Biochemical characterization of Wnt-frizzled interactions using a soluble, biologically active vertebrate Wnt protein.
Biochemical studies of Wnt signaling have been hampered by difficulties in obtaining large quantities of soluble, biologically active Wnt proteins. In this paper, we report the production in Drosophila S2 cells of biologically active Xenopus Wnt8 (XWnt8). Epitope- or alkaline phosphatase-tagged XWnt8 proteins are secreted by concentrated S2 cells in a form that is suitable for quantitative biochemical experiments with yields of 5 and 0.5 mg per liter, respectively. Conditions also are described for the production in 293 cells of an IgG fusion of the cysteine-rich domain (CRD) of mouse Frizzled 8 with a yield of 20 mg/liter. We demonstrate the use of these proteins for studying the interactions between soluble XWnt8 and various Frizzled proteins, membrane anchored or secreted CRDs, and a set of insertion mutants in the CRD of Drosophila Frizzled 2. In a solid phase binding assay, the affinity of the XWnt8-alkaline phosphatase fusion for the purified mouse Frizzled 8-CRD-IgG fusion is approximately 9 nM.
Hsieh
J C
JC
Department of Molecular Biology and Genetics, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA.
Rattner
A
A
Smallwood
P M
PM
Nathans
J
J
D000818:Animals; D001483:Base Sequence; D019556:COS Cells; D002460:Cell Line; D003598:Cytoskeletal Proteins; D029721:Drosophila Proteins; D004331:Drosophila melanogaster; D051157:Frizzled Receptors; D006801:Humans; D007700:Kinetics; D051379:Mice; D008969:Molecular Sequence Data; D017433:Protein Structure, Secondary; D011506:Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D043562:Receptors, G-Protein-Coupled; D017981:Receptors, Neurotransmitter / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D014162:Transfection; D014714:Vertebrates; D051153:Wnt Proteins; D014981:Xenopus; D029961:Zebrafish Proteins
D003598:Cytoskeletal Proteins; D029721:Drosophila Proteins; D051157:Frizzled Receptors; D011506:Proteins; D043562:Receptors, G-Protein-Coupled; D017981:Receptors, Neurotransmitter; D011994:Recombinant Proteins; D051153:Wnt Proteins; D029961:Zebrafish Proteins; C101703:fz2 protein, Drosophila; C094521:wnt8a protein, zebrafish