Quantitative Profiling of WNT-3A Binding to All Human Frizzled Paralogues in HEK293 Cells by NanoBiT/BRET Assessments.

Paweł Kozielewicz,Gunnar Schulte,Janine Wesslowski,Rawan Shekhani,Stefanie Moser,Carl-Fredrik Bowin,Gary Davidson

doi:10.1021/acsptsci.1c00084

Paweł Kozielewicz, Gunnar Schulte + Show 5 more

Open Access

https://doi.org/10.1021/acsptsci.1c00084

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Abstract

The WNT signaling system governs critical processes during embryonic development and tissue homeostasis, and its dysfunction can lead to cancer. Details concerning selectivity and differences in relative binding affinities of 19 mammalian WNTs to the cysteine-rich domain (CRD) of their receptors—the ten mammalian Frizzleds (FZDs)—remain unclear. Here, we used eGFP-tagged mouse WNT-3A for a systematic analysis of WNT interaction with every human FZD paralogue in HEK293A cells. Employing HiBiT-tagged full-length FZDs, we studied eGFP-WNT-3A binding kinetics, saturation binding, and competition binding with commercially available WNTs in live HEK293A cells using a NanoBiT/BRET-based assay. Further, we generated receptor chimeras to dissect the contribution of the transmembrane core to WNT-CRD binding. Our data pinpoint distinct WNT-FZD selectivity and shed light on the complex WNT-FZD binding mechanism. The methodological development described herein reveals yet unappreciated details of the complexity of WNT signaling and WNT-FZD interactions, providing further details with respect to WNT-FZD selectivity.

Highlights

The WNT signaling system governs critical processes during embryonic development and tissue homeostasis, and its dysfunction can lead to cancer
Using live cell analysis of transiently transfected HEK293A cells overexpressing HiBiT-tagged FZDs, we provide a comparative assessment of binding affinities of eGFP-WNT-3A to all human FZD paralogues using kinetic
The addition of the complementary LgBiT to the system allows rapid and high-affinity association to the HiBiT peptide, forming a stable NanoBiT moiety with a luciferase activity. This setup allows targeted analysis of cell surface receptors due to the cell impermeability of LgBiT, thereby providing a system with less intracellular background luminescence. This method is a modification of a well-established NanoBRET binding assay to study ligand−receptor association,[30] and has been lately employed to study ligand binding to Class A G proteincoupled receptors (GPCRs) and receptor tyrosine kinases.[31−34] Our results demonstrate that eGFP-WNT-3A interacts with full-length human FZDs transiently overexpressed in live HEK293A cells in a paralogue selective manner