title: Essential role of the Dishevelled DEP domain in a Wnt-dependent human-cell-based complementation assay.
abstract: Dishevelled (DVL) assembles Wnt signalosomes through dynamic head-to-tail polymerisation by means of its DIX domain. It thus transduces Wnt signals to cytoplasmic effectors including β-catenin, to control cell fates during normal development, tissue homeostasis and also in cancer. To date, most functional studies of Dishevelled relied on its Wnt-independent signalling activity resulting from overexpression, which is sufficient to trigger polymerisation, bypassing the requirement for Wnt signals. Here, we generate a human cell line devoid of endogenous Dishevelled (DVL1- DVL3), which lacks Wnt signal transduction to β-catenin. However, Wnt responses can be restored by DVL2 stably re-expressed at near-endogenous levels. Using this assay to test mutant DVL2, we show that its DEP domain is essential, whereas its PDZ domain is dispensable, for signalling to β-catenin. Our results imply two mutually exclusive functions of the DEP domain in Wnt signal transduction - binding to Frizzled to recruit Dishevelled to the receptor complex, and dimerising to cross-link DIX domain polymers for signalosome assembly. Our assay avoids the caveats associated with overexpressing Dishevelled, and provides a powerful tool for rigorous functional tests of this pivotal human signalling protein.
authors.affiliation: MRC Laboratory of Molecular Biology, Cambridge Biomedical Campus, Francis Crick Avenue, Cambridge CB2 0QH, UK melissag@mrc-lmb.cam.ac.uk mb2@mrc-lmb.cam.ac.uk.
authors.forename: Melissa V
authors.initials: MV
authors.lastname: Gammons
authors.affiliation: MRC Laboratory of Molecular Biology, Cambridge Biomedical Campus, Francis Crick Avenue, Cambridge CB2 0QH, UK.
authors.forename: Trevor J
authors.initials: TJ
authors.lastname: Rutherford
authors.affiliation: Leslie Dan Faculty of Pharmacy, Room 901, University of Toronto, 144 College Street, Toronto, Ontario, Canada M5S 3M2.
authors.forename: Zachary
authors.initials: Z
authors.lastname: Steinhart
authors.affiliation: Leslie Dan Faculty of Pharmacy, Room 901, University of Toronto, 144 College Street, Toronto, Ontario, Canada M5S 3M2.
authors.forename: Stephane
authors.initials: S
authors.lastname: Angers
authors.affiliation: MRC Laboratory of Molecular Biology, Cambridge Biomedical Campus, Francis Crick Avenue, Cambridge CB2 0QH, UK melissag@mrc-lmb.cam.ac.uk mb2@mrc-lmb.cam.ac.uk.
authors.forename: Mariann
authors.identifier: 0000-0002-7170-8706
authors.initials: M
authors.lastname: Bienz
mesh_terms: D001681:Biological Assay / Q000379:methods*; D000072261:Dishevelled Proteins / Q000737:chemistry* / Q000378:metabolism*; D015536:Down-Regulation / Q000187:drug effects; D051157:Frizzled Receptors / Q000378:metabolism; D055786:Gene Knockout Techniques; D057809:HEK293 Cells; D006801:Humans; D009154:Mutation / Q000235:genetics; D054731:PDZ Domains; D010455:Peptides / Q000378:metabolism; D055503:Protein Multimerization / Q000187:drug effects; D013329:Structure-Activity Relationship; D060449:Wnt Signaling Pathway / Q000187:drug effects; D060509:Wnt3A Protein / Q000494:pharmacology*; D051176:beta Catenin / Q000378:metabolism
chemical_list: D000072261:Dishevelled Proteins; D051157:Frizzled Receptors; D010455:Peptides; D060509:Wnt3A Protein; D051176:beta Catenin
keywords: DEP domain; Dishevelled; Frizzled; PDZ domain