title: Systematic mapping of WNT-FZD protein interactions reveals functional selectivity by distinct WNT-FZD pairs.
abstract: The seven-transmembrane-spanning receptors of the FZD1-10 class are bound and activated by the WNT family of lipoglycoproteins, thereby inducing a complex network of signaling pathways. However, the specificity of the interaction between mammalian WNT and FZD proteins and the subsequent signaling cascade downstream of the different WNT-FZD pairs have not been systematically addressed to date. In this study, we determined the binding affinities of various WNTs for different members of the FZD family by using bio-layer interferometry and characterized their functional selectivity in a cell system. Using purified WNTs, we show that different FZD cysteine-rich domains prefer to bind to distinct WNTs with fast on-rates and slow off-rates. In a 32D cell-based system engineered to overexpress FZD2, FZD4, or FZD5, we found that WNT-3A (but not WNT-4, -5A, or -9B) activated the WNT-β-catenin pathway through FZD2/4/5 as measured by phosphorylation of LRP6 and β-catenin stabilization. Surprisingly, different WNT-FZD pairs showed differential effects on phosphorylation of DVL2 and DVL3, revealing a previously unappreciated DVL isoform selectivity by different WNT-FZD pairs in 32D cells. In summary, we present extensive mapping of WNT-FZD cysteine-rich domain 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 toward different downstream signaling pathways, a phenomenon that could be of great importance in the design of FZD-targeting drugs.
authors.affiliation: From the Section of Receptor Biology and Signaling, Department of Physiology and Pharmacology, Karolinska Institutet, S-171 77 Stockholm, Sweden, the Laboratory of Cellular and Molecular Biology, Center for Cancer Research, NCI, National Institutes of Health, Bethesda, Maryland 20892.
authors.forename: Jacomijn P
authors.initials: JP
authors.lastname: Dijksterhuis
authors.affiliation: the Laboratory of Cellular and Molecular Biology, Center for Cancer Research, NCI, National Institutes of Health, Bethesda, Maryland 20892.
authors.forename: Bolormaa
authors.initials: B
authors.lastname: Baljinnyam
authors.affiliation: the Department of Early Discovery Biochemistry, Genentech, South San Francisco, California 94080, and.
authors.forename: Karen
authors.initials: K
authors.lastname: Stanger
authors.affiliation: the Laboratory of Cellular and Molecular Biology, Center for Cancer Research, NCI, National Institutes of Health, Bethesda, Maryland 20892.
authors.forename: Hakki O
authors.initials: HO
authors.lastname: Sercan
authors.affiliation: the Laboratory of Cellular and Molecular Biology, Center for Cancer Research, NCI, National Institutes of Health, Bethesda, Maryland 20892.
authors.forename: Yun
authors.initials: Y
authors.lastname: Ji
authors.affiliation: the Laboratory of Cellular and Molecular Biology, Center for Cancer Research, NCI, National Institutes of Health, Bethesda, Maryland 20892.
authors.forename: Osler
authors.initials: O
authors.lastname: Andres
authors.affiliation: the Laboratory of Cellular and Molecular Biology, Center for Cancer Research, NCI, National Institutes of Health, Bethesda, Maryland 20892.
authors.forename: Jeffrey S
authors.initials: JS
authors.lastname: Rubin
authors.affiliation: the Department of Early Discovery Biochemistry, Genentech, South San Francisco, California 94080, and hannoush.rami@gene.com.
authors.forename: Rami N
authors.initials: RN
authors.lastname: Hannoush
authors.affiliation: From the Section of Receptor Biology and Signaling, Department of Physiology and Pharmacology, Karolinska Institutet, S-171 77 Stockholm, Sweden, the Faculty of Science, Institute of Experimental Biology, Masaryk University, 611 37 Brno, Czech Republic gunnar.schulte@ki.se.
authors.forename: Gunnar
authors.initials: G
authors.lastname: Schulte
mesh_terms: D000818:Animals; D002460:Cell Line; D051157:Frizzled Receptors / Q000378:metabolism*; D051379:Mice; D010766:Phosphorylation; D025941:Protein Interaction Mapping; D060066:Protein Interaction Maps; D020033:Protein Isoforms / Q000378:metabolism; D051153:Wnt Proteins / Q000378:metabolism*; D060449:Wnt Signaling Pathway; D051176:beta Catenin / Q000378:metabolism
chemical_list: D051157:Frizzled Receptors; D020033:Protein Isoforms; D051153:Wnt Proteins; D051176:beta Catenin
keywords: 32D Cells; Disheveled; Frizzled; Functional Selectivity; LDL Receptor-related Protein 6; Myeloid Cell; Receptor; WNT Pathway; WNT Signaling; β-Catenin (B-catenin)