Systematic mapping of WNT-FZD protein interactions reveals functional selectivity by distinct WNT-FZD pairs.

Abstract The seven transmembrane spanning receptors of the Class Frizzled (FZD1-10) are bound and activated by the WNT family of lipoglycoproteins, thereby inducing a complex network of signaling pathways. Yet the specificity of interaction between mammalian WNT and FZD proteins and the subsequent signaling cascade downstream of the different WNT-FZD pairs has not been systematically addressed to date. In this report, we determine the binding affinities of various WNT proteins to different members of the FZD family by using biolayer interferometry and characterize their functional selectivity in a cellular system. Using purified WNT proteins, we show that different FZD cysteine rich domains (CRD) prefer to bind to distinct WNTs with fast on-rates and slow off-rates. In a 32D cell-based system engineered to overexpress FZD2, -4 or -5, we find that WNT-3A but not -4, -5A or -9B activates the WNT/β-catenin pathway through FZD2, 4, 5 as measured by phosphorylation of low-density-lipoprotein receptor-related protein 6 (LRP6) and β-catenin stabilization. Surprisingly, different WNT-FZD pairs show differential effects on phosphorylation of disheveled 2/3 (DVL2/3), revealing a previously unappreciated DVL isoform selectivity by different WNT-FZD pairs in 32D cells. In summary, we present extensive mapping of WNT/FZD CRD interactions complemented by analysis of WNT/FZD pair functionality in a unique cell system expressing individual FZD isoforms. Differential WNT-FZD binding and selective functional readouts suggest that endogenous WNT ligands evolved with an intrinsic natural bias towards different downstream signaling pathways, a phenomenon which could be of great importance in the design of FZD-target drugs.