Ligand Receptor Interactions in the Wnt Signaling Pathway inDrosophila (2002)

Secreted Wnt proteins have numerous signaling functions during development, mediated by Frizzled molecules that act as Wnt receptors on the cell surface. In the genome ofDrosophila, seven Wnt genes (includingwingless; wg), and five frizzledgenes have been identified. Relatively little is known about signaling and binding specificities of different Wnt and Frizzled proteins. We have developed an assay to determine the strength of binding between membrane-tethered Wnts and ligand binding domains of the Frizzled receptors. We found a wide spectrum of binding affinities, reflecting known genetic interactions. Most Wnt proteins can bind to multiple Frizzleds and vice versa, suggesting redundancy in vivo. In an extension of these experiments, we tested whether two different subdomains of the Wg protein would by themselves bind to Frizzled and generate a biological response. Whereas these two separate domains are secreted from cells, suggesting that they form independently folded parts of the protein, they were only able to evoke a response when co-transfected, indicating that both are required for function. In addition to the Frizzleds, members of the LRP family (represented by the arrow gene in Drosophila) are also necessary for Wnt signal transduction and have been postulated to act as co-receptors. We have therefore examined whether a soluble form of the Arrow molecule can bind to Wingless and Frizzled, but no interactions were detected. Secreted Wnt proteins have numerous signaling functions during development, mediated by Frizzled molecules that act as Wnt receptors on the cell surface. In the genome ofDrosophila, seven Wnt genes (includingwingless; wg), and five frizzledgenes have been identified. Relatively little is known about signaling and binding specificities of different Wnt and Frizzled proteins. We have developed an assay to determine the strength of binding between membrane-tethered Wnts and ligand binding domains of the Frizzled receptors. We found a wide spectrum of binding affinities, reflecting known genetic interactions. Most Wnt proteins can bind to multiple Frizzleds and vice versa, suggesting redundancy in vivo. In an extension of these experiments, we tested whether two different subdomains of the Wg protein would by themselves bind to Frizzled and generate a biological response. Whereas these two separate domains are secreted from cells, suggesting that they form independently folded parts of the protein, they were only able to evoke a response when co-transfected, indicating that both are required for function. In addition to the Frizzleds, members of the LRP family (represented by the arrow gene in Drosophila) are also necessary for Wnt signal transduction and have been postulated to act as co-receptors. We have therefore examined whether a soluble form of the Arrow molecule can bind to Wingless and Frizzled, but no interactions were detected. wingless frizzled cysteine-rich domain secreted Frizzled-related protein Frizzled-related protein low density lipoprotein-related protein alkaline phosphatase hemagglutinin neurotactin Among the many signaling events occurring during animal development, the interactions between members of the secreted Wnt protein family and their receptors, the Frizzleds, are prominent (1Bhanot P. Brink M. Samos C.H. Hsieh J.C. Wang Y. Macke J.P. Andrew D. Nathans J. Nusse R. Nature. 1996; 382: 225-230Crossref PubMed Scopus (1235) Google Scholar, 2Yang-Snyder J. Miller J.R. Brown J.D. Lai C.J. Moon R.T. Curr. Biol. 1996; 6: 1302-1306Abstract Full Text Full Text PDF PubMed Scopus (395) Google Scholar, 3Rocheleau C.E. Downs W.D. Lin R. Wittmann C. Bei Y. Cha Y.H. Ali M. Priess J.R. Mello C.C. 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Evangelista C.C. Ferraz C. Ferriera S. Fleischmann W. Fosler C. Gabrielian A.E. Garg N.S. Gelbart W.M. Glasser K. Glodek A. Gong F. Gorrell J.H., Gu, Z. Guan P. Harris M. Harris N.L. Harvey D. Heiman T.J. Hernandez J.R. Houck J. Hostin D. Houston K.A. Howland T.J. Wei M.H. Ibegwam C. et al.Science. 2000; 287: 2185-2195Crossref PubMed Scopus (4854) Google Scholar). Given that there are multiple genes for ligands and receptors, the phenotypic consequences of Wnt signaling in vivo are probably determined not only by where the genes are expressed but also by the strength of binding between these molecules. However, very few studies have examined to what extent different Wnt proteins bind to their receptors (1Bhanot P. Brink M. Samos C.H. Hsieh J.C. Wang Y. Macke J.P. Andrew D. Nathans J. Nusse R. Nature. 1996; 382: 225-230Crossref PubMed Scopus (1235) Google Scholar, 8Rattner A. Hsieh J.C. Smallwood P.M. Gilbert D.J. Copeland N.G. Jenkins N.A. Nathans J. Proc. Natl. Acad. Sci. U. S. 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Nature. 2000; PubMed Scopus Google to the protein from the of determine whether can bind to cell the protein to cells, by a In to the binding of to cells, we not binding of whether soluble Wg bind to soluble we Wg with the secreted to the in Wg not in Wg be by the as to whether a be we and However, a not be The of the binding between Wnts and Frizzleds are in with genetic in the we have not found of the Wnt molecules tested to bind to is a of the Frizzled family and is in in Wnt In the is not by an extracellular but by an with the protein as the binding Hynes M. M. Gu, Q. M.P. D. A. H. M. de F. A. Nature. 1996; PubMed Scopus Google Scholar, R.A. Y. J.M. C.J. Nature. 1996; PubMed Scopus Google Scholar). is a signal J. Chen Wang B. L. M.P. Nature. 2000; PubMed Scopus Google Scholar, Y. G. 1998; PubMed Google Scholar, D. L. S.M. Cell. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar, P.W. S. Y. Brown W. D. M. Curr. 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