# Quantification of Wnt3a, Wnt5a and Wnt16 Binding to Multiple Frizzleds Under Physiological Conditions Using NanoBit/BRET

## TL;DR

This study measures how three Wnt proteins bind to different Frizzled receptors in living cells, helping understand how Wnt signaling is controlled.

## Contribution

The study provides new physiological binding data for Wnt3a, Wnt5a, and Wnt16 with multiple Frizzled receptors using real-time NanoBRET.

## Key findings

- NanoBRET analysis reveals binding affinities of Wnt3a, Wnt5a, and Wnt16 to various Frizzled receptors.
- LRP6 is shown to regulate Wnt/FZD binding in the trimeric complex under physiological conditions.
- Low receptor expression in cells allows more accurate quantification of Wnt-FZD interactions.

## Abstract

Upon engagement of one of the nineteen secreted Wnt signaling proteins with one of the ten Frizzled transmembrane Wnt receptors (FZD1–10), a wide variety of cellular Wnt signaling responses can be elicited, the selectivity of which depends on the following: (1) the specific Wnt-FZD pairing, (2) the participation of Wnt co-receptors and (3) the cellular context. Co-receptors play a pivotal role in guiding the specificity of Wnt signaling, most notably between β-catenin-dependent and -independent pathways, where co-receptors such as LRP5/6 and ROR1/2/PTK7 play major roles, respectively. It remains less understood how specific Wnt/FZD combinations contribute to the selectivity of downstream Wnt signaling, and we lack accurate comparative data on their binding properties under physiological conditions. Here, using fluorescently tagged Wnt3a, Wnt5a and Wnt16 proteins and cell lines expressing HiBiT-tagged Frizzled, we build on our ongoing efforts to provide a complete overview of the biophysical properties of all Wnt/FZD interactions using full-length proteins. Our real-time NanoBRET analysis using living cells expressing low receptor levels provides more accurate quantification of binding and will help us understand how these binary engagements control Wnt signaling outputs. We also provide evidence that LRP6 regulates the binding affinity of Wnt/FZD interactions in the trimeric Wnt-FZD-LRP6 complex.