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 XWnt...

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Bibliographic Details
Published in:Proceedings of the National Academy of Sciences - PNAS 1999-03, Vol.96 (7), p.3546-3551
Main Authors: Hsieh, Jen-Chih, Rattner, Amir, Smallwood, Philip M., Nathans, Jeremy
Format: Article
Language:English
Subjects:
Animals
Base Sequence
Biochemistry
Biological Sciences
Cell Line
Cell lines
Codons
COS Cells
Cytoskeletal Proteins
Drosophila
Drosophila melanogaster
Drosophila Proteins
Frizzled Receptors
HEK293 cells
Heparin
Humans
Insects
Kinetics
Mice
Molecular biology
Molecular Sequence Data
Protein Structure, Secondary
Proteins
Proteins - chemistry
Proteins - genetics
Proteins - metabolism
Receptors, G-Protein-Coupled
Receptors, Neurotransmitter - chemistry
Receptors, Neurotransmitter - genetics
Receptors, Neurotransmitter - metabolism
Recombinant Proteins - biosynthesis
Recombinant Proteins - chemistry
Recombinant Proteins - metabolism
Sequence Alignment
Sequence Homology, Amino Acid
Transfection
Vertebrates
Wnt Proteins
Xenopus
Zebrafish Proteins
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Summary: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 ≈ 9 nM.
ISSN:0027-8424
1091-6490
DOI:10.1073/pnas.96.7.3546