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-FZD
Sections du résumé
BACKGROUND AND PURPOSE
OBJECTIVE
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, FZD
EXPERIMENTAL APPROACH
METHODS
SW480 cells were CRISPR-Cas9-edited to insert a HiBiT tag on the N-terminus of FZD
KEY RESULTS
RESULTS
With this new assay the binding of eGFP-Wnt-3a to endogenous HiBiT-FZD
CONCLUSIONS AND IMPLICATIONS
CONCLUSIONS
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-FZD
Types de publication
Journal Article
Langues
eng
Sous-ensembles de citation
IM
Subventions
Organisme : Cancerfonden
ID : CAN2018/715
Organisme : Cancerfonden
ID : 20 0264P
Organisme : Cancerfonden
ID : 20 1102 PjF
Organisme : Deutsche Forschungsgemeinschaft
ID : 504098926
Organisme : Deutsche Forschungsgemeinschaft
ID : 331351713-SFB 1324
Organisme : Knut och Alice Wallenbergs Stiftelse
ID : 2016.0087
Organisme : Novo Nordisk Fonden
ID : NNF22OC0078104
Organisme : Novo Nordisk Fonden
ID : NNF21OC00700
Organisme : Novo Nordisk Fonden
ID : NNF20OC0063168
Organisme : Stiftelsen Lars Hiertas Minne
ID : FO2020-0304
Organisme : Stiftelsen Lars Hiertas Minne
ID : FO2021-0127
Organisme : Vetenskapsrådet
ID : 2019-01190
Informations de copyright
© 2023 The Authors. British Journal of Pharmacology published by John Wiley & Sons Ltd on behalf of British Pharmacological Society.
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