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

Upon engagement of one of the 19 secreted Wnt signalling proteins with one of the 10 Frizzled transmembrane Wnt receptors (FZD _1-10 ), a wide variety of cellular Wnt signalling responses can be elicited, the selectivity of which depends on: 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.