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NanoBiT‐ and NanoBi...
NanoBiT‐ and NanoBiT/BRET‐based assays allow the analysis of binding kinetics of Wnt‐3a to endogenous Frizzled 7 in a colorectal cancer model
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- Grätz, Lukas (författare)
- Karolinska Institutet
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- Sajkowska‐Kozielewicz, Joanna J. (författare)
- Department of Medical Biochemistry and Biophysics Karolinska Institute Stockholm Sweden;Department of Organic and Physical Chemistry, Faculty of Pharmacy Medical University of Warsaw Warsaw Poland
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- Wesslowski, Janine (författare)
- Institute of Biological and Chemical Systems‐Functional Molecular Systems Karlsruhe Institute of Technology Karlsruhe Germany
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- Kinsolving, Julia (författare)
- Karolinska Institutet
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- Bridge, Lloyd J. (författare)
- Department of Computer Science and Creative Technologies University of the West England Bristol UK
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- Petzold, Katja, 1981- (författare)
- Uppsala universitet,Institutionen för medicinsk biokemi och mikrobiologi,Department of Medical Biochemistry and Biophysics, Karolinska Institute, Stockholm, Sweden,Petzold
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- Davidson, Gary (författare)
- Institute of Biological and Chemical Systems‐Functional Molecular Systems Karlsruhe Institute of Technology Karlsruhe Germany
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- Schulte, Gunnar (författare)
- Karolinska Institutet
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- Kozielewicz, Paweł (författare)
- Karolinska Institutet
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(creator_code:org_t)
- 2023
- 2023
- Engelska.
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Ingår i: British Journal of Pharmacology. - : John Wiley & Sons. - 0007-1188 .- 1476-5381.
- Relaterad länk:
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https://doi.org/10.1...
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https://urn.kb.se/re...
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https://doi.org/10.1...
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http://kipublication...
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Abstract
Ämnesord
Stäng
- Background and PurposeWnt 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 ApproachSW480 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 ResultsWith 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 ImplicationsBinding 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.
Ämnesord
- NATURVETENSKAP -- Biologi -- Biofysik (hsv//swe)
- NATURAL SCIENCES -- Biological Sciences -- Biophysics (hsv//eng)
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